Journal of Orthopaedic Science xxx (xxxx) xxx

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Journal of Orthopaedic Science

journal homepage: http://www.elsevier.com/locate/jos

Original Article

Facilitatory effects of artificial nerve filled with adipose-derived stem

cell sheets on peripheral nerve regeneration: An experimental study
Tadahiro Nakajima*, Kaoru Tada, Mika Nakada, Masashi Matsuta, Hiroyuki Tsuchiya
Department of Orthopedic Surgery, Graduate School of Medical Sciences, Kanazawa University, Ishikawa, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Background: We evaluated how artificial nerves filled with adipose-derived stem cell (ADSC) sheets
Received 28 August 2019 could facilitate peripheral nerve regeneration.
Received in revised form Methods: We prepared ADSC sheets following previously described protocols. We transected the sciatic
25 August 2020
nerve in 12-week-old Wistar rats, fixed the nerve ends to the artificial conduit, and prepared three
Accepted 16 September 2020
Available online xxx
groups: (1) conduits alone (control group); (2) conduits filled with ADSCs (ADSCs group), and (3) con-
duits filled with ADSC sheets (ADSC sheet group). We assessed the subjects 4 and 12 weeks post-
transplantation (n ¼ 24). We investigated bIII-tubulin and anti-S100 expression at 4 and 12 weeks
post-transplantation, in longitudinal- and cross-sections of the central portion in the regenerated tissues.
The vascular endothelial growth factor A (VEGFA) and neuregulin-1 expressions were analyzed using
real-time reverse-transcription polymerase chain reaction (real-time RT-PCR). We evaluated the tibialis
anterior muscle wet weight (affected/healthy sides, %) and sciatic function index (SFI) 12 weeks post-
transplantation.
Results: The ADSC sheet group comprised more S100-positive cells than the other groups. The regen-
erated axon length in the ADSC sheet group was markedly the longest among the studied groups. The
immunostaining revealed a positive area in the regenerated tissue center in all groups, tending to be the
largest in the ADSC sheet group. The muscle wet weight indicated that the ADSC sheet group exhibited
significantly higher weight than the control. The mean SFI showed that the ADSC sheet group exhibited
significantly better results than the control. The VEGFA expression was higher both in the ADSC and the
ADSC sheet group than in the control. The neuregulin-1 expression was higher both in the ADSC and the
ADSC sheet group than in the control.
Conclusions: The ADSC sheets could potentially support transplanting an adequate number of ADSCs at
the target site. Compared with the conventional method of attaching ADSCs, the use of ADSC sheets
promotes accelerated nerve regeneration.
© 2020 The Authors. Published by Elsevier B.V. on behalf of The Japanese Orthopaedic Association. This is
an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
4.0/).

1. Introduction
Although autologous nerve grafts have long been used to treat
peripheral nerve defects, persistent numbness or pain might
develop at the nerve harvesting site as healthy nerves are sacrificed
for this procedure. Therefore, Lundborg et al. proposed a tubuli-
zation principle that promotes nerve regeneration by bridging the
deficient site, other than the nerve, with a cylindrical chamber [1].
For nearly half a century since then, a lot of studies have
* Corresponding author. 13-1, Takaramachi, Kanazawa city, Ishikawa, 920-8641,
Japan. Fax.þ81 76 265 2374.
E-mail address: t_nnnkaji@yahoo.co.jp (T. Nakajima).
investigated the two nerve conduit elements, the material and
luminal fillers, in order to improve tubulization performance.
Schwann cells play a vital role in peripheral nerve regeneration
[2], and they might be ideal “fillers” for the nerve conduit. However,
it remains a technical challenge to secure an adequate number of
Schwann cells from cell cultures. Recently, Schwann cell functions
have been anticipated in various stem cell types and tested as
“fillers.” Therefore, we focused on adipose-derived stem cells
(ADSCs) as fillers due to their advantages, such as the safety and
ease of sampling. To date, ADSCs are safely used in clinical settings
for breast reconstruction, ischemic heart disease, and ischemic ul-
cers, without any serious complications. Furthermore, a large
number of ADSCs could be easily isolated from subcutaneous

https://doi.org/10.1016/j.jos.2020.09.014
0949-2658/© 2020 The Authors. Published by Elsevier B.V. on behalf of The Japanese Orthopaedic Association. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: T. Nakajima, K. Tada, M. Nakada et al., Facilitatory effects of artificial nerve filled with adipose-derived stem cell sheets
on peripheral nerve regeneration: An experimental study, Journal of Orthopaedic Science, https://doi.org/10.1016/j.jos.2020.09.014
T. Nakajima, K. Tada, M. Nakada et al.
adipose tissues, and the number of stem cells that could be sampled
is drastically higher than those derived from the bone marrow [3].
During cell transplantation, cells must be supported at the
target site. The ADSCs in the sheet are bridged with collagen and
the ascorbic acid properties of collagen synthesis [4]. In this study,
we aimed at assessing whether artificial nerves filled with ADSC
sheets promote peripheral nerve regeneration.
2. Materials and methods
2.1. Animals
This research was approved by the IRB of the authors’ affiliated
institutions. A total of 24 female Wistar rats (age, 10e12 weeks)
were used in this experiment. First, we randomly categorized 12-
week-old Wistar rats into three groups and prepared a 15-mm
sciatic nerve defect model in each group (n ¼ 8 per group). Four
of each were assessed at 4 and 12 weeks post-transplantation. Rats
used for the experiment were euthanized using an intraperitoneal
injection of 64.8 mg pentobarbital (Somnopentyl®, MSD K.K.,
Tokyo, Japan).
2.2. Isolation of ADSCs
ADSCs were isolated as previously reported [5]. First, approxi-
mately 1.5 g harvested from the left inguinal region of Wistar rats
was cut into smaller pieces with scissors. Thereafter, collagenase
(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was
adjusted to 0.12% in 20 mL and allowed to react with adipose tissues
in a water bath at 37  C for 45 min with continuous stirring every
15 min. Immediately after the reaction, 20 mL of Dulbecco's
Modified Eagle's Medium (DMEM; FUJIFILM Wako Pure Chemical
Corporation, Osaka, Japan) containing 10% fetal bovine serum (FBS;
Nichirei Biosciences Inc., Tokyo, Japan) and 1% penicillin-
streptomycin solution (P/S; FUJIFILM Wako Pure Chemical Corpo-
ration) was added to neutralize collagenase. The solution was
filtered and centrifuged for 6 min at 1300 rpm at 25  C. Precipitates
obtained were then cultured in a normal medium such that the
number of cells per 100-mm2 dish would be 1  106. After reaching
confluency, the cultures were sub-cultured three times; the cells
thus obtained were called ADSCs. These ADSCs were then cultured
for 1 week in a medium containing 50 mM ascorbate-2-phosphate
(FUJIFILM Wako Chemical Corporation, Miyazaki, Japan) added to
500 mL of DMEM with 10% FBS and 1% P/S, according to the method
proposed by Fang et al. [6] to create ADSC sheets (Fig. 1).
2.3. Surgical methods
Surgery was performed under sterile conditions and strict
adherence to the guidelines set by the Ethics Committee of the
International Association for the Study of Pain [7]. Rats were
anesthetized by intraperitoneal administration of 6.5 mg pento-
barbital (Somnopentyl®, MSD K.K., Tokyo, Japan), and the left
sciatic nerve was exposed. The sciatic nerve was then dissected 15-
mm in length, and 1-mm nerve stumps were fixed at both ends of
the 17-mm Nerbridge® (Toyobo, Osaka, Japan), which is an artificial
nerve made of a polyglycolic acid tube filled with collagen using
four nylon stitches by 9e0 mono-filament nylon, thus creating a 15-
mm nerve defect model.
The three experimental groups were as follows: (1) Nerbridge®
alone without any cells (control group). (2) ADSCs in 1 mL of
physiological saline (3e5  10⁶cells/mL) were injected into the
Nerbridge® after removal of the internal collagen material. (ADSC
group). (3) The ADSC sheet was filled into the Nerbridge® after
removal of the internal collagen material. (ADSC sheet group).
2
Journal of Orthopaedic Science xxx (xxxx) xxx
Fig. 1. Macroscopic observation of the ADSC sheet. Macroscopic appearance of an
ADSC sheet (10 cm dish).
Then, the gluteus muscles and skin were closed using 6e0 nylon
sutures.
2.4. Evaluated items
2.4.1. Immunohistochemical evaluation
After fixing in 10% formalin and embedding in paraffin, paraffin
sections were prepared containing longitudinal sections of the re-
generated tissues (n ¼ 4 at 4 weeks after surgery per group). After
deparaffinization in xylene (FUJIFILM Wako Chemical Corporation),
they were treated with antigen activation solution (Polysciences)
and blocked with blocking buffer (Agilent) for 10 min bIII-tubulin
(Anti-beta III Tubulin antibody®; Abcam, Cambridge, UK) was
added and incubated at 4  C overnight. The sections were then
incubated with Alexa Fluor 488elabeled goat derived anti-rabbit
antibody (ab150077, Abcam, Cambridge, UK) for 45 min at room
temperature. The regenerated axon length was measured from
1 mm inside the tube to the last visible positive areas. Then, the
presence of Schwann cells was analyzed immunohistochemically
(n ¼ 4 at 12 weeks after surgery per group). The nerve samples from
each group were fixed in 10% formalin for 24 h, embedded in
paraffin and cut into 2 mm thick transverse sections on a cryostat.
Sections were blocked with 5% normal goat serum for 30 min at
room temperature and incubated with S100 protein (Anti-S100
beta antibody®, Abcam, Cambridge, UK) for 1 h at room tempera-
ture. The localization of Schwann cells together with the negative
control was assessed. We also evaluated the ratio of S100 stained
area using Image J software (version.1.52, National Institutes of
Health, Bethesda, MD, USA).
2.4.2. Motor functional evaluation
In order to evaluate motor functional recovery, walking track
analysis was performed 12 weeks after surgery. As previously re-
ported by Bain et al. [8], the sciatic functional index (SFI) was
calculated according to the following: SFI ¼ 38.3 [(EPL e NPL)/
NPL] þ 109.5 [(ETS e NTS)/NTS] þ 13.3 [(EIT e NIT)/NIT] e 8.8. Print
length (PL) is the distance from the heel to the top of the 3rd toe, toe
spread (TS) is the distance between the 1st and 5th toe, and
intermediary toe spread (IT) is the distance from the 2nd to the 4th
toe. E and N in front of these abbreviations mean experimental and
T. Nakajima, K. Tada, M. Nakada et al. Journal of Orthopaedic Science xxx (xxxx) xxx

normal side, respectively. SFI zero means normal, and SFI -100
means complete dysfunction. Motor functional recovery was also
indexed with the tibialis anterior (TA) muscle weight ratio, defined
as the ratio of muscle wet weight of the affected side to the healthy
side was measured.

2.4.3. Real-time RT-PCR

Real-time RT-PCR was performed on regenerated tissues 12
weeks postoperatively using the methods outlined below. The
expression of neuregulin-1 and vascular endothelial growth factor
A (VEGFA) was analyzed. RNA was extracted using a NucleoSpin
RNA II kit (Takara Bio, Otsu, Japan). Each sample was harvested from
the center (proximal side) of the regenerated tissues with a margin
of approximately 5-mm and was disrupted and homogenized using
a syringe. The absorbance of the total RNA was measured at 260 nm
and the concentration was calculated. cDNA was synthesized using
a cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) using
a T100TM Thermal Cycler (Bio-Rad, Hercules, CA, USA). Treatment
was performed according to the manufacturer's protocol. Primers
for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
neuregulin-1, and VEGFA were purchased from Eurofins Genomics
Co., Tokyo, Japan. The following primers were used: GAPDH-F 5-
ACAGTCCATGCCATCACT-30 , -R50 -GCCTGCTTCACCACCTTC-30 ;
GAPDH-F 5 -TGGCCTTCCGTGTTCCTACCC-30 -R50 -CCGCCTGCTTCAC-
0

CACCTTCT-30 ; neuregulin-1-F 50 -GAAGGACCTGTCAAACCCGT-30 ,

-R50 -CTTCTGGTAGAGTTCCTCCGC-30 ; VEGF-F 50 -
GTGTGTGTGTGTATGAAATCTGTG-3 , 0 and 0
-R5 -GCA-
GAGCTGAGTGTTAGCAA-30 (Eurofins Genomics, Tokyo, Japan). Real- Fig. 2. bIII-tubulin staining on the longitudinal section of the regenerated segment
time RT-PCR was then performed using an ABI Prism 7900 appa- harvested at 4 weeks after implantation. A. Control group, B. ADSCs group, C. ADSC
ratus (Applied Biosystems, Foster City, CA, USA) with a SYBR Green sheet group. The bIII-tubulin staining (in green) highlights the Schwann cell migration
PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The from the proximal end of the tube.

amplification was carried out with an initial incubation at 95  C for

15 min, followed by 20 amplification cycles of 15 s at 94  C, 30 s at
60  C, and 30 s at 72  C. The reactions were completed in an
extended step of 7 min at 72  C and then stored at 4  C overnight.
Cycle threshold (Ct) values were calculated for each group. The
relative changes in expression were determined according to the
2DDCt method. The expression ratio of neuregulin-1 to GAPDH
and the expression ratio of VEGFA to GAPDH were calculated for
each sample.

2.5. Statistical analysis

All experimental results are expressed as the mean ± standard

deviation (SD). Statistical analysis was performed using one-way
analysis of variance (ANOVA) with the Bonferroni multiple com-
parison test (SPSS). Statistical than 0.05 was set at significant. Sig-
nificance was determined as *P < 0.05, **P < 0.01, and ***P < 0.001.

3. Results

3.1. Immunohistochemistry
Axonal regeneration distance was assessed using bIII-tubulin
immunohistochemistry (Fig. 2). The average distances of the re-
generated axons were 2.49±0.96, 2.68±0.75, and 4.45±1.3 mm in
the control, ADSC, and ADSC sheet groups, respectively. The ADSC
sheet group showed a significant improvement in axonal regener-
ation compared to the other groups. (Fig. 3). In the S100 staining, in
all three groups, the S100 protein-positive area at the center of
regenerated tissues was found. The positive area was the largest in
the ADSC sheet group compared with those in the control and ADSC
groups (Fig. 4), that is, more Schwann cells were suspected in the
regenerated tissues of the ADSC sheet group. The ratio of the S100
staining area (staining/total area) was 1.58 ± 2.0%, 4.54 ± 3.6%, and
3
Fig. 3. Quantification of regeneration distances of axonal regeneration at 4 weeks after
implantation. A. Control group, B. ADSCs group, C. ADSC sheet group. Axonal regen-
eration was evaluated quantitatively. Graphs show a significant improvement in axonal
regeneration in the ADSC sheet group than in the other groups.
13.2± 6.1% in the control, ADSC, and ADSC sheet groups, respec-
tively (Fig. 5). The ratio of the ADSC sheet group was highest
compared with those of the control and ADSC groups.
3.2. Evaluation of motor function
The TA muscle wet weight ratio (affected/healthy sides) was
24.8±4.2%, 43.6±6.3%, and 53.4±8.2% in the control, ADSC, and
T. Nakajima, K. Tada, M. Nakada et al. Journal of Orthopaedic Science xxx (xxxx) xxx

Fig. 4. Staining for S100 protein on the transverse section med portion of the regenerated segment harvested at 12 weeks after implantation with negative control (right side). A.
Control group, B. ADSCs group, C. ADSC sheet group. Paraffin sections containing cross-sections of the central regenerated tissues were prepared. The S100 positive area (brown,
circled with red) was the largest in the ADSC sheet group compared with the other groups.

Fig. 5. Quantification of the S100 staining area at 12 weeks after implantation. A. Fig. 6. Tibialis anterior muscle wet weight ratio at 12 weeks after implantation. A.
Control group, B. ADSCs group, C. ADSC sheet group. The ratio of the S100 staining area Control group, B. ADSCs group, C. ADSC sheet group. The tibialis anterior muscle wet
(staining/total area) was calculated. Graphs show a significantly higher ratio in the weight ratio (affected/healthy sides) in the ADSC sheet group was significantly higher
ADSC sheet group compared with the others. (p < 0.05) than in the control and ADSC groups.

ADSC sheet groups, respectively. The weight ratio in the ADSC sheet
group was significantly higher than those in the control and ADSC
groups (Fig. 6). The mean SFIs were 76.4±5.4%, 64.9±9.5%,
and 37.1±23.5% in the control group, ADSCs group, and ADSC
sheet group, respectively, with the ADSC sheet group showing
significantly higher results than the control group, and ADSC group
(Fig. 7).
3.3. Neuregulin-1 and VEGFA expression
In the regenerated tissue in all groups, VEGFA and neuregulin-1
expression was confirmed. VEGFA expression was significantly
higher in the ADSC sheet group than in the control and ADSC
groups but no significant difference was observed between the
control and ADSC groups (Fig. 8a). Furthermore, neuregulin-1

4
T. Nakajima, K. Tada, M. Nakada et al. Journal of Orthopaedic Science xxx (xxxx) xxx

Fig. 7. Sciatic function index at 12 weeks after implantation. A. Control group, B. ADSCs
group, C. ADSC sheet group. The sciatic function index, using walking track analysis,
showed that functional recovery was significantly greater in the ADSC sheet group
than in the control and ADSC groups.

expression was significantly higher in the ADSC sheet group, ADSC

group, and control group (Fig. 8b).

4. Discussion

In this study, we reported that the expression of humoral factors

was higher in the ADSCs and ADSC sheet groups that used ADSCs as
fillers than those in the control group that only used Nerbridge®.
Furthermore, the ADSC sheet group exhibited an increase in the
number of Schwann cells in the regenerated tissues and in the
length of regenerated axons compared with those in the ADSCs
group. In other words, filling the artificial nerve with the ADSC
sheet promoted peripheral nerve regeneration.
To date, several studies have investigated the material and filler
elements of cylindrical chambers for tubulization. In our study,
ADSCs were obtained by culturing heterogeneous populations of Fig. 8. Relative expression levels of VEGFA and Neuregulin-1 in the three groups at 12
adipose-derived regenerative cells (ADRCs) (Fig. 9), obtained from weeks after implantation. (a) VEGFA expression. (b) Neuregulin-1 expression. A.
adipose tissues through collagenase processing [9]. ADRCs were Control group, B. ADSCs group, C. ADSC sheet group.

identified as pluripotent cells in 2001 [10]. As ADSCs are easier to

harvest than bone marrow-derived stem cells, several fundamental
studies reported the use of ADSCs [11]. ADSCs differentiate into the
myocardium, bones, cartilages, nerves, and liver, and our depart-
ment has confirmed that ADSCs promote nerve [11], bone [12],
meniscus, and tendon regenerations.
ADSCs promote nerve regeneration. However, the underlying
mechanism remains unclear [13e16]. Initially, ADSCs were antici-
pated to play the functions of Schwann cells, and some studies have
reported that ADSCs will differentiate into cells similar to Schwann
cells [17,18]. However, Santiago et al. reported that ADSCs did not
differentiate into Schwann cells [13]. Suganuma et al. in our labo-
ratory have reported similar [19] results as those reported by
Santiago et al. However, no conclusions could be drawn on the
differentiation of administered ADSCs. Conversely, Luo et al. and Fig. 9. Schematic of ADSCs in the adipose tissue: ADSCs: adipose-derived stem cells.
Matsuda et al. have reported that ADSCs promote nerve regenera- ADRCs: adipose-derived regenerative cells.
tion by stimulating the growth of Schwann cells by releasing hu-
moral factors [20,21]. In this study, the expression of neuregulin-1,
VEGFA functions in vascular endothelial cells as well as in Schwann
a nerve growth factor, and VEGFA, vascular inducing factor, was
and neural stem cells, and it is closely associated with the vascular
markedly increased in the ADSC sheet group compared with that in
and nervous systems [24], especially in terms of promoting growth
the ADSC group at 12 weeks postoperatively. Neuregulin-1 strongly
and Schwann cell migration [25]. Thus, the results of our and pre-
promotes Schwann cell migration [22], perhaps via activation of the
vious studies indicate that ADSCs promote growth and Schwann
phosphatidylinositol 3-kinase and intracellular MEK pathways [23].
5
T. Nakajima, K. Tada, M. Nakada et al.
cell migration via neuregulin-1 and VEGFA expression, which in
turn promotes axon regeneration.
During cell transplantation, a support function is necessary to
maintain cells at the desired site. Methods to support transplanted
cells that promote nerve regeneration include sealing cells with
fibrin glue [26] or rolled collagen gel [27] as well as the use of
fibroin/chitosan [28]. Methods that support cells without changing
the environment surrounding the cells are desirable. Shen et al.
compared groups with cells sealed with collagen gel or sheets and
reported that the sheet was more suitable to administer cells as it
better supported the cells [29]. In this study, the number of
Schwann cells in the regenerated tissue increased to a higher extent
in the group using the ADSC sheet than in the groups with an
injected suspension of ADSCs. Moreover, the length of the regen-
erated axons was greater in the former group. Thus, the ADSC sheet
is effective in promoting the growth and support of transplanted
cells.
The present study has some limitations. First, we did not track
ADSCs that were administered; therefore, the distribution of ADSCs
in the regenerated tissues and their differentiation into similar cells
could not be assessed. Second, we assessed the expression of only
some humoral factors. Third, we did not assess the appropriate
number of cells in nerve regeneration. We determined the number
of cells based on previous reports [30,31]. We intended to assess the
impact of the number of ADSCs that are most effective for nerve
regeneration. Thus, the ADSC sheet as a filler promoted peripheral
nerve regeneration.
5. Conclusions
The ADSC sheet improved peripheral nerve regeneration, pro-
moting the effect of the artificial nerve. The mechanism is that the
number of locally retained ADSCs is increased by the ADSC sheet
and the effect of promoting the proliferation and migration of
Schwann cells.
Declaration of competing interest
There are no conflicts of interest to declare.
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