Shinshu Med J, 71⑶：167～177, 2023

Development of Bilayered Bone Marrow-derived Cell-Gelatin Grafts forAugmentation Cystoplasty and Reconstruction of Bladder Tissues in Rats
Yoshihiro Inoue, Tetsuya Imamura＊, Noriyuki Ogawa, Hiroaki Hara, Yuki Matsumoto
Manabu Ueno, Tomonori Minagawa, Teruyuki Ogawa and Osamu Ishizuka
Department of Urology, Shinshu University School of Medicine

Background : This study attempted to produce a novel graft composed of bone marrow-derived mesenchymal cell(BMC) layer-gelatin sheets for bladder augmentation cystoplasty. Then, we determined if the grafts could recon­
struct bladder tissues.
Methods : BMCs harvested from the femurs of green fluorescence protein (GFP)-transfected Sprague-Dawley(SD) rats were adherent and proliferating cells on collagen dishes. The cells were then cultured on temperatureresponsive culture dishes. Following this, the BMCs maintaining cell-cell contacts within the monolayer itself
were applied to a gelatin sheet. Two BMC layer-gelatin sheets were overlaid together with the cell sides juxta­
posed with one another (bilayered BMC-gelatin graft). Bladder top of SD rats were incised and transplantedwith the bilayered BMC-gelatin grafts. Similarly, urinary bladders irradiated with 2 Gy once a week for 5 weeks
were also conducted. As control, bilayered acellular-gelatin grafts were used. At 4 weeks after transplantation,
the bladders were histologically investigated.
Results : At 4 weeks after transplantation into either normal or radiation-injured urinary bladders, incised regions
closed. The closed regions of bladder top had reconstructed tissues that were formed with urothelium, andsmooth muscle layers. Within the reconstructed tissues, the thickness of the smooth muscle layers in the bilayered
BMC-gelatin graft-transplanted bladders were larger compared to controls. The GFP-positive transplanted
BMCs were detected. Some of the cells were simultaneously positive for smooth muscle or nerve cell markers.
Conclusion : This study showed that the bilayered BMC-gelatin grafts that were experimentally produced could
reconstruct bladder tissues. The grafts would be developed as grafts for bladder augmentation cystoplasty.
Shinshu Med J 71 : 167―177, 2023
(Received for publication January 6, 2023 ; accepted in revised form February 1, 2023)
Key words : bladder augmentation cystoplasty, bone marrow-derived mesenchymal cell, gelatin,
bladder tissue, rat

Ⅰ Introduction

plasty can improve storage function due to enlarged
bladder capacity. However, resulting from utilization

In urology, bladder augmentation cystoplasty isof a portion of intact ileum1）, the operation is highly

conducted to treat a contracted bladder which occurs

invasive and has several side effects : intestinal ste­

as a result of various diseases such as neurogenic

nosis, stone formation, metabolic and electrolyte dis­

bladders, radiation cystitis, bladder tuberculosis, orturbance, or neoplastic progression5）. Thus, in bladderinterstitial cystitis1）-4）. Bladder augmentation cysto­

augmentation cystoplasty, developments of novel＊

Corresponding author : Tetsuya Imamura
Department of Urology, Shinshu University School of
Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan
E-mail : imatetu@shinshu-u.ac.jp

No. 3, 2023

grafts that can replace the utilization of intact ileum
are urgently needed6）-8）.
Tissue engineering technologies can contribute toproduce the novel grafts for bladder augmentation
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Inoue・Imamura・Ogawa et al.

cystoplasty9）-11）. Previously, we showed that human12）amnion13）

or animal tissue-derived membranes

could

Ｂ Preparation of bone marrow-derived mesenchymal cells

be used as the grafts for bladder augmentation cysto­

GFP-transfected Tg-SD rats were anesthetized byplasty in rats. However, these membranes have someintramuscular injection of midazolam (2.0 mg/kg-bodylimitations such as immunological rejection, animal

weight, Sandoz International GmbH, Tokyo, Japan),origin, and/or xenobiotics materials in clinical appli­

medetomidine hydrochloride (0.15 mg/kg-body weight,

cation. To overcome those problems, gelatin sheets

Kyoritsu Seiyaku Co., Tokyo, Japan), and butorphanolthat have high biocompatibility and biodegradability

tartrate (2.5 mg/kg-body weight, Meiji Seika Pharma

14）

have been developed . In addition, our previous stud­Co., Ltd., Tokyo, Japan), and then further anesthetizedies indicated that autologous mesenchymal cells de­by inhalation of 2-3 ％ sevoflurane (Abbot Japan Co.,

rived from bone marrow or adipose tissues can pro­LTD., Tokyo, Japan). Both femurs of the donor GFP-

15）
-19）

. Thus, atransfected Tg-SD rats were harvested and both ends

combination of the gelatin sheets and the mesenchymal

were cut, and then bone marrow cells were flushedcells would provide highly efficient grafts for bladderwith 10 ml of culture medium composed of Dulbecco’s

augmentation cystoplasty.

Modified Eagle Medium (DMEM) high-glucose (Gibco,

mote reconstruction of bladder tissues

In this study, we experimentally produced 4-type

Thermo Fisher Scientific K.K., Kanagawa, Japan) sup­grafts composed of the bone marrow-derived mesen­

plemented with 15 ％ regular fetal bovine serum (Bio­chymal cells (BMCs) and the gelatin sheets, and de­west, Nuaille, France) and 0.1 ％ penicillin-streptomycin

termined which BMC-gelatin grafts could be devel­

solution (Gibco) from one side of the femurs. After

oped as clinical grafts to replace the utilization of

flushing, the cell-solution was centrifuged at 1,000

intact ileum in normal rat urinary bladders. Then, by

rpm for 4 minutes, and then the pelleted cells wereusing our established rat radiation-injured urinary

suspended in the flesh culture medium. After passing

bladders that imitated human radiation cystitis-through a cell strainer (Life Science, Corning, NY,

induced contracted bladder15）-17）, we determined ifUSA), the suspended bone marrow cells were cul­

the grafts combined with BMCs and gelatin sheets

tured on type I collagen-coated 10-cm culture dishes

could reconstruct bladder tissues.

(AGC Techo Glass, Shizuoka, Japan) at 37 ℃ in humid

Ⅱ
Ａ

Material and Methods

Animals
Twenty-eight female 10-week-old Sprague-Dawley(SD) rats (Japan SLC Inc., Shizuoka, Japan) were used

air with 5 ％ CO2 for 7-10 days. The medium waschanged completely every day to wash off unattachedcells. We used the attached and proliferating cells on
the type I collagen as BMCs15）-18）.
Ｃ

Production of bilayered BMC-gelatin grafts

as recipients. Five male 17-week-old green fluores­

The adherent proliferating BMCs were dissociatedcence protein (GFP)-transfected SD rats (Tg-SD rats ;with 0.25 ％ trypsin (Gibco). After suspension at 4.0×

Japan SLC Inc.) were used as cell donors. All of the105 cells/ml in culture medium, the cells were seeded

rats were maintained with freely available food and(1 ml) onto temperature-responsive culture dishes

water under a 12-hour alternating light-dark cycle.

(4.0×105 cells/ml/well, UpCell®35-mm dish, CellSeed

After each experiment, the rats were euthanized byInc., Tokyo, Japan), and then Culture medium (2 ml)

an overdose of pentobarbital sodium solution (Kyoritsu

was added. The cells were cultured for 7-10 days,

Seiyaku Co., Tokyo, Japan). All animals were treated inreaching over-confluence in which more than 80 ％ ofaccordance with National Institutes of Animal Healtheach dish surface was covered by a monolayer of cells.

Care Guidelines and the guidelines approved by the

To harvest the cells after reaching over-confluence,

Animal Ethics Committee of Shin­shu University School

the culture medium was removed, and the tempera­of Medicine.

ture of the culture dishes was reduced to 20 ℃. This
procedure enables detachment of the BMCs main­

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Bilayered BMC grafts

taining cell-cell contacts within the layer itself from
16）

bivalve (Fig. 1C). The experimental grafts : bilayered

the culture dish . The detached BMC layers wereacellular-gelatin graft, monolayer BMC-gelatin graft,then applied to a wetting gelatin hydrogel sheet withbilayered BMC-acellular gelatin graft or bilayered

dimensions of approximately 12-mm in diameter and

BMC-gelatin graft were put on the incised region (napproximately 0.5-mm in thickness (gelatin sheet ;＝4, in each), and then a biodegradable polyglycolic

Genocel®: dry dimensions of 8-mm in diameter andacid (PGA) nonwoven fabric that was designed to be

0.3-mm in thickness ; Nikke Medical Co., Inc. Osaka,

resorbed after about 2 weeks (Nikke Medical Co.,

14）

Japan) . Two BMC layer-gelatin sheets, each sup-Inc.) was put on the experimental grafts. Then, at the

porting a BMC layer, were then overlaid together

3, 6, 9, and 12-o’clock position, the bladder and bothwith the cell sides juxtaposed with one another (bi-the experimental graft and PGA nonwoven fabric werelayered BMC-gelatin graft, Fig. 1A). In addition, we

sutured with a 5-0 silk thread by mattress suture

produced another 3-type grafts ; two acellular-gelatin

techniques (Fig. 1D).

sheets were overlaid together (bilayered acellular-

Similarly, transplantation of the bilayered BMC-

gelatin graft), the BMC layer-gelatin sheet was notgelatin grafts into the radiation-injured urinary blad-

overlaid with another gelatin sheet (monolayer BMC-

ders was conducted (n＝5). As control, transplanta-

gelatin graft), and the BMC layer-gelatin sheet wastion of bilayered acellular-gelatin grafts was performed

overlaid with an acellular-gelatin sheet (bilayeredin the radiation-injured urinary bladders (n＝7).

BMC-acellular gelatin graft). These grafts are indi-

Ｆ Histological and immunohistochemical investiga-

cated in Fig. 1A.

tions

The bilayered BMC-gelatin grafts and other type

At 4 weeks after transplantation, the urinary blad-

grafts were incubated without culture medium at 37

ders were removed and fixed with 4 ％ paraformal-℃ in humid air with 5 ％ CO2 for 60 minutes. After thedehyde solution. Then the samples were embedded

incubation, these grafts were cultured with 10 ml R-

in paraffin and cut in 5-μm thick serial longitudinal-

STEM (Rohto Pharmaceuticals, Osaka, Japan) at 37 ℃

sections from bladder top to trigone. For histologicalin 5 ％ CO2 for 3 days. One of the cultured bilayered

investigations, the sections were deparaffinized with

BMC-gelatin grafts that were not used in a transplan-

xylene, rehydrated with ethanol, and rinsed threetation (as below) received Masson staining to histologi-

times with phosphate-buffered saline (PBS). Eachcally examine the structural organization (Fig. 1B).

section was stained with Masson trichrome (ScyTek

Ｄ Production of radiation-injured urinary bladders

Laboratories, Inc., Logan, UT, USA) according to the

Twelve female 10-week-old SD rats were anesthe-

protocol of the manufacturer. The Masson trichrome-

tized as above, and then protected with an iron shieldstained samples were observed and photographed

except for a 1-cm diameter circle bordering on thewith a common optical microscope. To measure thepubic bone to allow radiation of the pelvic region con-thickness of smooth muscle layers at both bladdertaining the bladder. The exposed area was irradiatedtops that were transplanted with the grafts, andwith 2 Gy once a week for 5 weeks. Following the last

original intact bladder walls that were not incised,radiation exposure, the rats were maintained for 2

the Masson trichrome-stained smooth muscle layers

15）
-17）

weeks

.

Ｅ Transplantation of experimental grafts into

were randomly viewed and measured in 5-10 locations per tissue sample with a x20 objective lens byurinary bladders

using image analysis software (cellSens, Olympus Co.,

Sixteen female 10-week-old SD rats were anesthe-Tokyo, Japan).

tized as above, and then their urinary bladder was

For immunohistochemistry, some of the deparaf-

exposed through a midline lower abdominal incision.

finized sections were immersed in 10 mM sodium

The bladder top received a transverse incision, andcitrate (pH 6.0) and microwaved at 100 ℃ for 10 min-was divided into anterior and posterior wall, such as autes for antigen retrieval. The sections were blocked

No. 3, 2023

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Fig. 1 Production of 4-type experimental grafts and transplantation into urinary bladders. (A) This study ex­perimentally produced 4-type grafts, bilayered acellular-gelatin graft ; two acellular-gelatin sheets wereoverlaid, monolayer BMC-gelatin graft ; a BMC layer-gelatin sheet was not overlaid with a gelatin sheet,bilayered BMC-gelatin graft ; a BMC layer-gelatin was overlaid with an acellular gelatin sheet, bilayered
BMC-gelatin sheet ; two BMC layer-gelatin sheets were overlaid together with the cell sides juxtaposedwith one another. (B) At 3 days after overlaying, the BMC layers between gelatin sheets tightly attachedto gelatin fibers (asterisks), and fully contacted each other. Bar : 100μm. (C) Just prior to transplantation,
urinary bladders received a transverse incision, and were divided into anterior and posterior wall. (D) For
transplantation, the experimental sheets (arrowheads) were put on the incised region, and then a PGA
nonwoven fabric (arrow) was put on the sheets.
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with 1.5 ％ non-fat milk in PBS for 1 hour at 4 ℃ .

Ｂ Bladder tissues reconstructed by transplantation

Each section was incubated with GFP-antibody (1 :of 4-type experimental grafts into normal uri-

1000, goat polyclonal, abcam, Cambridge, UK) to de­nary bladders

tect the BMCs, and smooth muscle actin (SMA, 1 : 100

At 4 weeks after transplantation, incised regions

mouse monoclonal ; Progen Biotechnik GmbH, Heidel­that were treated with bilayered acellular-gelatinberg, Germany) to identify mature smooth muscle(Fig. 2A), monolayer BMC-gelatin (Fig. 2B), bilay­

cells, desmin (1 : 150 rabbit polyclonal ; Progen Bio­ered BMC-acellular gelatin (Fig. 2C), or bilayered

technik GmbH) a marker for immature smooth muscle

BMC layer-gelatin (Fig. 2D) grafts were well closed,

cells, or Pax7 (1 : 1000, rabbit polyclonal ; Lifespan Bio­and the treated bladders were similar in appearance

sciences, Inc., Seattle, WA, USA) as a marker for myo­to the pre-operated bladders. While small fragments

blasts, or calcitonin gene-related peptide (CGRP, 1 :of PGA nonwoven fabric or thread were still present

500, guinea pig polyclonal, Progen Biotechnik GmbH)

within the bladder lumen, at the closed regions of

as a marker for afferent nerve cells for 12 hours at 4

bladder tops, those experimental grafts were re­℃. After the primary antibody reactions, the sections

placed by reconstructed tissues. The reconstructed

were incubated with the respective secondary antibod­

tissues were formed with urothelium arranged into a

ies consisting of donkey anti-goat conjugated withmulti-layered form, smooth muscle layers integrated

Alexa Fluor 488 (1 : 250 ; Molecular Probes, Eugene,

into small cluster form, and filamentous gelatin fiber-

OR, USA) and donkey anti-mouse, rabbit, or guinea

like materials that might be derived from biodegrad­

pig each conjugated with Alexa Fluor 594 (1 : 250 ;ed gelatin sheets (Fig. 2E-H). The urothelia were

Molecular Probes) for 1 hour at 4 ℃. After rinsing,similar to the normal ones, and did not show any dif­they were counterstained with 5 μg/ml 4’, 6-diamidi­

ferences among the experimental grafts (Fig. 2E-H).

no-phenylindole dihydrochloride (DAPI ; Molecular

The thickness of the smooth muscle layers within

Probes). The immunohistochemically stained samples

the tissues of bladder top transplanted with the bi­

were observed with a fluorescence microscope (Key­layered BMC-gelatin grafts was the greatest com­ence, Osaka, Japan).

pared to the tissues transplanted with the bilayered

Ｇ

acellular-gelatin, the monolayer BMC-gelatin, or theStatistical analysis
Results were expressed as means±standard devi­

bilayered BMC-acellular gelatin graft (Table 1). Thus,

ations. Statistical differences were determined using

this study made a decision to investigate the effects

the Excel®Statistics program (Esumi Co., Ltd. Tokyo,of the bilayered BMC-gelatin grafts in radiation-

Japan). Comparisons were made by non-repeatedinjured urinary bladders.

measures analysis of variance (ANOVA) or t-tests.

Ｃ Bladder tissues reconstructed with bilayered

P-values less than 0.05 were considered statisticallyBMC-gelatin grafts into radiation-injured urinary

significant.

bladders
Ⅲ Results

Ａ Histological investigations of bilayered BMC-

At 4 weeks after transplantation into radiationinjured urinary bladders, incised regions that weretransplanted with control bilayered acellular- (Fig.

gelatin grafts

3A) or bilayered BMC- (Fig. 3B) gelatin grafts were

Masson stain samples of bilayered BMC-gelatinclosed. While small fragments of PGA nonwoven fabricgraft were observed. Between the gelatin sheets, theor thread were still present within the bladder lumen,

BMC layers tightly attached to gelatin fibers, and fullyat the closed regions of bladder tops, either the control

contacted each other. Some of the BMCs migrated

grafts (Fig. 3C) or the bilayered BMC-gelatin grafts

along the gelatin fibers (see Fig. 1B).

(Fig. 3D) were replaced by reconstructed tissues thatwere formed with urothelium, smooth muscle layers,
and filamentous gelatin fiber-like materials. These

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Fig. 2 At 4 weeks after transplantation of experimental sheets into normal urinary bladders. (A-D) Incised re­gions transplanted with bilayered acellular-gelatin (A), monolayer BMC-gelatin (B), bilayered BMCacellular (C), or bilayered BMC-gelatin (D) grafts were well closed. These transplanted experimental grafts
were replaced by reconstructed tissues (arrows). (E-H) At the closed regions of bladder top, the tissuesreconstructed with bilayered acellular-gelatin (E), monolayer BMC-gelatin (F), bilayered BMC-acellular(G), or bilayered BMC-gelatin (H) grafts were formed with urothelium (UT), smooth muscle layers (aster­isks), and filamentous gelatin fiber-like materials (blue). Bars : 100μm.
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Table 1 Thickness of smooth muscle layers within reconstructed tissues of bladder tops at 4 weeks after
transplantation of experimental grafts into normal urinary bladders

Smooth muscle layers(μm)

Bilayered acellulargelatin graft

Monolayer BMCgelatin graft

acellular gelatin graft

Bilayered BMC-

Bilayered BMCgelatin graft

50.59±5.82

53.78±9.1449.63±1.21164.54±13.93＊§†＊

P＜0.01 ; compared to bilayered acellular-gelatin graft, §P＜0.01 ; compared to monolayer BMC-gelatin graft,†P＜0.01 ; compared to bilayered BMC-acellular gelatin graft.

results were similar to the transplantation into normal

bilayered acellular-gelatin grafts were also closed,

urinary bladders. At the closed regions of bladderand then the control closed regions similarly had re­tops, the thickness of the smooth muscle layers with­constructed tissues. These results showed that thein the tissues reconstructed with the bilayered BMC-

reconstructed tissues were formed with not only thegelatin grafts was greater than that reconstructedtransplanted BMCs, but also the cells derived fromwith the control bilayered acellular-gelatin grafts (Pthe origin recipient bladder tissues.

＜0.01, Fig. 3E). In addition, the rate of smooth muscle

Ⅳlayer thickness to origin recipient ones within the in­

Discussion

tact bladder walls in the bilayered BMC-gelatin graft-

Previously, we showed that the bone marrow-transplanted bladders was higher than that in thederived cells had the potential to reconstruct func­

control graft-transplanted bladders (P＜0.01, Fig. 3F).

tional urinary bladders. To deliver the cells into re­Ｄ Survival and differentiation of transplanted

cipient bladders, we performed two methods, theBMCs

direct-injection of single cells17）-19）and the patch-trans­

At the 4 weeks closed regions of bladder tops, GFP-

plantation of cell sheets16）. However, these methods cannot

positive transplanted BMCs, which formed the mono­

be applied for bladder augmentation cystoplasty.

layer BMC-gelatin, bilayered BMC-acellular-gelatin,

Thus, this study attempted to develop based-tech­or bilayered BMC-gelatin grafts, were detected in theniques of grafts combining both the mesenchymal

reconstructed tissues (data not shown). These results

cells and the gelatin sheets for bladder augmentation

showed that the BMCs survived in the recipientcystoplasty.

bladders.

This study experimentally produced 4-type grafts

In the radiation-injured urinary bladders, the GFP-

composed of BMCs and the gelatin sheet (see Fig. 1A).

positive transplanted BMCs that formed the bilayered

We overlaid two BMC layer-gelatin sheets together

BMC-gelatin grafts were present in the reconstructedwith the cell sides juxtaposed with one another. The

tissues at the closed regions of bladder tops (Fig.

3D-conformation provides naturally occurring cell-

4A-D). In the same observed position in each, there

to-cell contacts, and each cell can increase its activa­

were also SMA-positive mature smooth muscle cells

tion15）. In contrast, over 3-layers cannot supply enough(Fig. 4E), desmin-positive immature smooth muscle

nutritious substances and oxygen to the cells arranged

cells (Fig. 4F), Pax7-positive myoblasts (Fig. 4G), or

within inside-center regions. To estimate those ex­

CGRP-positive afferent nerve cells (Fig. 4H). Some of

perimental grafts, we transplanted each type of graft

the transplanted GFP-positive BMCs were simulta­

onto incised bladder top in normal or radiation-in­

neously positive for these makers (Fig. 4I-L).

jured urinary bladders. Four weeks later, the bilay­

In contrast, the reconstructed tissues had numerous

ered BMC-gelatin graft-transplanted bladders showed

GFP-negative and smooth muscle markers-positive

the greatest thickness of smooth muscle layers amongcells that were derived from recipient tissues. In ad­

the other experimental grafts-transplanted bladders.

dition, the incised regions transplanted with control

Thus, the reconstructed tissues in the bilayered

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Inoue・Imamura・Ogawa et al.

Fig. 3 At 4 weeks after transplantation of bilayered BMC-gelatin grafts into radiation-injured urinary bladders.
(A and B) Incised regions transplanted with control bilayered acellular- (A) or bilayered BMC- (B) gelatin
grafts were closed. The closed regions of bladder top had reconstructed tissues (arrows). (C and D) The
reconstructed bladder tissues with control bilayered acellular- (C) or bilayered BMC- (D) gelatin grafts
were formed with urothelium (UT), smooth muscle layers (asterisks), and filamentous gelatin fiber-likematerials (blue). Bars : 100μm. (E) At the bladder top, thickness of the smooth muscle layers of thebilayered BMC-gelatin graft-transplanted bladders was greater than that of the control bilayered
acellular-gelatin graft-transplanted bladders (P＜0.01). (F) Rate of smooth muscle layer thickness to origin
recipient ones within the intact walls in the bilayered BMC-gelatin graft-transplanted bladders washigher than that in the control bilayered acellular-gelatin graft-transplanted bladders (P＜0.01).

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Fig. 4 Differentiation of transplanted BMCs. (A-D) GFP-positive transplanted BMCs (green) were present
within the reconstructed tissues. (E-H) In the same observed position in each, SMA- (E, red), desmin- (F,red), Pax7- (G, red), or CGRP- (H, red) positive cells were present. (I-L) Some of the GFP-positive BMCs
were simultaneously positive for these marker antibodies (arrows, yellow). Bars : 10 or 20μm.

BMC-gelatin graft-transplanted bladders tended totransplanted BMCs were present. Some of the GFP-

be thicker than those of the other control graft-positive BMCs differentiated into SMA-positive ma­transplanted bladders.

ture smooth muscle cells, desmin-positive immature

Here, this study histologically investigated thesmooth muscle cells, Pax7-positive myoblasts, or GFP-

mechanisms of these tissue recoveries. Within thepositive afferent nerve cells. These results showedreconstructed tissues at the bladder top, GFP-positivethat the bilayered BMC-gelatin grafts might contrib­

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Inoue・Imamura・Ogawa et al.

ute as cell sources. Our previous study showed thatanimals such as rabbits, and conduct measurements

BMC monolayer-sheets could provide 24 kinds ofof leak point pressure that are able to estimate thegrowth factors and/or cytokines that were muscle

bladder capacity and compliance without inserting

tissue development-, peripheral nerve system devel­

the bladder catheter.

opment-, angiogenesis-, apoptosis regulator-, cellIn the process to clinical use, the bilayered BMC-

differentiation-, morphogenesis-, immune response-,

gelatin grafts have some advantages. One is easy

and ureteric bud development-related bioactive sub­scale-up to human size by using large gelatin sheets.

16）

stances in radiation-injured bladders . Thus, these

Secondly, this bilayered BMC-gelatin graft is able to

growth factors and/or cytokines provided from BMC

be produced with simple handling. Thirdly, comparedlayers might also promote tissue recovery. In addi­

to collagen, the gelatin is generally considered to havetion, GFP-negative and SMA-positive cells were also

a high migration of cells. Finally, while the universal

present within the reconstructed tissues of the blad­collagen-based materials were extracted with a low-

der top. Thus, it is likely that these cells migrated

temperature acid solution, the gelatin hydrogel sheetsfrom origin recipient tissues. In contrast, the control

that were used in this study were produced throughrats receiving the control bilayered acellular-gelatin

alkali-thermal decomposition. Thus, manufacturing

grafts also showed the reconstruction of tissues. This

processes of the gelatin sheets can avoid contamina­suggested that the recipient cells might have migrat­tion risks of infection or toxicity compared to these ofed through the gelatin sheets. These results showedcollagen sheets14）20）. These advantages might contrib­that the reconstructed tissues were formed with not

ute to developing the bilayered BMC-gelatin grafts

only the transplanted BMCs, but also the cells mi­in future.

grated from origin recipient tissues. Therefore, thegrafts combined with both BMC layers and gelatin
sheets could be highly effective in reconstructing
bladder tissues.

Ⅴ

Conclusions

Bilayered BMC-gelatin grafts were produced byoverlaying two BMC layer-gelatin sheets. At 4 weeks

To develop the bilayered BMC-gelatin grafts forafter transplantation into normal or radiation-injured

clinical use, we need to overcome some limitations. In

urinary bladders, incised regions transplanted withthis study, we provided strength to suture the ex­

the bilayered BMC-gelatin grafts were closed withperimental grafts on incised region of bladders withreconstructed tissues that were formed with urothe­

PGA nonwoven fabric. While the PGA nonwoven

lium, smooth muscle layers, and filamentous gelatinfabrics were designed to be resorbed after about 2

fiber-like materials. Also, the thickness of the smoothweeks in the body, small fragments of PGA nonwo­

muscle layers in the bilayered BMC-gelatin graft-

ven fabric were still present within the bladder lu­

transplanted bladders was the greatest compared tomen of almost all recipient rats. Thus, we would

other control graft-transplanted bladders. Some GFP-

need to develop a replacement for the PGA nonwoven

positive transplanted BMCs differentiated into smoothfabric, or increase the mechanical strength of the gel­muscle- or afferent nerve-marker positive cells.

atin sheets. The main aims of bladder augmentation

These results showed that the bilayered BMC-gelatin

cystoplasty are enlargement of both bladder capacity

grafts could reconstruct the bladder tissues.

and compliance ; however, this study did not measurethese functions in the graft-transplanted rats. To mea­
sure these parameters, cystometric investigations in

Disclosure of Potential Conflicts of Interest
The authors have no conflicts of interest to disclose.

which a catheter is inserted from the bladder top areneeded. Because this study histologically focused on
the bladder tops, the cystometric investigations were
not conducted. As then next steps, we will use larger
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