![Extent of SF degradation in vivo. Photomicrographs of H&E-stained, residual fragments of SF in central regions of regenerated bladder domes supported by bilayer BAMG-SF scaffold following 2 weeks (A), 4 weeks [B], 12 weeks (C) of implantation. ( ⁄ ) denotes residual SF fragments. 200Â, scale bars = 200 mm; (D) Statistical analysis of the extent of SF degradation displayed in (A-C).](https://www.researchgate.net/profile/Dongdong-Xiao/publication/277777239/figure/fig1/AS:722373535875073@1549238680207/Extent-of-SF-degradation-in-vivo-Photomicrographs-of-H-E-stained-residual-fragments-of_Q320.jpg)
Content uploaded by Dongdong Xiao
Author content
All content in this area was uploaded by Dongdong Xiao on Feb 04, 2019
Content may be subject to copyright.
Time-dependent bladder tissue regeneration using bilayer bladder
acellular matrix graft-silk fibroin scaffolds in a rat bladder augmentation
model
Yang Zhao
a,3
, Yi He
b,3
, Jian-hua Guo
a
, Jia-sheng Wu
c
, Zhe Zhou
a
, Ming Zhang
a
, Wei Li
c
, Juan Zhou
a
,
Dong-dong Xiao
a
, Zhong Wang
a,2
, Kang Sun
c
, Ying-jian Zhu
d,1
, Mu-jun Lu
a,
⇑
a
Department of Urology, Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200011, China
b
Department of Urology, Jiaxing First Hospital, Jiaxing, Zhejiang Province 314001, China
c
State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, Shanghai 200240, China
d
Department of Urology, Shanghai First People’s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200080, China
article info
Article history:
Received 19 November 2014
Received in revised form 22 May 2015
Accepted 28 May 2015
Available online 3 June 2015
Keywords:
Bladder acellular matrix graft
Silk fibroin
Bladder augmentation
Tissue engineering
abstract
With advances in tissue engineering, various synthetic and natural biomaterials have been widely used in
tissue regeneration of the urinary bladder in rat models. However, reconstructive procedures remain
insufficient due to the lack of appropriate scaffolding, which should provide a waterproof barrier function
and support the needs of various cell types. To address these problems, we have developed a bilayer scaf-
fold comprising a porous network (silk fibroin [SF]) and an underlying natural acellular matrix (bladder
acellular matrix graft [BAMG]) and evaluated its feasibility and potential for bladder regeneration in a rat
bladder augmentation model. Histological (hematoxylin and eosin and Masson’s trichrome staining) and
immunohistochemical analyses demonstrated that the bilayer BAMG-SF scaffold promoted smooth mus-
cle, blood vessel, and nerve regeneration in a time-dependent manner. At 12 weeks after implantation,
bladders reconstructed with the BAMG-SF matrix displayed superior structural and functional properties
without significant local tissue responses or systemic toxicity. These results demonstrated that the
bilayer BAMG-SF scaffold may be a promising scaffold with good biocompatibility for bladder regenera-
tion in the rat bladder augmentation model.
!2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Various congenital and acquired urinary tract diseases, such as
bladder exstrophy, bladder cancer, and neurogenic bladder dys-
function, require bladder reconstruction [1]. Currently, bladder
reconstruction is still one of the greatest surgical challenges in
the field of urology. While enterocystoplasty is still considered
the gold standard and has played a major role in bladder recon-
struction for decades [2], it is associated with a series of complica-
tions, such as recurrent urinary tract infections, electrolyte
imbalance, urinary incontinence, perforation and urolithiasis,
severely impacting the quality of life of patients [3].
Tissue engineering (TE) approaches provide a potential strategy
to minimize these consequences and have great promise for blad-
der repair and reconstruction [4,5]. In recent studies, different
types of scaffolds, constructed mainly from naturally derived
materials and synthetic polymers, are used widely for bladder
reconstruction [6,7]. Bladder acellular matrix grafts (BAMGs) are
intact collagen-based xenogenetic biomaterials derived from pigs
that are prepared using rigorous techniques to remove all cells
[8]. Moreover, BAMGs exhibit good biocompatibility and support
tissue regeneration [9], and several studies have indicated that
BAMGs are the suitable scaffold for bladder reconstruction
[6,10,11]. In addition, BAMGs have also been shown to prevent per-
meation of luminal contents into the abdominal cavity [12].
Although multilayered urothelial tissues have been shown to be
generated by implantation of a simple BAMG scaffold in the blad-
der, these dense and smooth BAMGs cannot promote satisfactory
smooth muscle regeneration, vessel formation, and nerve tissue
regeneration, particularly for seeding cells or host tissue penetra-
tion into the scaffold [10,13]. Silk fibroin (SF) is a unique natural
http://dx.doi.org/10.1016/j.actbio.2015.05.032
1742-7061/!2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
⇑
Corresponding author. Tel.: +86 (021) 63131855; fax: +86 (021) 63087768.
E-mail addresses: zhongwang2000@sina.com (Z. Wang), zhuyingjian_sjtu@126.
com (Y.-j. Zhu), lumujun@163.com (M.-j. Lu).
1
Co-corresponding author. Tel.: +86 (021) 63240090; fax: +86 (021) 63240825.
2
Co-corresponding author. Tel.: +86 (021) 23271699; fax: +86 (021) 63136856.
3
These authors contributed equally to this work and should be viewed as co-first
authors.
Acta Biomaterialia 23 (2015) 91–102
Contents lists available at ScienceDirect
Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat
fibrous protein that has a unique array of properties, including high
structural strength and elasticity, diverse processing plasticity, and
tunable biodegradability [14]. Unfortunately, the simple and com-
pact SF structure is not easily degraded and increases the risk of
urinary stones [15,16].
The ideal scaffold for the regeneration of the urinary bladder
must fulfill specific requirements pertaining to three-dimensional
(3-D) architecture, microenvironmental needs of the various types
of cells in the bladder wall, barrier function, and structural integ-
rity of the bladder [17]. To reach this goal, we have developed a
bilayer scaffold by adding SF directly on the BAMG and lyophilizing
the sample, resulting in the SF-dependent generation of an internal
interconnected porous architecture; this structure was then shown
to increase the extent of degradation and infiltration of host
bladder tissue [16]. We believe that this bilayer BAMG-SF scaffold
may represent a novel promising biomaterial for bladder
reconstruction.
Therefore, in the present study, we examined the feasibility of
the bilayer BAMG-SF scaffold for bladder reconstruction in rats.
The morphological features of the bilayer scaffold were character-
ized by scanning electron microscopy (SEM), and biological perfor-
mance of the bilayer scaffold was analyzed at 2, 4, and 12 weeks
after implantation into the rat bladder. Furthermore, we also eval-
uated the extent of SF scaffold degradation in vivo, inflammatory
responses, and systemic safety.
2. Materials and methods
2.1. Fabrication of bladder acellular matrix grafts (BAMGs)
All animal procedures were approved by the Institutional
Animal Care and Use Committee of the Affiliated 9th People’s
Hospital of Shanghai Jiao Tong University School of Medicine.
Porcine bladder tissues were harvested from 3-month-old pigs
and rinsed with phosphate-buffered saline (PBS; pH 7.2–7.4) to
remove blood. The bladder samples were then transported in
ice-cold PBS to the laboratory immediately. Fat tissues and fascia
around the urinary bladder were removed with scissors. The
urothelium, muscle, and serosal layers were grossly removed by
surgical delamination and washed in distilled water in a stirring
flask (200 rpm) for 48 h at 4 "C, followed by treatment with
0.03% trypsin for 1 h. The bladders were then soaked for 7 days
at 37 "C in 0.2% Triton X-100 (Sigma, St. Louis, MO, USA) and
0.1% (v/v) ammonium hydroxide. The solution was refreshed every
day. The resulting matrix was washed with distilled water for
2 days at 4 "C and sterilized with 75% ethanol. The complete elim-
ination of cellular nuclei in the BAMG was confirmed by histolog-
ical evaluation and quantification of residual DNA content.
2.2. Evaluation of the decellularization efficacy
The BAMG were fixed in 10% buffered formalin, dehydrated, and
embedded in paraffin. For histological examinations, sections
(5 mm) of the BAMG were deparaffinized and stained with H&E
and Masson’s trichrome stainings to evaluate the cellular nucleus
and ECM components. The total DNA in freeze-dried native porcine
bladders and BAMG was extracted using a DNA isolation kit for tis-
sues (Roche Applied Sciences). The samples were weighed, finely
minced using scissors, and digested with RNase and proteinase K.
The remaining DNA was collected and quantified using the
Quant-iT™ PicoGreen
#
dsDNA Assay Kit according to the manufac-
turer’s protocol. The fluorescence of test solution was measured on
a fluorescence microplate reader (excitation, 480 nm; emission,
520 nm; Thermo Scientific Multiskan FC). The amount of DNA in
the native porcine bladders and BAMG was quantified against a
DNA standard curve and expressed as
l
g/mg dry weight of the test
samples. We randomly and respectively selected 6 native porcine
bladders and BAMGs to carry out the DNA content assay. All the
evaluations were repeated 3 times.
2.3. Fabrication of the bilayer BAMG-SF scaffold
The bilayer BAMG-SF scaffold was composed of 3-D porous SF
and BAMG. First, the BAMG was washed with deionized water 10
times to remove the alcohol component, which could negatively
affect the adhesion of SF on the BAMG. Then, the BAMG was
trimmed into 15 mm !15 mm square pieces and placed in a pre-
pared rectangular casting vessel (bottom: 15 mm !15 mm), where
the muscle layer surface of the BAMG is directly exposed to the SF
solution. Preparation of silk fibroin solution and fabrication of highly
interconnected porous silk fibroin scaffolds were accomplished by
following the procedure described previously [18,19]. Next,
300
l
L of SF solution (2% w/v) was slowly poured into the vessel.
The whole material was transferred to the refrigerator and frozen
at "20 "C for 2 days. The frozen composite was then lyophilized to
remove the water and the other solvents to form a porous SF scaf-
fold. Finally, the scaffolds were dried at room temperature under
vacuum for 2 days, which were then sterilized in 75% ethanol, rinsed
in PBS, and subjected to surgical procedures described below.
2.4. SEM
Structural analysis of the matrix prior to implantation was per-
formed by SEM in order to gain insights into the scaffold surface
morphology, interfiber space, and thickness. The samples were fro-
zen with liquid nitrogen, cut into small pieces, and vacuum dried
overnight. The samples were then sputter-coated (Balzers Union
07120/135, Germany) with 10 nm of platinum/gold, and the
images were recorded with a JEOL 6360 LV microscope (Tokyo,
Japan) at 20–25 kV with different magnifications. The average scaf-
fold thickness was measured at random sites through the analysis
of cross-section, top-view, and bottom-view SEM images.
2.5. Mechanical testing
Uniaxial tensile tests were performed as previously described
[16] on an Instron5542 testing frame (Norwood, MA, USA)
equipped with a 1000 N capacity load cell and Biopuls pneumatic
clamps. We randomly selected 6 pieces of BAMGs to evaluate the
mechanical testing. The bilayer BAMG-SF (N= 6) were trimmed
into rectangular strips (40 mm !10 mm) and hydrated in PBS for
at least 24 h to reach a swelling equilibrium prior to testing. Each
material was cut into a dog-bone shape before testing. Test sam-
ples were submerged in a temperature-controlled testing con-
tainer (Biopuls) filled with PBS (37 "C). Tensile testing was
performed by clamping each prepared specimen into a custom fab-
ricated tensile tester with a 50-N load cell. The displacement con-
trol mode with a crosshead displacement rate of 10 mm/min was
used. The initial elastic modulus (EM), ultimate tensile strength
(UTS), and % elongation to failure (ETF) were calculated from
stress/strain plots. The EM was calculated using a least squares
fit within the linear elastic region. The UTS was determined as
the highest stress value attained during the test, and the %ETF
was the last data point before the load decreased by more than
10%. Data were presented as the mean ± standard deviation.
2.6. Animals
Biomaterials were evaluated in a bladder augmentation model
using adult male Sprague Dawley rats (8 weeks old) following
IACUC-approved protocols, as previously described [20]. The
92 Y. Zhao et al. /Acta Biomaterialia 23 (2015) 91–102
animals were divided into two groups: the cystotomy control
group (suture closure; n= 6) and the bilayer BAMG-SF group
(n= 18). Animals in the bilayer BAMG-SF group were sacrificed at
2, 4, and 12 weeks after implantation (n= 6 rats per time point).
Rats in the cystotomy control group were sacrificed at 12 weeks
postimplantation.
2.7. Surgical technique for rat bladder augmentation
Briefly, animals were anesthetized using isoflurane inhalation
and then shaved to expose the surgical site. A low midline laparo-
tomy incision was then made, and the underlying tissue (rectal
muscle and peritoneum) was dissected to expose the bladder. A
1.5-cm longitudinal cystotomy incision was then made at the dome
of the bladder using fine scissors, creating a bladder defect. A
square piece of the bilayer BAMG-SF scaffold (10 mm !10 mm)
was then anastomosed to this site using 7–0 polypropylene absorb-
able sutures. Animals receiving a cystotomy alone were treated
similarly. A watertight seal was confirmed by filling the bladder
with sterile saline via instillation through a 30-gauge hypodermic
needle. After the bladder was augmented by the BAMG-SF scaffold,
the bladder capacity increased by at least 50%. The bladder capacity
of the animals receiving the cystotomy only was similar to that of
normal Sprague Dawley rats. The two groups were assessed inde-
pendently for 2, 4, and 12 weeks after implantation.
2.8. Cystography and urodynamics
Radiographic cystograms and urodynamic studies were serially
performed in the cystotomy control and BAMG-SF scaffold groups
at 2, 4, and 12 weeks after implantation. In each time point, 3 rats
were subjected to radiographic cystograms and another 3 rats
were used for bladder capacity and compliance. All the evaluations
were repeated 3 times. The cystography was performed under gen-
eral anesthesia (pentobarbital, 30 mg/kg i.p.). The bladder was
emptied, and 1–2 mL of contrast medium (30% iopamidol; Polfa,
Poland) was then injected into the bladder through intravesical
instillation under fluoroscopic control. One X-ray film was
obtained for each experimental subject. All animals underwent
urodynamic testing under general anesthesia (pentobarbital,
30 mg/kg i.p.). The bladder was emptied by manual abdominal
pressure. A sterile 2-Fr transurethral polyurethane catheter was
advanced retrograde into the bladder and connected by a
T-shaped tube to a pressure transducer and peristaltic pump for
a constant-rate continuous infusion of saline solution (at room
temperature) into the urinary bladder. The rate of infusion of phys-
iological saline was 100
l
L/min. The urodynamic parameters were
recorded continuously. Bladder compliance was computed from
the equation:
D
V/
D
P, where
D
Pis the threshold pressure (pressure
that triggered voiding) minus resting bladder pressure and
D
Vis
the maximal bladder capacity.
2.9. Histological and Immunofluorescence analyses
At 2, 4, and 12 weeks after implantation, animals implanted
with the BAMG-SF scaffold were euthanized by CO
2
asphyxiation,
and bladders were excised for standard histological processing.
Before being euthanized, cystography and urodynamic analyses
were performed. Briefly, the bladders were rinsed with PBS to
remove blood and then embedded in optimum cutting tempera-
ture compound (OCT) in an optimal orientation to capture the
regenerated and normal tissue regions of the bladder dome within
each section. Sections (6 mm) were cut and then stained with
hematoxylin and eosin (H&E) or Masson’s trichrome (MTS) as pre-
viously described [16]. We made use of the Image-Pro Plus soft-
ware to measure the percentage that the residual SF accounted
for the entire field of vision. We randomly selected 6 fields in both
the peripheral and central regions of regeneration bladder to calcu-
late the percent of residual SF. For immunofluorescence analysis,
contractile smooth muscle markers (
a
-smooth muscle actin
[
a
-SMA] and SM22a); the urothelial-associated protein cytokeratin
(CK) AE1/AE3; and the neuronal and endothelial markers NeuN and
CD31 were detected using the following primary antibodies:
anti-
a
-SMA (Sigma–Aldrich, cat. #A2457, 1:150 dilution),
anti-SM22a (Abcam, Cambridge, MA, USA, cat. #ab14106, 1:150
dilution), anti-cytokeratin AE1/AE3 (Abcam, cat. #ab174707,
1:100 dilution), anti-NeuN (Abcam, cat. #ab104225, 1:150 dilu-
tion), and anti-CD31 (Abcam, cat. #ab228364, 1:100 dilution).
Sections were then incubated with species-matched
Cy3-conjugated secondary antibodies (Millipore, Billerica, MA,
USA), and nuclei were counterstained with 4
0
,6-diamidino-2-phe
nylindole. Specimens were visualized using a Nikon Eclipse
TE2000-U fluorescence microscope (Nikon Instruments Inc.,
Melville, NY, USA). Representative images were acquired using
Axiovision software (version 4.8). We randomly selected 6 fields
of vision to carry out corresponding calculation in each slide. All
the evaluations were repeated 3 times using different slides.
2.10. Local tissue response
Giemsa staining (for detection of eosinophils) and immunohis-
tochemical staining (for detection of macrophages) were used to
assess acute and chronic inflammatory cell infiltration, respec-
tively. Giemsa staining (Sigma–Aldrich) was performed using stan-
dard protocols. For immunohistochemical staining, markers of
chronic inflammation response and macrophages were detected
using mouse anti-rat CD68 monoclonal antibodies (Abcam, cat.
#ab31630, 1:150 dilution). The sections were then incubated with
goat anti-mouse biotinylated secondary antibodies. Antibody bind-
ing was observed after incubation with stable diaminobenzidine
for 2 min, and color development was terminated by washing with
distilled water. We randomly selected 6 fields of vision to calculate
the number of eosinophils and macrophages in each slide. All the
evaluations were repeated 3 times using different slides.
2.11. Systemic safety evaluation
For hematological studies, routine blood tests, serum chemistry
measurements, and serum electrolyte measurements were per-
formed at 1, 2, 4, 8, and 12 weeks postimplantation. For urinalysis
and urine chemistry samples, routine urinalysis was performed at
1, 2, 4, 8, and 12 weeks postimplantation. All experiments were
performed in the clinical laboratory of the 9th People’s Hospital
affiliated with Shanghai Jiao Tong University School of Medicine.
2.12. Statistical analysis
All statistical evaluations were performed with GraphPad Prism
v5.0 Software. Quantitative measurements for the DNA content,
urodynamic and immunofluorescence parameters were analyzed
with an independent student t-tests and the inflammatory cell
influx was analyzed by one-way analysis of variance (ANOVA) with
a Bonferroni post hoc test (parametric). All data were expressed as
means ± standard deviations. Differences with Pvalues of less than
0.05 were considered statistically significant.
3. Results
3.1. Evaluation of the decellularization efficacy
Evaluation of the decellularization efficacy of the bladder acel-
lular matrix grafts (BAMGs) was shown (Fig. 1A–D). Histological
Y. Zhao et al. /Acta Biomaterialia 23 (2015) 91–102 93
specimen of the BAMG was white and high water content extracel-
lular matrix (ECM) shown (Fig. 1A). H&E and Masson’s trichrome
stainings of the BAMG revealed that the cellular nuclei were almost
eliminated and ECM composed mainly of collagen fibers was pre-
served (Fig. 1B and C). The results from the analysis of the DNA
content of BAMG and the native porcine bladder confirmed the evi-
dence from histological analysis. In detail, the DNA content of the
BAMG (0.027 ± 0.008
l
g/mg) was significantly reduced compared
with native bladders (2.638 ± 0.313
l
g/mg) (p< 0.05) (Fig. 1D).
3.2. SEM and mechanical analyses
Structural analyses of the bilayer BAMG-SF scaffold (Fig. 1E–H)
revealed the formation of a bilayer structure with distinct materi-
als. The thickness of bilayer BAMG-SF scaffold was approximately
4 mm which consisted of a dense BAMG matrix layer and a porous
SF layer. The SF layer was tightly interfaced with the underlying
BAMG tissue. The SF layer resembled a foam configuration with
large pores (pore size, approximately 100–200
l
m) interconnected
by a network of smaller pores dispersed along their periphery. This
SF layer was generated by lyophilizing silk solutions and was
located on the external face of the relatively smooth, nonporous
BAMG layer (2 mm thick).
The mechanical properties of bilayer BAMG-SF scaffolds prior to
implantation were assessed by determining the EM, UTS, and %ETF
(Fig. 1I). The bilayer BAMG-SF scaffold showed a UTS of
0.39 ± 0.09 MPa, an EM of 3.50 ± 0.61 MPa, and a %ETF of
88.17% ± 18.16%.
3.3. Rat bladder augmentation
Augmentation cystoplasty was a feasible approach for surgical
integration of the bilayer BAMG-SF scaffold into bladder defects
(Fig. 2A and B). Gross tissue evaluations revealed host tissue
ingrowth spanning the entire area of the original implantation site
in animals implanted with the BAMG-SF graft, with trivial contrac-
tion observed between the marking sutures (Fig. 2C). In addition,
urinary crystals and stones were discovered by macroscopic exam-
ination in the BAMG-SF and cystotomy groups after euthanasia.
3.4. Cystography
Retrograde cystography in the cystotomy control and BAMG-SF
scaffold groups was carried out at 2, 4, and 12 weeks after implan-
tation (Fig. 3). Over the duration of the study, we observed increas-
ing smooth contour profiles in the reconstructed bladders in the
bilayer BAMG-SF group (Fig. 3A–C). Furthermore, cystography
showed that bladders reconstructed with the bilayer BAMG-SF
scaffolds exhibited adequate capacity, regular shape, and smooth
configuration at 12 weeks postimplantation, similar to the obser-
vations in the cystotomy group (Fig. 3C and D). Finally, there was
no evidence of contrast extravasation or fistulas at each time point.
3.5. Extent of SF degradation in vivo
Over the course of the 12-week study period, the bilayer
BAMG-SF scaffolds underwent degradation gradually (Fig. 4). The
majority of the residual SF scaffold was located within the lamina
propria of the bladder, and the SF matrix was highly fragmented. At
2 weeks, the host tissue began to permeate into the SF layer, where
the bulk of the SF scaffolds was found, and SF accounted for
56 ± 9.9% of the total area within the lamina propria of the bladder.
At 4 weeks, the volume of residual SF began to shrink, and SF
accounted for only 34.1 ± 10.1% of the total area. Finally, at
12 weeks, little residual SF was found (with SF accounting for only
3.6 ± 1.6% of the total area) throughout the regenerated tissue.
3.6. Evaluation of inflammatory cell influx
At week 2 after implantation, 18.5 ± 7.9 eosinophils/mm
2
and
35.5 ± 12.0 macrophages/mm
2
were identified in the grafts.
However, there was a significant decrease in the number of eosino-
phils in the grafts at week 4 (8.0 ± 3.7 cells/mm
2
,P< 0.05), and
eosinophil counts continued to decrease, reaching
7.2 ± 2.7 cells/mm
2
at week 12. The number of macrophages
rapidly increased to 85.7 ± 21.6 cells/mm
2
(P< 0.05) at week 4;
however, there was a significant decrease in macrophage numbers
at 12 weeks (29.5 ± 10.0 cells/mm
2
,P< 0.05; Fig. 5).
3.7. Histological examination
Gross histological examination of bladder tissue sections (H&E
and MTS staining) in the BAMG-SF group from 2 weeks to 12 weeks
postimplantation demonstrated that ingrowth of connective tissue
from the host bladder wall into both the radial peripheral and cen-
tral regions of the original implantation sites was increased over
time (Fig. 6). In addition, longitudinal sections of organization were
observed throughout the de novo bladder wall in the bilayer
BAMG-SF group, with distinct regions including a multilayer
urothelium, an extracellular matrix-rich lamina propria, and an
outer layer of smooth muscle bundles that resembled the tissue
architecture of native control tissue.
3.8. Immunofluorescence analyses
Immunofluorescence assessments (Fig. 7A) revealed regenera-
tion of the urothelium. The percentage of CK area/total area at
2 weeks after BAMG-SF scaffold implantation was lower than that
in the cystotomy group (2.8 ± 1.3% versus 8.3 ± 2.0%, respectively,
P< 0.05). After 4 or 12 weeks in vivo, the percentages of CK area/-
total area were both significantly higher than that in the cystotomy
group (21 ± 4.9% and 18.2 ± 4.7% versus 8.3 ± 2.0%, respectively,
P< 0.05; Fig. 7B). In addition, the expression of
a
-SMA in the recon-
stituted smooth muscle layers supported by the BAMG-SF scaffold
indicated that smooth muscle regeneration had occurred, similar
to the results in the cystotomy group (Fig. 7A). At 2 weeks after
implantation, little smooth muscle infiltration was observed with
an irregular distribution along the edges and near luminal surface
of the BAMG-SF scaffold from the host bladder wall into the radial
peripheral region of the original implantation site. At 4 weeks after
implantation, we observed a more regular arrangement of regener-
ated smooth muscle that had started to grow into the central zone
of the original implantation site. Regenerated smooth muscle in
the BAMG-SF group improved over time. Histomorphometric anal-
ysis (Fig. 7C) revealed that the number of de novo
a
-SMA-positive
smooth muscle bundles supported by the BAMG-SF scaffold was
significantly lower than that in the cystotomy group (7.3 ± 2.4%
and 19.8 ± 5.1%, versus 50.1 ± 7.0% respectively, P< 0.05). The
number of
a
-SMA-positive smooth muscle bundles increased con-
tinuously with time after bladder reconstruction with the
BAMG-SF scaffold, reaching values similar to those of the cysto-
tomy control after 12 weeks (46.1 ± 7.3% versus 50.1 ± 7.0%,
respectively, P> 0.05). Expression of the neuronal marker NeuN
was found to be localized to the suburothelial region of the regen-
erated bladder walls in each group (Fig. 7A). However, the density
of NeuN-positive cells in the BAMG-SF group at 2, 4, and 12 weeks
after surgery was still significantly lower than that in the cysto-
tomy group (P< 0.05), reaching only 6.6% (32.2 ± 12.4/mm
2
), 20%
(97.3 ± 24.3/mm
2
), and 50.2% (245.7 ± 76.0/mm
2
) that of the cysto-
tomy control (488.0 ± 83.2/mm
2
), respectively (Fig. 7D).
Additionally, immunofluorescence analyses revealed that the de
novo vascularization observed in the regenerated tissues was ves-
sels containing prominent CD31-positive endothelial cells present
94 Y. Zhao et al. /Acta Biomaterialia 23 (2015) 91–102
Fig. 1. Evaluation of the decellularization efficacy and structural and mechanical analyses of the bladder acellular matrix graft-silk fibroin (BAMG-SF) scaffold. (A) The gross
view of the BAMG scaffold. Scale bar = 1 cm. (B–C) Photomicrographs of H&E (B) and Masson’s trichrome (C) staining of the BAMG. Scale bar = 200
l
m. (D) Statistical analysis
of DNA content of the native bladder and BAMG. (E) The gross view of BAMG-SF scaffold prior to implantation. Scale bar = 1 cm. (F–H) Photomicrographs of representative
scanning electron microscopy images demonstrating cross-sectional, top and bottom views of scaffold configurations. F, scale bar = 800
l
m; G/H, scale bar = 200
l
m. (I)
Evaluation of ultimate tensile strength (UTS), elastic modulus (EM), and % elongation to failure (ETF) in bilayer BAMG-SF scaffold. Means ± standard deviation per data point.
Fig. 2. Photomicrographs of various surgical stages of bladder augmentation with bilayer BAMG-SF scaffold and gross morphology of regenerated tissue and urinary stones.
(A) Extrusion and defect creation of bladder. (B) Anastomosis of bi-layer BAMG-SF scaffold (1 cm !1 cm) into the bladder defect. (C) Regenerated tissues present in vivo
within the original implantation sites supported by bi-layer BAMG-SF scaffold after 12 weeks of implantation. (D) Gross appearance and urinary stones following 12 weeks
post-op. The curve marks the location of the incision. The arrows point out original marking sutures between native bladder and the reconstructed area. Scale bar = 1 cm.
Fig. 3. Retrograde cystography at different postoperative time points. Retrograde cystography in cystotomy group (A) and BAMG-SF scaffold group at 2 weeks (B), 4 weeks (C)
and 12 weeks (D) post-op respectively. Scale bar = 1 cm.
Y. Zhao et al. / Acta Biomaterialia 23 (2015) 91–102 95
in all groups (Fig. 7A). The number and diameter of vessels contain-
ing CD31-positive endothelial cells in the BAMG-SF group
improved over time and was significantly lower than that in the
cystotomy group at 2 and 4 weeks after implantation (BAMG-SF
group at 2 weeks and 4 weeks versus cystotomy group: number
of vessels, 9.3 ± 3.1/mm
2
and 36.5 ± 8.8/mm
2
versus
60.8 ± 10.7/mm
2
, respectively, P< 0.05; diameter of vessels,
23.5 ± 5.2
l
m and 55.2 ± 18.8
l
m versus 117.3 ± 37.9
l
m, respec-
tively, P< 0.05). The number and diameter of vessels increased
continuously after bladder reconstruction with the BAMG-SF scaf-
fold, reaching values similar to those in the cystotomy control after
12 weeks (BAMG-SF group at 12 weeks versus cystotomy group:
number of vessels, 65.5 ± 14.3/mm
2
versus 60.8 ± 10.7/mm
2
,
respectively, P> 0.05; diameter of vessels, 92.5 ± 24.0
l
m at
12 weeks versus 117.3 ± 37.9
l
m, respectively, P> 0.05; Fig. 7E).
3.9. Bladder capacity and compliance evaluation
The time course of functional status following bladder replace-
ment with the BAMG-SF scaffold or cystotomy was evaluated with
urodynamic studies at baseline (pre-implantation) and at 2, 4, and
12 weeks after implantation with triplicate detection (Fig. 8).
Baseline bladder capacity was 0.40 ± 0.04 mL, and bladder compli-
ance was 7.48 ± 0.94 mL/cm H
2
O. In the cystotomy group, bladder
capacity did not differ significantly in comparison with
pre-implantation values at different postimplantation time points
(P> 0.05). However, the bladder capacity increased continuously
with time after bladder reconstruction with the BAMG-SF scaffold,
reaching 183% of that of the pre-implantation value after 12 weeks,
and differences appeared to be significant (0.73 ± 0.21 mL versus
0.40 ± 0.04 mL, respectively, P< 0.05). In addition, bladder
Fig. 4. Extent of SF degradation in vivo. Photomicrographs of H&E-stained, residual fragments of SF in central regions of regenerated bladder domes supported by bilayer
BAMG-SF scaffold following 2 weeks (A), 4 weeks [B], 12 weeks (C) of implantation. (
⁄
) denotes residual SF fragments. 200!, scale bars = 200 mm; (D) Statistical analysis of
the extent of SF degradation displayed in (A–C).
Fig. 5. Differential inflammatory responses elicited by BAMG-SF scaffold groups at different time. (A–D) Giemsa staining for eosinophils (acute inflammation response,
eosinophils denoted by arrows) at week 2, 4, 12; (
⁄
) denotes P< 0.05. (E–H) CD68 staining for macrophages (chronic inflammation response, macrophages denoted by
triangle) at week 2, 4, 12. 200!, scale bar = 200 mm.
96 Y. Zhao et al. / Acta Biomaterialia 23 (2015) 91–102
compliance in the cystotomy group increased continuously over
time, although the differences were not significant (P> 0.05). In
the BAMG-SF group, bladder compliance at 2 weeks was signifi-
cantly lower than that at baseline (4.98 ± 1.53 mL/cm H
2
O versus
7.48 ± 0.94 mL/cm H
2
O, respectively, P< 0.05) and recovered to
baseline levels at 4 and 12 weeks (6.62 ± 2.42 mL/cm H
2
O and
10.13 ± 2.79 mL/cm H
2
O, respectively, P> 0.05).
3.10. Hematological and urinalysis findings
Serum chemistry (Table 1) and urinalysis studies (Table 2) were
performed at weeks 1, 2, 4, 8, and 12 postimplantation. Leukocyte
counts initially rose 1 week after surgery and normalized at
4 weeks in the two groups. The development of neutrophil seg-
ment cells was similar between groups. Routine blood tests per-
formed at 1, 4, 8, and 12 weeks postimplantation showed no
significant differences between the cystotomy and BAMG-SF
groups. However, leukocyte (neutrophil) counts were higher in
the BAMG-SF group than in the cystotomy group at 2 weeks
postimplantation. Serum chemistry and serum electrolytes
remained within normal limits in the two groups throughout the
study period. In addition, gross hematuria was found in the first
2–3 days after surgery and subsequently disappeared. Urinalysis
in both groups revealed increased red blood counts up to 2 weeks
after implantation; red blood cell counts were then normalized by
4 weeks postimplantation. A rise in white blood counts was
observed in the cystotomy group at up to 2 weeks after implanta-
tion. However, white blood cell counts were normalized by
8 weeks postimplantation. Red and white blood cell counts were
higher in the BAMG-SF group than in the cystotomy group at 1
and 2 weeks. Other urine parameters did not show abnormal find-
ings at any time point.
Fig. 6. Histological evaluations (H&E and MTS analyses) of bladder tissue regeneration augmented with BAMG-SF scaffolds at 2, 4, 12 weeks after surgery as well as non-
augmented cystotomy control. (1st and 2nd rows) Photomicrographs of gross bladder (longitudinal section of H&E and MTS-staining). 40!, scale bars = 3 mm. Brackets
represent sites of original scaffold implantation. (3rd rows) Magnification of global tissue regeneration bracketed in the 1st row. 40!, scale bars = 1 mm. (4th and 5th rows)
Magnified de novo urothelium (UE) and smooth muscle (SM) tissue formation displayed in the 3rd row. 40!, scale bars = 200
l
m.
Y. Zhao et al. / Acta Biomaterialia 23 (2015) 91–102 97
Fig. 7. Immunofluorescence and histomorphometric assessments of regenerated bladder domes supported by the BAMG-SF scaffold at 2, 4, 12 weeks after surgery as well as
cystotomy control group. (A) Photomicrographs of cytokeratins (CK); protein expression of smooth muscle contractile markers (
a
-SMA); Neuronal Marker (NeuN); blood
vessel endothelial marker (CD31) in the regenerated bladder tissue. UE = urothelium; SM = smooth muscle; V denotes vessels and arrows denotes neuronal lineages. For all
panels, respective marker expression is displayed in red (Cy3 labeling) and blue denotes DAPI nuclear counterstain. 200!, scale bars in all panels = 200um.
Histomorphometric analysis of the extent of regenerated CK + epithelium (B),
a
-SMA + smooth muscle bundles (C), NeuN + neuronal boutons (D) and CD31 + vessels (E)
present in the original surgical sites of control and scaffold groups. (
⁄
) denotes P< 0.05 in comparison with cystotomy group.
98 Y. Zhao et al. / Acta Biomaterialia 23 (2015) 91–102
4. Discussion
A suitable scaffold plays an important role during the tissue
engineering bladder regeneration. Previous studies found the sim-
ple acellular matrices like BAMG and SIS scaffold could induce fast
urothelium regeneration but not the smooth muscle cells, espe-
cially in large area defect [21,22]. The dense structure of BAMG
can impede penetration of urine into the abdominal cavity [6].
The porous structure of synthetic polymers like PGA, PLGA and silk
fiber could provide a sufficient space for seeding or surrounding
cells ingrowth [23–25]. But the porous biopolymers also have the
drawbacks of urine leakage directly into smooth muscle layer
and the abdominal cavity and fragile mechanical strength not
afforded to suture. Recently, some new bilayered scaffold combi-
nes the advantages of each scaffold layer for bladder TE and serves
as a barrier between urine and the viscera while accommodating
sufficient numbers and various types of cells in the regenerated
bladder wall, facilitating bladder reconstruction [17,26]. In this
article, we prepared a BAMG-SF bilayer scaffold, which exhibited
characteristics of a bilayered scaffold described above. SEM analy-
ses of the bilayer BAMG-SF scaffold showed that it consisted of a
dense BAMG matrix layer and a porous SF layer. The dense
BAMG layer mainly plays a role in waterproofing, while the porous
SF layer provides an excellent structure for cell ingrowth and
proliferation. Thus, our data demonstrated that this bilayer
BAMG-SF scaffold may have applications in bladder tissue regener-
ation in the future.
The important goal of a decellularization protocol is to com-
pletely remove cellular materials for eliminating the undesirable
immune-mediated rejection. We think that more than 99% of the
cells are removed by our decellularization protocol and there are
almost no residual cells. However, the adopted decellularization
protocol appears time-consuming (10 days). In the last few years,
decellularization techniques have been greatly developed. Recent
literatures prove that a quick decellularization protocol reduces
the adverse effects of the decellularizing agents upon ECM pro-
teins, tissue 3D architecture and tissue biomechanical properties
[27–29]. These quick decellularization protocols may lay a solid
basis for further optimizing the scaffold.
Of the initial 24 rats in this study, three died before the end of
the study period. The survival rate in the BAMG-SF group was
slightly lower than that in the cystotomy group (83.3% [15/18 rats]
versus 100% [6/6 rats], respectively). All spontaneous animal
deaths occurred within the first week postimplantation.
Postmortem analyses provided evidence of either scaffold perfora-
tion or dehiscence at the suture line between the implant and the
native bladder wall in all three dead animals, and urinary ascites
were evident (data not shown). The remaining animals could void
Fig. 8. Bladder capacity (A) and compliance (B) were evaluated at pre-operation time and at 2, 4 and 12 weeks after surgery in both cystotomy control and BAMG-SF groups.
(
⁄
) denotes P< 0.05 in comparison with pre-operation value.
Table 1
Serum chemistry in the cystotomy and BAMG-SF groups at different time points. ALT: alanine aminotransferase; AST: aspartate aminotransferase; BUN: blood urea nitrogen; Cr:
creatinine.
Time (weeks) Leukocyte (10
9
/L) Neutrophil (10
9
/L) ALT (U/L) AST (U/L) BUN (mmol/L) Cr (
l
mol/L) Na (mmol/L) K (mmol/L) Cl (mmol/L)
Preop
Cystotomy group 5.9 ± 0.9 0.6 ± 0.1 49 ± 13 118 ± 14 5.8 ± 1.0 49 ± 5 142 ± 2 5.3 ± 0.4 105 ± 2
BAMG-SF group 6.1 ± 0.8 0.6 ± 0.1 52 ± 12 115 ± 16 5.6 ± 0.8 47 ± 4 142 ± 2 5.1 ± 0.3 105 ± 2
1 weeks
Cystotomy group 7.9 ± 1.0 0.9 ± 0.2 52 ± 10 116 ± 12 5.6 ± 1.0 47 ± 3 141 ± 2 5.3 ± 0.2 105 ± 2
BAMG-SF group 9.0 ± 0.9 1.1 ± 0.4 55 ± 9 111 ± 15 5.6 ± 0.9 48 ± 4 140 ± 1 5.2 ± 0.3 105 ± 1
2 weeks
Cystotomy group 9.2 ± 1.1 1.0 ± 0.1 53 ± 15 121 ± 12 5.4 ± 0.8 50 ± 5 140 ± 1 5.3 ± 0.4 105 ± 1
BAMG-SF group 11.6 ± 1.3 1.3 ± 0.2 52 ± 12 117 ± 13 5.7 ± 1.1 48 ± 3 143 ± 1 5.2 ± 0.2 106 ± 1
4 weeks
Cystotomy group 6.3 ± 1.5 0.6 ± 0.2 53 ± 9 118 ± 9 5.5 ± 0.7 52 ± 4 142 ± 2 5.2 ± 0.3 105 ± 1
BAMG-SF group 6.5 ± 1.3 0.7 ± 0.1 54 ± 13 115 ± 12 5.8 ± 0.9 51 ± 4 142 ± 1 5.2 ± 0.1 105 ± 1
8 weeks
Cystotomy group 6.5 ± 0.9 0.7 ± 0.1 55 ± 12 123 ± 15 5.7 ± 0.7 48 ± 3 143 ± 2 5.2 ± 0.2 106 ± 2
BAMG-SF group 6.6 ± 1.1 0.6 ± 0.1 53 ± 11 116 ± 10 5.3 ± 0.6 47 ± 5 142 ± 2 5.3 ± 0.1 106 ± 1
12 weeks
Cystotomy group 6.1 ± 1.0 0.6 ± 0.2 51 ± 8 114 ± 12 6.0 ± 1.0 49 ± 3 140 ± 3 5.2 ± 0.3 106 ± 1
BAMG-SF group 6.3 ± 0.7 0.7 ± 0.1 54 ± 12 118 ± 16 5.7 ± 0.6 49 ± 4 143 ± 2 5.2 ± 0.5 105 ± 1
Y. Zhao et al. / Acta Biomaterialia 23 (2015) 91–102 99
spontaneously without a catheter. Gross examination of the blad-
ders revealed adhesions of the bladder to adjacent fat and intestine
to varying degrees. Negligible scar formation and graft shrinkage
were observed in the bladder tissue following augmentation with
the BAMG-SF scaffold.
In this study, we used a scaffold that was 10 mm !10 mm in
size and augmented the bladder capacity by at least 50%.
Therefore, the greater tendency for animal death from scaffold
urine leaks observed in this study was presumably related to the
increase in scaffold area used between the rat and murine models
[16]. In addition, bladder stone formation was first found at the
second week after implantation. Previous literatures had reported
that the rough surface of simple scaffold and incompletely
absorbed matrix fragments within the bladder lumen could serve
as niduses for stone formation, which also was induced by the
inflammatory response caused by the nude biologic scaffold
[30,31]. So we suppose the urothelium seeding on the BAMG scaf-
fold before in vivo implantation can make the luminal surface of
the scaffold smoother and hamper the urine crystals precipitate
and the direct inflammatory response on the scaffold, conse-
quently reducing the incidence of stone formation.
During the 12-week implantation period, the volume and area
of residual SF remnants significantly decreased. In addition, exten-
sive fragmentation of residual SF remnants was noted, with quali-
tatively higher levels of residual scaffold remnants present within
the central bladder wall in comparison with the radial peripheral
areas. These results suggested that degradation of the SF layer pro-
ceeded in a radial fashion from the border of the original implanta-
tion site toward the center regions as host tissue integration
proceeded. This effect was presumably related to the highly porous
structure of the SF configuration, allowing for more efficient expo-
sure to proteolytic enzymes and subsequent polymer hydrolysis
due to increases in overall surface area [16,32]. Thus, this bilayer
BAMG-SF scaffold reached a balance between degradation and
regeneration simultaneously during the process of bladder aug-
mentation since scaffolds should initially reinforce the defect site,
but gradually dissipate and be replaced by regenerated host tissue.
Complex wound healing and tissue regeneration involved mul-
tiple cells and cytokines acting together in concert and via a correct
sequence. The first phase of any wound healing process and even-
tual regeneration involved hemostasis and inflammation. In this
study, we focused on the inflammatory changes that coexist with
regeneration during the 12-week implantation period. Early eosi-
nophilic infiltration was identified, responded rapidly, and
dissipated over time in the subepithelial and lamina propria
regions of the de novo bladder wall with the BAMG-SF scaffold.
Eosinophil counts decreased significantly after week 2 and then
continued to decrease steadily from weeks 4 to 12. These results
are consistent with our observations that urothelial regeneration
was minimal at 2 weeks; however, hyperplasia and hypertrophy
of the urothelium appeared at 4 weeks and 12 weeks after implan-
tation. This feature may be explained by the observation that
active eosinophils may impede epithelialization and exude toxic
eosinophil peroxidase, which can alter urothelial integrity and per-
meability [33–35]. In addition, macrophage infiltration was not
substantial at week 2 after implantation. However, macrophage
counts rose prominently from week 2 to week 4 and decreased sig-
nificantly from week 4 to week 12. These data revealed that macro-
phages may promote proper bladder regeneration cascades, as
they are known to phagocytose degrading proteins and coordinate
tissue repair [36]. Therefore, we hypothesize that macrophages
function primarily during later stages of bladder remodeling.
Immunofluorescence and histomorphometric analysis demon-
strated extensive regeneration of the epithelium, smooth muscle
bundles, nerves, and vessels in regenerated bladders in the
BAMG-SF group at each time point. According to previous study
[37], there are some urothelial-associated proteins in the urothe-
lium such as cytokeratins (CK, localizated in the cell membrane),
uroplakin III(UP III, localizated in the endoplasmic reticulum mem-
brane) and p63 (a marker for epithelial stem cells, localizated in
the nucleus). We chose cytokeratins (CK) as a molecular marker
in urothelium in order to test the growth and proliferation of
urothelium. After 2 weeks in vivo, a small number of urothelial
cells were found on the luminal surface of the BAMG-SF scaffolds,
with an irregular and disperse distribution. However, by weeks 4
and 12, a CK-positive, multilayered, completed urothelial lining
was present across the entire luminal surface of the de novo blad-
der walls supported by BAMG-SF scaffolds, consistent with the
presence of urothelial hyperplasia and hypertrophy. This feature
may reflect incomplete urothelial maturation since normalization
of basal/intermediate cell proliferation is required during wound
healing for native tissue stratification to be achieved [16,38].
Therefore, urothelial normalization of the original defect sites sup-
ported by BAMG-SF scaffolds would likely require more time than
12 weeks. Both regenerated smooth muscle and vessels improved
over time in the de novo bladder walls supported by the
BAMG-SF scaffold, reaching values similar to those of the cysto-
tomy control at 12 weeks postimplantation. These results
Table 2
Urine studies in the cystotomy and BAMG-SF groups at different time points. Neg: negative.
Time (weeks) PH White blood counts/HP Red blood cells/HP Ketones (mg/dL) Protein (mg/dL)
Preop
Cystotomy group 6.5 0–5 0–3 Neg Neg
BAMG-SF group 7.0 0–5 0–3 Neg Neg
1 weeks
Cystotomy group 7.0 10–15 10–20 Neg Neg
BAMG-SF group 7.0 20–25 30–40 Neg Neg
2 weeks
Cystotomy group 6.5 5–10 4–10 Neg Neg
BAMG-SF group 6.5 10–15 10–20 Neg Neg
4 weeks
Cystotomy group 6.0 0–5 0–3 Neg Neg
BAMG-SF group 6.5 0–5 0–3 Neg Neg
8 weeks
Cystotomy group 7.0 0–5 0–3 Neg Neg
BAMG-SF group 6.5 0–5 0–3 Neg Neg
12 weeks
Cystotomy group 6.5 0–5 0–3 Neg Neg
BAMG-SF group 6.5 0–5 0–3 Neg Neg
100 Y. Zhao et al. / Acta Biomaterialia 23 (2015) 91–102
demonstrated that bladder augmentation and construction with
the BAMG-SF scaffold could achieve excellent regeneration of
smooth muscle and vessels.
In our study, we found that the density of NeuN+ cells increased
continuously with time after bladder reconstruction with the
BAMG-SF scaffold; however, even after 12 weeks, apparent inner-
vation was still significantly lower as compared to the cystotomy
group (reaching only 50.2% that of the cystotomy control). These
results were consistent with a previous report [39] showing that
the expression of nerve markers in the de novo bladder wall sup-
ported by the BAMG increased continuously after grafting, reach-
ing 60% of normal values by 6 months. The presence of nerve
markers throughout the entire graft confirmed effective
re-innervation of the reconstructed bladder wall, which could rep-
resent complete functional regeneration of the graft. However,
nerve regeneration in the de novo bladder wall is slow and may
require much longer than the 12-week period used in this study.
Taken together, from the above results, although the innervation
problem remains unaddressed in TE reconstruction of the bladder,
the BAMG-SF scaffold described in this study was capable of sup-
porting satisfactory regeneration of urothelium, smooth muscle,
and blood vessels to levels similar to those of the cystotomy con-
trol in a rat model of bladder defect repair.
In our study, bladder capacity was augmented at least 50% with
the BAMG-SF scaffold (10 mm !10 mm). However, there was no
significant augmentation of bladder capacity in the BAMG-SF
group at weeks 2 or 4. This result may be explained by the bladder
stimulation and temporary bladder dysfunction following the
implantation [21,22]. Importantly, compliance of the engineered
neobladder was found to increase over time to normal levels.
The parameters of hematological findings remained within the
normal range, except for leukocyte (neutrophil) counts, which
exceeded the upper reference level during weeks 1 and 2 postim-
plantation and returned to normal levels at 4 weeks in the
BAMG-SF and cystotomy groups. Leukocyte (neutrophil) counts
were higher in the BAMG-SF group than in the cystotomy group
at 2 weeks postimplantation. Moreover, red and white blood
counts were higher in the BAMG-SF group than in the cystotomy
group at weeks 1 and 2. Thus, these changes may reflect an acute
response to surgery. More importantly, the BAMG-SF scaffold
may stimulate the local inflammatory response, which causes
microscopic hematuria [2,40]. Other serum chemistry and elec-
trolyte parameters remained within the normal range during the
entire study period. These observations are consistent with the
lack of significant alterations in serum chemistry levels in all
groups over the course of the study, suggesting that animals
retained normal kidney function and liver function. Therefore, in
summary, the BAMG-SF scaffold can be engineered to produce
anatomically and functionally normal neobladders for augmenta-
tion without significant local tissue responses or systemic toxicity
in our long-term observational study.
The limitation of our study is lack of comparable result between
simple BAMG, SF and bilayer scaffold. Optimization of BAMG-SF
bilayer scaffold will be a future goal in order to reduce the inci-
dence of stone formation and enhance innervation. Future studies
also will focus on evaluation of pre-seeding of bilayer scaffold with
stem cells and long-term and efficacy assessments of BAMG-SF
biomaterials in large animal models of bladder reconstruction in
order to achieve translation potential of this scaffold technology
toward clinical application.
5. Conclusions
The data presented in this report demonstrate the feasibility of
developing a bilayer scaffold by combining the unique functions of
a porous network and underlying natural acellular matrix. This
bilayer scaffold provided a natural barrier function and supported
the microenvironmental needs of the various types of cells found
in the bladder wall. Structural measurements showed that
BAMG-SF scaffolds could support regeneration of the urothelium,
smooth muscle, blood vessels, and innervated tissues in a rat
model of bladder augmentation repair. Furthermore, functional
parameters showed that bladder compliance improved gradually
over time, recovering to normal levels, while bladder capacity
was augmented at least 50%. Moreover, there were no significant
local tissue responses or systemic toxicity in the neobladders engi-
neered by the BAMG-SF scaffold in a long-term observational
study. Optimization of the BAMG-SF bilayer scaffold will be a
future goal in order to reduce the incidence of stone formation
and enhance innervation. Future studies will also focus on evalua-
tion of preseeding of the bilayer scaffold with stem cells and
long-term and efficacy assessments of BAMG-SF biomaterials in
large animal models of bladder reconstruction in order to deter-
mine the translational potential of this scaffold technology in clin-
ical applications.
Acknowledgements
This study was supported by the National Natural Science
Foundation (81070605, 81370860) and the Biomedical
Engineering Research Fund of Shanghai Jiao Tong University
(YG2011MS14). Juan Zhou and the staff at the Shanghai Tissue
Engineering Key Laboratory are acknowledged for technical assis-
tance with tissue processing for histological analyses.
Appendix A. Figures with essential colour discrimination
Certain figures in this article, particularly Figs. 1–7, are difficult
to interpret in black and white. The full colour images can be found
in the on-line version, at doi: http://dx.doi.org/10.1016/j.actbio.
2015.05.032.
References
[1] C. Feng, Y.M. Xu, Q. Fu, W.D. Zhu, L. Cui, J. Chen, Evaluation of the
biocompatibility and mechanical properties of naturally derived and
synthetic scaffolds for urethral reconstruction, J. Biomed. Mater. Res. A 94
(2010) 317–325.
[2] T.G. Kwon, J.J. Yoo, A. Atala, Local and systemic effects of a tissue engineered
neobladder in a canine cystoplasty model, J. Urol. 179 (2008) 2035–2041.
[3] H. Krueger, V.K. Noonan, L.M. Trenaman, P. Joshi, C.S. Rivers, The economic
burden of traumatic spinal cord injury in Canada, Chronic Dis. Inj. Can. 33
(2013) 113–122.
[4] A. Atala, S.B. Bauer, S. Soker, J.J. Yoo, A.B. Retik, Tissue-engineered autologous
bladders for patients needing cystoplasty, Lancet 367 (2006) 1241–1246.
[5] G.S. Jack, R. Zhang, M. Lee, Y. Xu, B.M. Wu, L.V. Rodriguez, Urinary bladder
smooth muscle engineered from adipose stem cells and a three dimensional
synthetic composite, Biomaterials 30 (2009) 3259–3270.
[6] L. Song, S.V. Murphy, B. Yang, Y. Xu, Y. Zhang, A. Atala, Bladder acellular matrix
and its application in bladder augmentation, Tissue Eng. B Rev. (2013).
[7] F. Ajalloueian, S. Zeiai, M. Fossum, J.G. Hilborn, Constructs of electrospun PLGA,
compressed collagen and minced urothelium for minimally manipulated
autologous bladder tissue expansion, Biomaterials 35 (2014) 5741–5748.
[8] B. Brown, K. Lindberg, J. Reing, D.B. Stolz, S.F. Badylak, The basement
membrane component of biologic scaffolds derived from extracellular
matrix, Tissue Eng. 12 (2006) 519–526.
[9] J. Kim, S.Y. Jeong, Y.M. Ju, J.J. Yoo, T.L. Smith, G. Khang, et al., In vitro osteogenic
differentiation of human amniotic fluid-derived stem cells on a poly(lactide-
co-glycolide) (PLGA)-bladder submucosa matrix (BSM) composite scaffold for
bone tissue engineering, Biomed. Mater. 8 (2013) 014107.
[10] Y. Mitsui, H. Shiina, T. Hiraoka, N. Arichi, H. Yasumoto, R. Dahiya, et al.,
Simultaneous implantation of bilateral ureters into bladder acellular matrix
graft after partial cystectomy in a porcine model, BJU Int. 110 (2012). E1212-7.
[11] W.D. Zhu, Y.M. Xu, C. Feng, Q. Fu, L.J. Song, L. Cui, Bladder reconstruction with
adipose-derived stem cell-seeded bladder acellular matrix grafts improve
morphology composition, World J. Urol. 28 (2010) 493–498.
[12] D. Eberli, R. Susaeta, J.J. Yoo, A. Atala, Tunica repair with acellular bladder
matrix maintains corporal tissue function, Int. J. Impot. Res. 19 (2007) 602–
609.
Y. Zhao et al. / Acta Biomaterialia 23 (2015) 91–102 101
[13] A. Atala, Tissue engineering of human bladder, Br. Med. Bull. 97 (2011) 81–
104.
[14] U.J. Kim, J. Park, H.J. Kim, M. Wada, D.L. Kaplan, Three-dimensional aqueous-
derived biomaterial scaffolds from silk fibroin, Biomaterials 26 (2005) 2775–
2785.
[15] J.R. Mauney, G.M. Cannon, M.L. Lovett, E.M. Gong, D. Di Vizio, P. Gomez 3rd,
et al., Evaluation of gel spun silk-based biomaterials in a murine model of
bladder augmentation, Biomaterials 32 (2011) 808–818.
[16] P. Gomez 3rd, E.S. Gil, M.L. Lovett, D.N. Rockwood, D. Di Vizio, D.L. Kaplan,
et al., The effect of manipulation of silk scaffold fabrication parameters on
matrix performance in a murine model of bladder augmentation, Biomaterials
32 (2011) 7562–7570.
[17] M. Horst, S. Madduri, V. Milleret, T. Sulser, R. Gobet, D. Eberli, A bilayered
hybrid microfibrous PLGA–acellular matrix scaffold for hollow organ tissue
engineering, Biomaterials 34 (2013) 1537–1545.
[18] R. Nazarov, H.J. Jin, D.L. Kaplan, Porous 3-D scaffolds from regenerated silk
fibroin, Biomacromolecules 5 (2004) 718–726.
[19] M. Zhu, K. Wang, J. Mei, C. Li, J. Zhang, W. Zheng, et al., Fabrication of highly
interconnected porous silk fibroin scaffolds for potential use as vascular grafts,
Acta Biomater. 10 (2014) 2014–2023.
[20] D.D. Tu, A. Seth, E.S. Gil, D.L. Kaplan, J.R. Mauney, C.R. Estrada Jr., et al.,
Evaluation of biomaterials for bladder augmentation using cystometric
analyses in various rodent models, J. Vis. Exp. (2012).
[21] L. Zhou, B. Yang, C. Sun, X. Qiu, Z. Sun, Y. Chen, et al., Coadministration of
platelet-derived growth factor-BB and vascular endothelial growth factor with
bladder acellular matrix enhances smooth muscle regeneration and
vascularization for bladder augmentation in a rabbit model, Tissue Eng. A
(2012). 121116104322001.
[22] M.J. Jayo, D. Jain, B.J. Wagner, T.A. Bertram, Early cellular and stromal
responses in regeneration versus repair of a mammalian bladder using
autologous cell and biodegradable scaffold technologies, J. Urol. 180 (2008)
392–397.
[23] J.P. Zambon, L.S. de Sa Barretto, A.N. Nakamura, S. Duailibi, K. Leite, R.S.
Magalhaes, et al., Histological changes induced by polyglycolic-acid (PGA)
scaffolds seeded with autologous adipose or muscle-derived stem cells when
implanted on rabbit bladder, Organogenesis 10 (2014) 278–288.
[24] M. Horst, V. Milleret, S. Notzli, S. Madduri, T. Sulser, R. Gobet, et al., Increased
porosity of electrospun hybrid scaffolds improved bladder tissue regeneration,
J. Biomed. Mater. Res. A 102 (2014) 2116–2124.
[25] N. Kasoju, U. Bora, Silk fibroin in tissue engineering, Adv. Healthcare Mater. 1
(2012) 393–412.
[26] D. Eberli, L. Freitas Filho, A. Atala, J.J. Yoo, Composite scaffolds for the
engineering of hollow organs and tissues, Methods 47 (2009) 109–115.
[27] P.M. Crapo, T.W. Gilbert, S.F. Badylak, An overview of tissue and whole organ
decellularization processes, Biomaterials 32 (2011) 3233–3243.
[28] S.F. Badylak, D. Taylor, K. Uygun, Whole-organ tissue engineering:
decellularization and recellularization of three-dimensional matrix scaffolds,
Annu. Rev. Biomed. Eng. 13 (2011) 27–53.
[29] F. Consolo, S. Brizzola, G. Tremolada, V. Grieco, F. Riva, F. Acocella, et al., A
dynamic distention protocol for whole-organ bladder decellularization:
histological and biomechanical characterization of the acellular matrix, J.
Tissue Eng. Regen. Med. (2013).
[30] Y. Zhang, D. Frimberger, E.Y. Cheng, H.K. Lin, B.P. Kropp, Challenges in a larger
bladder replacement with cell-seeded and unseeded small intestinal
submucosa grafts in a subtotal cystectomy model, BJU Int. 98 (2006) 1100–
1105.
[31] M.I. Bury, N.J. Fuller, J.W. Meisner, M.D. Hofer, M.J. Webber, L.W. Chow, et al.,
The promotion of functional urinary bladder regeneration using anti-
inflammatory nanofibers, Biomaterials 35 (2014) 9311–9321.
[32] Y. Wang, D.D. Rudym, A. Walsh, L. Abrahamsen, H.J. Kim, H.S. Kim, et al., In
vivo degradation of three-dimensional silk fibroin scaffolds, Biomaterials 29
(2008) 3415–3428.
[33] N.M. Itano, R.S. Malek, Eosinophilic cystitis in adults, J. Urol. 165 (2001) 805–
807.
[34] T.J. Kleine, G.J. Gleich, S.A. Lewis, Eosinophil peroxidase increases membrane
permeability in mammalian urinary bladder epithelium, Am. J. Physiol. 276
(1999). C638-47.
[35] J. Yang, A. Torio, R.B. Donoff, G.T. Gallagher, R. Egan, P.F. Weller, et al.,
Depletion of eosinophil infiltration by anti-IL-5 monoclonal antibody (TRFK-5)
accelerates open skin wound epithelial closure, Am. J. Physiol. 151 (1997)
813–819.
[36] G. Broughton 2nd, J.E. Janis, C.E. Attinger, The basic science of wound healing,
Plast. Reconstr. Surg. 117 (2006) 12S–34S.
[37] E.J. Pechriggl, M. Bitsche, M.J. Blumer, H. Fritsch, The male urethra:
spatiotemporal distribution of molecular markers during early development,
Ann. Anat. 195 (2013) 260–271.
[38] W.I. de Boer, A.G. Schuller, M. Vermey, T.H. van der Kwast, Expression of
growth factors and receptors during specific phases in regenerating
urothelium after acute injury in vivo, Am. J. Pathol. 145 (1994) 1199–1207.
[39] A.M. Kajbafzadeh, S. Payabvash, A.H. Salmasi, Z. Sadeghi, A. Elmi, K. Vejdani,
et al., Time-dependent neovasculogenesis and regeneration of different
bladder wall components in the bladder acellular matrix graft in rats, J. Surg.
Res. 139 (2007) 189–202.
[40] Manuel J. Jayo1, Deepak Jain1, J.W. Ludlow1, Long-term durability, tissue
regeneration and neo-organ growth during skeletal maturation with a neo-
bladder augmentation construct, Future Med. (2008).
102 Y. Zhao et al. / Acta Biomaterialia 23 (2015) 91–102
