Shinshu Med J, 68⑹：357～370, 2020

Recovery of Cryo-injured Rabbit Urethras by Biofabricated C-shaped
Adipose-derived Mesenchymal Cell Structures
Sudha Silwal Gautam1）, Tetsuya Imamura1）＊, Mitsuru Shimamura2）, Tomonori Minagawa1）
Masaki Nakazawa1）, Teruyuki Ogawa1） and Osamu Ishizuka1）
1） Department of Urology, Shinshu University School of Medicine
2） Cyfuse Biomedical K.K.

Background : Urethral tissue damage can cause stress urinary incontinence or urinary retention, and there are
few long-term, effective treatments. For structural and functional recovery of urethral tissues, we designed andconstructed C-shaped structures composed of adipose-derived mesenchymal cells (AMCs) by using a threedimensional (3D)-bioprinter system, which could be transplanted in damaged urethras without obstructing thelumen.
Methods : AMCs were harvested from New Zealand White rabbits, labeled with PKH26 for later identification,and cultured to form spheroids. The spheroids were assembled on a custom-designed C-shaped support by athree-dimensional bioprinter, and perfusion cultured for 7 days. Urethras of female rabbits were cryo-injured byspraying with liquid nitrogen for 20 seconds. The damaged region was incised, and the biofabricated structure
was autologously transplanted into the cryo-injured region (n＝4). Control rabbits were treated similarly but
without transplantation of a C-shaped structure (n＝4). Two and four weeks after surgery, the urethras were
harvested for analyses.
Results : Prior to transplantation, the AMCs within the biofabricated structures expressed mesenchymal cellmarkers and extracellular matrices. Two and four weeks after surgery, the control urethras were partiallyconstricted ; however, the structure-transplanted urethras were patent. The AMCs within the structures had
differentiated into skeletal muscle, smooth muscle, or nerve cells. Some cells contained growth factors andcytokines. In addition, they expressed decreased fibroblast markers, hypoxia markers, and apoptotic markers.
Conclusions : The transplantation of biofabricated C-shaped AMC structure has the potential to be an effective
treatment for urethral recovery due to enhanced reconstruction of muscle, and nerve cells. Shinshu Med J
68 : 357―370, 2020
(Received for publication May 21, 2020 ; accepted in revised form July 7, 2020)
Key words : biofabrication, adipose-derived mesenchymal cell, urethra, cryo-injury, rabbit

Ⅰ Introduction

voiding dysfunction. Long-term, safe, and effective
treatments for the urethral tissue damage have notPatients with urethral tissue damage due to aging

been developed. Recently, mesenchymal stem cell-often complain about stress urinary incontinence

based therapies have been attempted as treatments(SUI), which is one manifestation of urine storage

for SUI. In our previous study, we showed that injec‑

dysfunction. Also, urethral injury or post-operation

tion of autologous adipose-1） or bone marrow-derived

stricture can induce urinary retention, which is amesenchymal cells2） into the cryo-injured urethra of＊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. 6, 2020

rabbits reconstructed the functional urethra. Clinicalresearch suggests that injection of autologous adi‑pose-derived cells into the urethra of male patientswith SUI has the potential to provide significant ben‑357

Silwal Gautam・Imamura・Shimamura et al.

efits3）4）. Thus, autologous cells including somaticJapan) and medetomidine hydrochloride (0.5 mg/kg ;mesenchymal stem cells, such as adipose- or bone

Zenoaq, Orion Pharma, Orion Corporation Espoo,

marrow-derived mesenchymal stem cells, have theFinland) and further anesthetized by inhalation ofpotential to repair injury-damaged urethras and thus

3 ％ sevoflurane (Abbot Japan Co., LTD., Tokyo,

warrant further study.

Japan). Previously, we established methods of isola‑

However, there are some limitations to the cell in‑tion and harvesting autologous adipose-derived

jection methods. The cells can be damaged during

cells1）. Briefly, intraperitoneal adipose tissue (approxi‑

the harvesting and handling procedures. The mostmately 1 g) was harvested from an abdominal inci‑

serious limitation is the low survival rate of the in‑sion. The adipose tissue was digested with 0.2 ％ col‑jected cells in the recipient tissues. Previously, welagenase type I (Wako Pure Chemical Industries,

reported that biofabricated structures improve cellLtd., Osaka, Japan) for 90 minutes at 37 ℃ with mod‑survival rates during harvesting and handling and inerate rotary shaking. After shaking, the solution was

5）6）

the recipient tissues. In this study, we designed

centrifuged at 1,000 rpm for 4 minutes, and then thenovel biofabricated structures composed of adipose-

cells were suspended in culture medium composed

derived mesenchymal cells (AMCs) and constructedof 4.5 g/L glucose-Dulbecco’s modified Eagle’s medi‑them with a three-dimensional (3D)-bioprinter. Afterum (Gibco, Thermo Fisher Scientific K.K., Kanagawa,construction, the structures were inserted into theJapan) supplemented with 15 ％ regular fetal bovine

injured urethras of rabbits where they promoted

serum (Biowest, Nuaille, France) and 0.1 ％ penicil‑

healing of the damaged tissues. By design, the struc‑lin-streptomycin (Gibco). After passing through a celltures were C-shaped to avoid blocking the lumen ofstrainer (Life Science, Corning, NY, USA), primary

the urethra and potentially impeding the flow of

cultures of the suspended cells were established onurine. Here we report the biofabrication methodolo‑type I collagen-coated 10-cm culture dishes (AGCgy, the transplantation of the C-shaped structures

Techo Glass, Shizuoka, Japan) and incubated at 37 ℃

into the damaged urethras, and the reconstruction of

in humid air with 5 ％ CO2 for 5-7 days. The medium

the damaged urethral tissues through cellular prolif‑

was completely changed every day to wash off unat‑

eration and differentiation. We also report evidencetached cells. After reaching confluence, the attachedof paracrine stimulation through the presence ofand proliferating cells were sub-cultured to increase

growth factors and cytokines within transplanted

the number of cells in a Collagen Type I-coated T

structures and adjacent tissues.

225 cm2 Flask (AGC Techno Glass).

Ⅱ Methods
Ａ

Animals

To track the attached and proliferating cells in
passages 2 or 3, a lipophilic red fluorescent dye thatbinds to cell membranes, PKH26 (Sigma Aldrich, St.

Eight female New Zealand White rabbits (2.5-3.0

Louis, MO, USA), was applied according to the man‑

kg ; Japan SLC, Inc., Shizuoka, Japan) at postnatal

ufacturer’s protocol. The labeled cells were re-week 10 were used in this study. The rabbits weresuspended with Procul AD®that was designed fortreated in accord with the Guide for the Care andhuman adipose mesenchymal stem cells (Rohto Phar‑Use of Laboratory Animals published by the US

maceuticals, Osaka, Japan). To form cell spheroids,

National Institutes of Health. The Animal Ethics

100-μl volumes of 4×104 cells/well were seeded inCommittee of Shinshu University School of Medicine

ultra-low attachment, U-shaped, 96-well plates (3-5approved the protocol used in this study.

plates for each rabbit, Sumitomo Bakelite, Tokyo,

Ｂ Preparation of AMCs and spheroids for bio-

Japan) and allowed to form cell aggregates at 37 ℃

fabrication of C-shaped structuresin 5 ％ CO2 for 5 days.

Rabbits were anesthetized by intramuscular injec‑Ｃtion of midazolam (1.5 mg/kg ; Astellas, Tokyo,
358

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Biofabricated structures for reconstruction of urethra

Fig. 1 Autologous C-shaped AMC structure, urethral cryo-injury, and transplantation into the injured urethra. (A) At 4
days during circulating culture, the mounted spheroids that were held in place by the microneedle array were forming
spheroid-spheroid contacts. (B) Biofabricated C-shaped AMC structure after removal from microneedle array. The
structure was approximately 3.5 (wide)×3.5 (depth)×1.0 (high) mm, Scale bar : 1 mm. (C) The intact urethra wassprayed with liquid nitrogen for 20 seconds (arrowheads). (D) A small incision of approximentally 5 mm was createdaround the cyroinjured regions (arowheads). Asterisk, catheter in the urethral lumen. (E) Biofabriacted C-shaped AMC
structure (arrow) was implanted into the incised urethra.

formed on a 3D-bioprinter (Regenova, Cyfuse Bio‑Ｄ Production of cryo-injured urethral tissues

medical K.K., Tokyo, Japan) designed to create 3D-

and transplantation of the biofabricated C-

5）
6）

structures consisting only of cells. Briefly, single

shaped AMC structures

spheroids, which were composed of the PKH26-

The rabbits from which the adipose tissue waslabeled AMCs, were recovered from each well of theharvested were anesthetized as above. A urethral

96-well plates, and mounted on a custom designed,

catheter (Fr. 8, Terumo, Yokohama, Japan) was in‑

C-shaped 9x9 microneedle array. The formation ofserted, and then the urethra was exposed througheach biofabricated structure required 234 spheroids.

a midline lower abdominal incision. The anterior

The mounted spheroids, which were held in place bywall of the urethra was sprayed with liquid nitrogenthe microneedle array, were cultured with circulating(Cryo Pro®, Cortex Technology, Hudsund, Denmark)Procul AD®and allowed to form spheroid-spheroidfor 20 s (Fig. 1C). The frozen region was naturally

contacts at 37 ℃ in 5 ％ CO2 for 7 days (Fig. 1A). On

thawed within 2-3 minutes by body temperature

the seventh day of culture, the microneedle array

and/or room temperature. In this study, we define

was removed from the assembled biofabricated C-

the frozen and thawed area as the cryo-injured re‑shaped structure (Fig. 1B).

gions. The cryo-injured regions were incised until

No. 6, 2020359

Silwal Gautam・Imamura・Shimamura et al.

the urethral catheter was visualized (Fig. 1D). After

markers : myoglobin (1 : 200, rabbit polyclonal ; Spring

incision, the autologous biofabricated C-shaped AMC

Bioscience, Inc., Pleasanton, CA, USA), smooth mus‑structure was placed around the urethral catheter

cle actin (SMA, 1 : 100 mouse monoclonal ; Progen(Fig. 1E). The wounded edges of the urethral tissue

Biotechnik GmbH, Heidelberg, Germany), desmin (1 :were sewed to the inserted C-shaped structure to

200 rabbit polyclonal ; Progen Biotechnik GmbH), pax

secure them together and prevent the structure

7 (1 : 1000, rabbit ; Lifespan Biosciences, Inc., Seattle,

from falling freely into the urethral lumen or from

WA, USA), S100 (1 : 100, mouse monoclonal ; Abcam,

falling out of the lumen. Finally, the abdominal inci‑

Cambridge, United Kingdom), or calcitonin gene-sion was closed, and the urethral catheter removed.

related peptide (CRGP, 1 : 800, guinea pig polyclonal ;Four other rabbits served as sham-operated controls

Progen Biotechnik GmbH).

in which a urethral catheter was inserted, and the

To detect the presence of growth factors and cy‑

urethra was exposed and cryo-injured. The injured

tokines in the AMCs of the biofabricated structures,

region was incised until the urethral catheter was

the sections were incubated for 12 h at 4 ℃ with an‑visualized, and then the wounded edges were sewn

tibodies for each of the following markers : vascular

together without the insertion of a C-shaped struc‑endothelial growth factor (VEGF, 1 : 100, rabbit poly‑ture. At two and four weeks after surgery (n＝2/clonal ; Bioss, Woburn, MA, USA), nerve growth fac‑group in each week), the urethras were harvested,tor (NGF, 1 : 500, rabbit polyclonal ; Abcam), trans‑

and the rabbits were sacrificed with the injection of

forming growth factor-β1 (TGF-β1, 1 : 100, rabbit

an overdose of pentobarbital sodium solution

polyclonal ; Abcam), and tumor necrosis factor-α(Kyoritsu Seiyaku Co., Tokyo, Japan).

(TNF-α, 1 : 100, rabbit polyclonal ; Abcam). OtherＥ Histological and immunofluorescence evalua-sections were similarly incubated antibodies fortion

P4HB (1 : 50 : mouse monoclonal, Novus Biologicals,

At two and four weeks after transplantation orInc.) or HIF1α (1 ; 50, rabbit polyclonal, Proteintech

sham operation, the harvested urethras were fixed inGroup, Inc., Rosemont, IL, USA) for 12 h at 4 ℃.

4 ％ paraformaldehyde for 12 hours. Then, the fixed

After the primary antibody reactions, the sections

samples were embedded in paraffin and cut in 5-μm

were incubated with the respective secondary anti‑

thick serial sections. The sections were deparaffin‑bodies consisting of donkey anti-rabbit, -mouse, or

ized with xylene, rehydrated with ethanol, and-guinea pig, each conjugated with Alexa Fluor 488

rinsed three times with phosphate-buffered saline.

(1 : 250 ; Molecular Probes, Eugene, OR, USA) for 1 h

Each section was stained with Masson trichrome, pi‑

at 4 ℃. After rinsing, they were counterstained withcrosirious red, or ApopTag®ISOL fluorescence apop‑

5 μg/ml 4’, 6-diamidino-phenylindole dihydrochloride

tosis detection kit (Merck Millipore, Merck KGaA,(DAPI ; Molecular Probes).

Darmstadt, Germany) according to the manufactur‑
er’s protocol.

Biofabricated structures that were not used in atransplantation operation were immunohistochemi‑

For immunohistochemistry, some of the deparaf‑

cally stained as described above with antibody forfinized sections were immersed in 10 mM sodium

mesenchymal cell marker ; STRO-1 (1 : 100, animal

citrate (pH 6.0) and microwaved at 100 ℃ for 10 min

poly- or mono-, R&D System, Inc., Minneapolis, MN,

for antigen retrieval. The sections were blocked withUSA) and the extracellular markers anti-integrin β1

1.5 ％ non-fat milk in phosphate-buffered saline for 1(1 : 50, rat monoclonal ; R&D Systems, Inc.) and N-

h at 4 ℃.

cadherin (1 : 100, rabbit polyclonal ; Bioss).

To detect differentiated cells derived from the
AMCs within the biofabricated structures, each sec‑tion was incubated with one of the following antibod‑
ies for 12 h at 4 ℃ to identify the following cellular
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Ⅲ

Results

Ａ Characterization of AMCs within biofabricated C-shaped structures
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Biofabricated structures for reconstruction of urethra
SS
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Fig. 2 Expression of mesenchymal and extracellular matrix markers of AMCs within biofabricated structures. (A-C) The
PKH26-labeled AMCs within the biofabricated structures (A, red, arrowheads) were positive for the mesenchymal cellmarker STRO-1 (B, green, arrowheads ; C, merged image, arrowheads). (D-F) The PKH26-labeled AMCs (D, red,arrowheads) were positive for the extracellular marker integrin (E, green, arrowheads ; F, merged image, arrowheads).
(G-I) The PKH26-labeled AMCs (G, red, arrowheads) were positive for the extracellular marker cadherin (H, green,arrowheads ; I, merged image, arrowheads). Blue : DAPI-labeled nuclei, Scale bar : 20 μm.

Before urethral transplantation, the PKH26-labeledthras had developed strictures (Fig. 3A). Further‑

AMCs (approximately 10 μm) within the biofabri­more, in the sham-operated regions, there werecated structures (Fig. 2A) were positive for mesenchymal

skeletal and/or smooth muscle cells within the ure‑cell marker STRO-1 antibody (Fig. 2B, C). They alsothral tissues, but they were sparsely distributed andexpressed integrin β1 (Fig. 2D-F) and N-cadherin

disorganized (Fig. 3B). In contrast, at two weeks(Fig. 2G-I) in the structures. However, other mark‑after surgery, the urethras transplanted with a C-

ers, such as those for skeletal muscle (myoglobin),shaped biofabricated structure were wider and straightsmooth muscle (smooth muscle actin), and nerve cells

compared to the controls (Fig. 3C). The urethral(S100), and vascular endothelial cell (vWF), were nottissues in the biofabricated structure-transplanted

present.

regions also had skeletal and smooth muscle cells

Ｂ Recovery of urethral tissues after transplanta-(Fig. 3D).

tion of biofabricated C-shaped structures

At four weeks after the sham operation, the con‑

Two weeks after the sham operation, the cryo-trol rabbits continued to have urethral strictures

injured and incised regions of the sham control ure‑that were similar to those seen at two weeks afterNo. 6, 2020361

Silwal Gautam・Imamura・Shimamura et al.

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Biofabricated structures for reconstruction of urethra

surgery (Fig. 3E). While the controls had some skele‑

At both two and four weeks after the sham opera‑

tal and smooth muscle cells, they were not organized

tion, some cells in the control regions contained

into muscle layers (Fig. 3F). However, the biofabri‑

VEGF (Fig. 6A). Similarly, some cells in the trans‑

cated structure-transplanted urethras, at four weeks

plantation regions were also positive for VEGF (Fig.

after surgery, did not have urethral strictures or

6B). In addition, the cells labeled with PKH26, indi‑

other obstructions (Fig. 3G). Similar to the findings at

cating that they were derived from the AMCs, also

two weeks, there were numerous skeletal andcontained VEGF (Fig. 6B). Another growth factor,

smooth muscle cells, but these were now organized

NGF, was also detected within some cells in both theinto layered muscle-like structures (Fig. 3H).

control (Fig. 6C) and transplantation (Fig. 6D) re‑Ｃ Survival and differentiation of AMCs withingions. The control and transplanted regions also con‑the biofabricated structures after transplanta-

tained the cytokines TGF-β1 (Fig. 6E, F) and TNF-αtion(Fig. 6G, H). In the transplanted regions, someAt both two and four weeks after transplantation,

PKH26-labeled AMCs were also positive for NGF

PKH26, the red fluorescent dye embedded in the(Fig. 6D), TGF-β1 (Fig. 6F), and TNF-α (Fig. 6H).

membranes of the AMCs before spheroid formation,

Ｅ Histological changes in biofabricated struc-

was present in AMCs of the transplanted structures.

ture-transplanted urethral tissues

At four weeks after transplantation, some of the

We observed cryo-injured urethral tissues at the

PKH26-labeled cells were positive for antibodies toborders of the wound in sham-operated regions andthe skeletal muscle marker myoglobin (Fig. 4A-C) or

between the biofabricated AMC structures and thethe smooth muscle marker SMA (Fig. 4D-F). Also,

recipient tissues in the transplanted regions. At twosome PKH26-labeled cells were positive for antibod‑

and four weeks after sham surgery in which no bio‑

ies to the immature skeletal muscle cell marker de‑

fabricated structure was transplanted, the urethralsmin (Fig. 4G-I) or the myoblast progenitor cellwound site contained collagen fibers in the extracel‑marker pax7 (Fig. 4J-L). Moreover, some of thelular matrix along with damaged skeletal muscle andPKH26-labeled cells had migrated from the trans‑smooth muscle layers (Fig. 7A). In contrast, in ure‑

planted structures into the surrounding urethral re‑thras containing the transplanted biofabricatedgions (data not shown).

structures, the collagen fibers were located between

Some of the PKH26-labeled AMCs were positive

the recovering skeletal and smooth muscle layers

for the nerve cell marker S100 (Fig. 5A-C) or the af‑(Fig. 7B). The control urethras had numerous fibro‑

ferent nerve marker CGRP (Fig. 5D-F).

blast P4HB-positive cells, an indicator of hypoxia,

Ｄ AMCs growth factors and cytokines within thewithin the collagen fibers (Fig. 7C) compared to thebiofabricated structures after transplantation

structure-transplanted urethras (Fig. 7D). The con‑Fig. 3 Urethral tissues transplanted with and without biofabricated structures at two and four weeks after surgery. (A)At two weeks after the sham operation, the gross appearance of the control urethra was narrowed and somewhat
tortuous (arrowheads). (B) The skeletal muscle cells and/or smooth muscles cells within the control urethra weresparsely distributed and disorganized (asterisks). Scale bar : 100 μm. (C) At two weeks after transplantation of an AMC
biofabricated structure, the gross appearance of the urethra was wider and straighter compared to the sham-operated
control (arrows). (D) At two weeks, a urethra transplanted with a biofabricated structure had skeletal (arrows) andsmooth muscle (arrowheads) cells. (E) At four weeks after sham surgery, the urethra had strictures that were similar to
those at two weeks after surgery (arrowheads). (F) The walls of the sham-operated controls had some skeletal andsmooth muscle cells, but they did not form typical muscle layers (asterisk). (G) At four weeks after surgery, thestructure-transplanted urethra had a wider lumen (arrows) that was straighter than in the controls. (H) At four weeks
after structure transplantation, the uretha had numerous skeletal (arrows) and smooth muscle (arrowheads) cells thatwere organized into layered tissue structures. Scale bar : gross images (A, C, E, and G) : 1 mm ; Masson trichrome
staining (B, D, F, and H) : 50 μm ; UT, urothelium.
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Silwal Gautam・Imamura・Shimamura et al.

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Fig. 4 Differentation of AMCs within the transplanted biofabricated structures into muscle marker-positive cells at fourweeks after transplantation. (A-C) The PKH26-labeled cells (A, red, asterisks) were positive for the skeletal muscle
marker myoglobin (B, green, arrowheads ; C, merged image, asterisks). (D-F) The PKH26-labeled cells (D, red, asterisks)were positive for the smooth muscle marker SMA (E, green, asterisks ; F, merged image, asterisks). (G-I) The PKH26labeled cells (G, red, asterisks) were positive for the immature skeletal muscle cell marker desmin (H, green, asterisks ; I,
merged image, asterisks), (J-L) The PKH26-labeled cells (J, red, asterisk) were positive for the myoblast progenitor cellmarker pax7, (K, green, asterisk ; L, merged image, asterisks). Blue : DAPI-labeled nuclei, Scale bar : 20 μm.

trol urethras also had numerous HIF1α-positive cells,
another indicator of hypoxia (Fig. 7E), and apoptotic

Ⅳ Discussion

cells (Fig. 7G). In contrast, the structure-transplanted

Biofabrication has emerged as one of the novel

urethra had few HIF1α-positive or apoptotic cells

technologies for constructing tissues and organs in(Fig. 7F, H).

tissue engineering and regenerative medicine. The

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Biofabricated structures for reconstruction of urethra

P
K

Mer
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Fig. 5 Differentation of AMCs within transplanted biofabricated structures into nerve marker-positive cells at fourweeks after transplantation. (A-C) The PKH26 labeled cells (A, red arrows) were positive for the nerve cell marker
S100 (B, green, arrows ; C, merged image, arrows). Scale bar : 15 μm. (D-F) The PKH26 labeled cells (D, red arrows)
were positive for the afferent nerve marker CGRP (E, green, arrows ; F, merged image, arrows). Scale bar : 20 μm.
Blue : DAPI-labeled nuclei, Scale bar : 20 μm.

process holds promise for diseases and injuries that

Cell-cell interactions play an important role in cell

are currently difficult to treat due to lack of suitabledifferentiation, and indeed, two and four weeks after

therapies. In this study, we used a 3D-bioprinter totransplantation, the biofabricated structures ex‑biofabricate structures from AMC-derived spher‑pressed markers typically found in normal urethras.

5）6）. The structures were of a suitable size, shape,Thus, the AMCs within the transplanted urethraland autologous cellular composition to be transplant‑

biofabricated structures underwent differentiation

ed into the cryo-injured urethras of rabbits. Thus,

into myoglobin-positive skeletal muscle cells, SMA-

biofabricated structures have the potential to over‑positive smooth muscle cells, S100- and CGRP-

come limitations of the cell-injection methods. Addi‑

positive nerve cells. These observations suggest thattionally, we designed the biofabrications as C-shapedcell-cell interactions within the biofabricated struc‑

structures that would prevent urinary retention af‑tures might be a possible mechanism to enhance dif‑

ter transplantation. To our knowledge, the C-shaped

ferentiation capacity after transplantation.

oids

structures are a unique and novel construct for a

In addition to undergoing differentiation to form

biofabricated structure and have not been reported.

the highly specialized cells and tissues typical of the

AMCs that were attached and proliferating on thenormal urethra, the AMCs in the transplanted tissue

collagen-coated dishes were used to make the bio‑produced important signal molecules in the forms offabricated structures. The cells were positive for thegrowth factors and cytokines that are probably es‑

mesenchymal cell marker STRO-1, but they did notsential for cellular differentiation to occur. For in‑exhibit differentiation markers typical of muscle, andstance, VEGF, which promotes myogenesis, muscle

nerve, and vascular cells that are present in the nor‑regeneration, and vascularization7）8）, was present, as

mal urethra. After the C-shaped structures were

was NGF, which could promote reconstruction offormed, the AMCs expressed integrin and/or cad‑

nerve cells and/or formation of layered muscle struc‑herin, which are the cell-cell adhesion molecules.

tures9）. In addition, there might be direct or indirect

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Fig. 6 Growth factors and cytokines within the sham-operated control and transplanted regions and in the PKH26labeled AMCs at four weeks after surgery. The sham-operated control and transplanted regions were identified
macroscopically by the presence of suture marks. (A, B) Within the control (A) and transplanted regions (B), some cells
contained VEGF (green, arrows). In addition, the PKH26-labeled AMCs also contained VEGF (B, yellow, arrowheads).
(C-H) Similarly, some cells within the control (C, E, G) and transplanted regions (D, F, H) contained NGF (C and D,
green, arrows), TGF-β1 (E and F, green, arrows), and TNF-α (G and H, green, arrows). Some of the PKH26-labeled
AMCs in the transplanted regions contained NGF (D, yellow, arrowheads), TGF-β1 (F, yellow, arrowheads) and TNF-α(H, yellow, arrowheads). Blue : DAPI-labeled nuclei, Scale bar : A, B : 20 μm ; C-H : 15 μm.
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Fig. 7
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Silwal Gautam・Imamura・Shimamura et al.
Fig. 7 Histological changes in sham-operated control urethral tissues and biofabricated structure-transplanted tissues atfour weeks after surgery. (A-B) Within the control regions (A), the collagen fibers had invaded the spaces of thedamaged skeletal muscle or smooth muscle layers (asterisks). In contrast, in the structure-transplanted regions (B), thecollagen fibers were located between the recovering skeletal and smooth muscle layers (arrowheads). Picrosirus red
staining for collagen. Scale bar : 100 μm. (C-D) In the control urethra, some cells within the collagen fibers were positive
for P4HB antibody, an indicator of hypoxia (C, green, arrows). However, the transplanted regions had few P4HBpositive cells (D, green or yellow, arrows). Red : SMA-positive smooth muscle cells. (E-F) The control regions had
numerous HIF1α-positive cells, another indicator of hypoxia (E, green, arrows). In contrast, the transplanted regions hadvery few HIF1α-positive cells. (F, yellow, arrows). Red : SMA-positive smooth muscle cells. (G-H) The control regions
had numerous apoptotic cells (G, green, caspase-independent apoptotic cells ; red, caspase-dependent apoptotic
cells ; yellow, overlapping red-labeled caspase-dependent and green-labeled caspase-independent apoptotic cells,
arrows). However, in the transplanted regions (H), there were very few apoptotic cells. Unless stated otherwise,Blue : DAPI-labeled nuclei, Scale bar : 50 μm.

roles for TGF-β1 and TNF-α that were present with‑that could impede the voiding of urine. Within thein the cells of the injured regions during the AMC-

sham-operated control urethras, collagen fibers in‑mediated reconstruction of the urethral muscle cells,

vaded the spaces of the damaged skeletal muscle

blood vessels and nerve cells. The cytokine TGF-β isand smooth muscle layers. There were also numer‑

important in muscle regeneration in the absence ofous P4HB- and HIF1α-positive cells, indicating a sig‑

10）

TNF-α , while TNF-α alone promotes proliferation
11）

nificant degree of hypoxia in the damaged region. In

and fusion of myoblasts . However, in our study thecontrast, in the transplanted urethras, the collagentransplanted cells contained both cytokines, suggest‑fibers were located between the recovering skele‑

ing the presence of a strong paracrine effect intal and smooth muscle layers, and there were few

which the two cytokines could act synergistically.

P4HB- and HIF1α-positive cells present. In addition,

Moreover, some cells that were originally surround‑the structure-transplanted urethras had fewer apop‑

ing the transplanted regions also produced growth

totic cells compared to the sham-operated urethras.

factor and cytokines. Rapid conversion of the proin‑

We did not quantify the number of P4HB- and HIF1α-

flammatory TNF-α-producing macrophages to an

positive cells or, for that matter, the number of skele‑

anti-inflammatory TGF-β-producing phenotype may

tal and smooth muscle cells or other cell types inbe critical to the long-term survival of stem and pro‑the sham-operated or the AMC-transplanted urethras.

12）
-14）

. Further‑

However, the absence of structural recovery in themore, some AMCs from the transplanted biofabricat‑

sham-operated urethras and the presence of structural

ed structure migrated from the transplanted regions

recovery in the transplanted urethras was verified

into the tissues surrounding the transplanted areas

by histological and immunohistological investigations.

genitor cell populations in most tissues

and helped in reconstruction of the injured areas

Therefore, we hypothesize that tissue recovery medi­(data not shown).

ated by the AMC-transplants inhibits the formation ofCells derived from AMCs also expressed desmin

urethral strictures.

or pax7 at two and four weeks after transplantation.

We were unable to measure urethral physiological

Desmin is a muscle-specific intermediate filament

functions in this study. The most readily measured

protein present in early myogenesis and is essentialfunction is the leak point pressure (LPP) at which the15）

for the structural integrity and function of muscle .

bladder pressure forces urine to leak through the

Pax7-positive cells might also play roles in regenera‑

urethra. However, in the sham-operated controls, thetion and integration of muscle cells within the recon‑presence of strictures narrowed the urethras, thus

structed muscle layers.

artificially raising the LPP, especially in comparison

Urethras transplanted with the C-shaped AMC-

to the transplanted urethras that did not develop

biofabricated structures did not develop strictures

strictures. Thus, comparison of LPPs for the control

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Biofabricated structures for reconstruction of urethra

and transplanted animals was not valid. Neverthe‑

inhibited by the biofabricated AMC-transplants.

less, recovery of the cryo-injured urethras by trans‑

Therefore, transplantation of biofabricated structures

plantation of the biofabricated C-shaped AMC struc‑derived from AMCs has the potential to provide

tures was strongly supported by the gross anatomy

novel and effective treatments for urine storage or

and microanatomy observations.

voiding dysfunctions associated with damaged ure‑thras.

Ⅴ Conclusion

Ⅵ Acknowledgements

We used 3-D printing to manufacture novel bio‑
fabricated C-shaped structures composed of AMCs.

This study was supported by a Grant-in-Aid for

The AMCs within the structures transplanted into

Scientific Research (C) from Japan Society for thethe cryo-injured rabbit urethras survived and pro‑

Promotion of Science (16K11043).

moted recovery through differentiation of muscle,
and nerve cells. In addition, these cells contained

Funding

growth factors and/or cytokines that likely enhancedNone

myogenesis, and neurogenesis. The development ofConflicts of Interest

urethral strictures during the recovery process was

The authors declare no conflicts of interest.

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（2020. 5. 21 received ; 2020. 7. 7 accepted）

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