- Email: [email protected]
Repairing sciatic nerve injury with an EPO-loaded nerve conduit and sandwiched-in strategy of transplanting mesenchymal stem cells
Accepted Manuscript Repairing sciatic nerve injury with an EPO-loaded nerve conduit and sandwiched-in strategy of transplanting mesenchymal stem cells Wei Zhang, Lihai Zhang, Jianheng Liu, Licheng Zhang, Jian Zhang, Peifu Tang PII:
S0142-9612(17)30423-4
DOI:
10.1016/j.biomaterials.2017.06.024
Reference:
JBMT 18144
To appear in:
Biomaterials
Received Date: 5 January 2017 Revised Date:
9 June 2017
Accepted Date: 19 June 2017
Please cite this article as: Zhang W, Zhang L, Liu J, Zhang L, Zhang J, Tang P, Repairing sciatic nerve injury with an EPO-loaded nerve conduit and sandwiched-in strategy of transplanting mesenchymal stem cells, Biomaterials (2017), doi: 10.1016/j.biomaterials.2017.06.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Repairing sciatic nerve injury with an EPO-loaded nerve conduit
2
and sandwiched-in strategy of transplanting mesenchymal stem
3
cells
RI PT
1
Wei Zhang 1, Lihai Zhang 1, Jianheng Liu 1, Licheng Zhang1, jian
5
Zhang 2, Peifu Tang 1*
6
1 Department of Orthopedics, General Hospital of Chinese PLA,
7
Beijing, 100853, China
8
2 Department of Orthopedics, General Hospital of Liaoyuan Mining
9
Group, Jilin Province, 136201, China
M AN U
SC
4
10
*Correspondence should be addressed to Peifu Tang;
11
pftang [email protected]
12 13
17 18 19 20 21 22 23 24
EP
16
AC C
15
TE D
14
25 26 27
1
ACCEPTED MANUSCRIPT
Abstract: Transplantation of a biosynthetic nerve conduit carrying
2
neuro-protective cytokines is promising for treating peripheral nerve
3
injury. Here we developed a novel strategy for repairing sciatic nerve
4
injury by EPO-loaded Chitosan nerve conduit (EPO/Chi) and
5
sandwiched-in transplantation of mesenchymal stem cells (MSCs). Then,
6
the beneficial effect of EPO/Chi+MSCs on nerve regeneration was also
7
further investigated by in vitro cellular experiments. In vivo experiment
8
showed that combination of EPO-loaded Chitosan nerve conduit with
9
MSCs could significantly accelerate nerve healing and improve
10
morphological repair. Furthermore, the in vitro cellular experiments
11
results demonstrated that the loaded EPO in nerve conduit could
12
significantly reinforce the repair performance of both MSCs and
13
Schwann cells, which may also contribute to the therapeutic outcome of
14
the EPO/Chi + MSCs strategy. Collectively, the EPO-loaded nerve
15
conduit and sandwiched-in transplantation of MSCs we reported in the
16
study may represent a new potential strategy for peripheral nerve
17
reconstruction.
18
Key words: Sciatic nerve injury; Mesenchymal Stem Cells (MSC);
19
Nerve regeneration or repair; Erythropoietin (EPO); Chitosan
AC C
EP
TE D
M AN U
SC
RI PT
1
2
ACCEPTED MANUSCRIPT 1
1.Introduction Despite the certain degree of self-regenerative capability, repair of
3
long-distance peripheral nerve transection injury was still a major
4
challenge in clinic and the therapeutic outcome was usually weak [1, 2].
5
Thus, tissue engineering-based efforts, such as biomaterials supplemented
6
with or without stem cells or neurotrophic factors, were proposed as
7
alternative promising ways to repair the injured peripheral nerve[3, 4].
8
Among
these
approaches,
the
RI PT
2
artificial
nerve
guidance
conduit-based surgery was considered as one of the most promising
10
strategies for treating peripheral nerve injury. These biodegradable
11
guidance conduits, with single/multi-channel hollow tubes [5] or more
12
complicated structure for bio-mimicking themacro- and micro-structures
13
of nerves [6, 7] demonstrated promising therapeutic benefits. A number
14
of materials, including the synthetic Poly-L-Lactide Acid (PLLA),
15
Polycaprolactone (PCL), and some natural biomaterials such as collagen
16
or fibrin, had been used for the guidance conduits development [2, 8, 9].
M AN U
SC
9
Recently, the natural derivative chitosan based porous and
18
biodegradable scaffolds were proved to be a potentially effective
19
candidate conduit material for nerve regeneration, especially for
20
promoting the long-distance regeneration of peripheral nerves [6, 10].
21
However, the biological response of single synthetic chitosan conduit
22
using is still underactive, especially for lacking of seeding cells for nerve
23
regeneration. In addition to single nerve conduit, supplemention of
24
growth-promoting and neuronal regulation cytokines, such as neurotropic
25
factors glial cell line derived neurotrophic factor (GDNF) and nerve
26
growth factor (NGF), were proved to further enhance the nerve
27
regeneration[2]. Recently, the normal known hematopoietic cytokine
28
erythropoietin (EPO) was demonstrated with neuro-protective on the
29
peripheral nerve system by targeting Schwann cells after nerve injury
30
[11-15]. Previously, we have demonstrated that sustained release of EPO
31
by poly (lactide-co-glycolide) (PLGA) microspheres could significantly
AC C
EP
TE D
17
3
ACCEPTED MANUSCRIPT 1
promote the recovery of injuried peripheral nerve in rats [16], and thus
2
we hypothesized in the study that the loading exogenous EPO in the
3
synthetic nerve conduits might increase their performance for peripheral
4
nerve repair. The application of stem cells therapy would provide not only large
6
numbers of exogenous seeding cells, but also adequate amounts of
7
paracrine secreted bioactive cytokines[17, 18]. However, traditional stem
8
cell transplantation was usually accompanied with excessive loss and
9
death of transplanted cells, which hampered the therapeutic efficacy for peripheral nerve injury.
SC
10
RI PT
5
The present study aimed to construct a simpler intraluminal modified
12
nerve guidance conduits and develop a sandwiched-in strategy for stem
13
cells transplantation. Based on the tissue engineering approach, the EPO
14
was loaded in the Chitosan scaffold for sustained release to further
15
enhance nerve regeneration and the functional repair of the sciatic nerve
16
injury. The immunohistochemical and histological monitoring methods
17
were performed to evaluate the performance. Finally, the in vitro cellular
18
experiments were performed to investigate beneficial effects of
19
EPO-loading Chitosan scaffolds on the repair performance of MSCs and
20
Schwann cells, as well as the potential underlying mechanism.
21
2. Materials and methods
22
2.1. Cell culture
TE D
EP
AC C
23
M AN U
11
Rat adipose-derived Mesenchymal Stem Cells (Ad-MSC or MSC)
24
were bought from Cyagen Co. (China) and cultured at 37 °C in a
25
humidified atmosphere containing 5% carbon dioxide. The culture
26
medium of Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen
27
Co., USA) with 10% (v/v) fetal bovine serum (FBS, Thermo scientific,
28
Logan, USA), 100 units/ml Penicillin and 100 µg/ml streptomycin was
29
used. 4
ACCEPTED MANUSCRIPT 1
2.2. Preparation of the EPO-Chitosan nerve guidance
2
conduit EPO-Chitosan nerve conduits were prepared as previously described
4
[8]. Briefly, 10 mg/mL (w/v) of chitosan in hydrochloric acid aqueous
5
solution (pH 5.8) with 1000 U/mL EPO were prepared and injected into a
6
casting mold with an outer sleeve (diameter = 3.8 mm) and an inner
7
sleeve (diameter = 1.2 mm) (Figure. 1 A). The solution was further
8
solidified under minus 20 oC overnight and dried by using a vacuum
9
freeze-drying machine with a FDP80 Oil Vacuum Pumps (Thermo
10
Scientific™, USA). The prepared nerve conduits were then washed with
11
phosphate-buffered saline (pH 7.4) and sterilized with 25 kGy Cobalt 60
12
prior to use. The Scanning Electron Microscope (SEM) was performed to
13
characterize the scaffold by using Mira3 LMH Schottky FE-SEM (Tescan,
14
Czech).
15
2.3.
16
guidance conduit
M AN U
SC
RI PT
3
of
TE D
Characterization
the
EPO-Chitosan
nerve
To characterize the in vitro release of the EPO from the conduit, 10
18
mg of EPO-Chitosan nerve conduits were emerged into 1mL 10 mM PBS and incubated at 37 °C with shaking at 110 rpm. The released supernatant with EPO at day 0.5, 1, 2, 4, 7, 10, 14, 18 and 21 were obtained via centrifugation at 3000 rpm for 2 min and replaced the same volume of PBS after collecting the samples. The concentration of EPO protein in the supernatant was measured using an EPO ELISA kit. The cumulative release (%) of EPO was calculated and 5 samples were included. To determine the effective swelling ratio, the dry weights (Wdry) of lyophilized Chitosan (Chi) and EPO loaded Chitosan (EPO/Chi) conduits were measured. After that, the conduits were immersed in 10 mM PBS
20 21 22 23 24 25 26 27 28
AC C
19
EP
17
5
ACCEPTED MANUSCRIPT
and incubated for 24 hours at 37 °C to reach equilibrium swelling state.
2
Five samples of each group were obtained and the weight of conduit after
3
swelling (Wwet) was measured. The swelling ratio (SR) was calculated
4
by the following equations: SR=( Wwet- Wdry)/ Wdry*100%. The
5
elastic modulus of conduits was measured using a sans CMT700 type
6
universal testing machine. The conduit were incubated in PBS for 24
7
hours at 37 °C and loaded on the machine. The elastic modulus of
8
conduits was at a stretching speed of 10 mm/min.
9
2.4.
injury
treatment.
animal
model
SC
nerve
and
M AN U
10
Peripheral
RI PT
1
The effect of EPO-loaded nerve conduit with MSCs on nerve
12
regeneration was examined in an experimental sciatic nerve defect injury
13
animal model employing specific pathogen-free (SPF) Sprague-Dawley
14
(SD) rats. The rats (about 220 g) were purchased from the Experimental
15
Animal Center, Academy of Military Medical Science (Beijing, China).
16
All animal experiments were approved by the local institution of Animal
17
Experimental Ethics Committee of Experimental Animal Center,
18
Academy of Military Medical Science (Beijing, China).
TE D
11
Animals were prepared and underwent surgery using the method as
20
described [7]. Briefly, zoletil (500 µg per animal) and rompun 2% (10 µL
21
per animal) of intramuscular injection were used to anesthetize the animal.
22
In the central area underneath the gluteus maximus, the sciatic nerve in
23
the right hind limb was exposed and transected by a scissor cut at a
24
constant point and a 5mm nerve defect was created. After that, four
25
different treatment approach were applied for the injury healing: (1) For
26
Control group, simple saline treatment was applied; (2) for Gel/MSC
27
group, 20uL Matrigel mixed with 2×105 MSC was injected to the defect
28 29
area (3) For Conduit group, only EPO- Chitosan conduit was applied for surgery transplantation; (4) for Conduit + Gel/MSC groups, 20uL
30
Matrigel mixed with 2×105 MSC was injected into the conduit after
AC C
EP
19
6
ACCEPTED MANUSCRIPT 1
Conduits implantation and then applied for the defect replacement. For
2
tracking of the exogenous MSC, the cells were labeled by DiI
3
(Invitrogen). There were 12 rats in every experimental group at each time
4
points.
5
2.5.
6
examination
analysis
and
electrophysiological
RI PT
Functional
Four and eight weeks after surgery, the walking track test for
8
foot-print analysis was applied for the all animals for functional recovery
9
of never injury as described [13, 19]. Briefly, a confined walking track
10
system (7 × 50 cm2) of an open and a darkened end corridor was used
11
respectively. The rats with black ink dyed paws were allowed to walk on
12
it to obtain the tractable paws prints. The footprint parameter sciatic
13
functional index (SFI) was calculated accordingly [13, 19] and an SFI
14
value (-100% - 0) stands for the incomplete nerve function recovery.
15
To further evaluate the motor performance of the rat, the extensor
16
postural thrust (EPT) and withdrawal reflex latency (WRL) were also
17
measure at the end of week 8. For EPT analysis, the entire body of rat
18
was wrapped in a surgical towel and held upright except for its one hind
19
limb. The foot extension of the rat was induced via lowering the rat
20
toward the surface a platform. The extension force to the platform of a
21
digital balance was recorded in grams. The EPT of normal hind limb
22
(EPT_nor) and experimental hind limb (EPT_exp) from every rat were
23
measured. The reduction in the extensor muscle force was set as the
24
indicative of a deficit in the motor function. The deficit percentage was
25
calculated with the formula EPT (%) = (EPT_nor – EPT_exp) / EPT_nor
26
*100%. For WRL measurement, the rat was fixed and the hind paw was
27
placed on a 56 oC platform. The WRL time was measured as the required
28
time for the rat to withdraw the paw. If the rat remain still for more than
29
12 seconds, removed the rat from the hot platform and the maximal WRL
30
of 12 seconds was assigned to the animal. Detailed descriptions of the
AC C
EP
TE D
M AN U
SC
7
7
ACCEPTED MANUSCRIPT 1
methods could be found in many relative published papers. The electrophysiological examination was further conducted to obtain
3
the Motor nerve conduction velocities (MNCV) of right sciatic nerve
4
using MEB-7102 instrument (Nihon Kohden,Osaka, Japan). The distance
5
between the stimulating and recording electrodes was then divided by the
6
latency of the distal and proximal to calculate the MNCV.
7
2.6.
8
examination.
histological
vivo
and
immunohistological
SC
In
RI PT
2
The sciatic nerves or targeting gastrocnemius muscles were harvest
10
from each group at relatively time points after surgery and fixed in 4%
11
paraformaldehyde for at least 24 hours. The tissue was embedded in
12
paraffin and 3-5 µm of paraffin sections were then prepared. After that,
13
hematoxylineosin (H&E) and Masson trichrome staining were applied for
14
the histological examination of nerve regeneration. An immunohisto-
15
chemical method was then performed to localize the expression of PGP
16
9.5 protein (protein gene product 9.5, 1:200, Abzoom, Wuppertal,
17
Germany). Modified Bielschowsky silver staining was also used and the
18
axon numbers were measured accordingly. In order to measure the
19
diameter of axons and thickness of myelin sheaths, the samples were also
20
observed by atomic force microscope (AFM) and calculated as previously
21
reported [13, 20]. For each section, at least 6 - 8 of random fields were
22
selected for the number counting and quantitative analysis.
23
2.7. In vitro cell viability and wound-healing migration
24
assay
25
2.7.1 Preparation of EPO/Chi coated glass substrates
AC C
EP
TE D
M AN U
9
26
In order to investigate the migratory response of MSCs to EPO/Chi,
27
the wound-healing migration assay was performed. For the in vitro 8
ACCEPTED MANUSCRIPT
wound-healing assay, glass slides with a size of 1 × 1 cm2 were used. The
2
glass slides substrates with different coating were prepared using a
3
slightly modified approach as reported previously [21]. Briefly, the glass
4
substrate was firstly ultrasonic cleaned with acetone for 15 minutes,
5
followed by 15 minutes of ethanol for two times. At last, the slides were
6
further ultrasonic treated with distilled water for 15 minutes. The cleaned
7
glass surface was then covered with a small amount of the chitosan
8
solution (10 mg/mL, w/v) with or without 1000 U/mL EPO and squeezed
9
a clean glass on the top to form a thin film. After solidification process,
10
the top glass was gently removed away and the thin film coated slide was
11
dried in a freeze-drying machine over night at a temperature of − 80 °C.
12 13
Before the cell seeding the coated slides were firstly incubated with culture medium to neutralize acidic film.
14
2.7.2 Cell migration (wound-healing) assay
M AN U
SC
RI PT
1
For wound-healing assay, the slides covered with chitosan (Chi) or
16
EPO loaded chitosan (EPO/Chi) were seeded with Ad-MSCs at the
17
density of 2×104/cm2, and the glass substrates without coating were used
18
as control. When the cells reach to 80–90% confluence, an artificial
19
wound was created by using a 10µL pipette tip by longitudinally straight
20
scratching the confluent cell monolayer. After washed two times with
21
PBS solution to removed the un-attached cells, the cells were then
22
incubated in DMEM containing 1% FBS for 12 hours. The cells at 0 h
23
and 12 h time points after the scratching were stained with fluorescein
24
diacetate (FDA) for observation and the images were obtained using an
25
inverted microscope (Olympus IX71, Japan). The total migrated cells
26
were then calculated by counting at least five random separate fields from
27
each sample (n=5) and then the cell numbers were normalized to control
28
groups.
29
2.7.3 Cell viability measurement
AC C
EP
TE D
15
30
The cell viability of Ad-MSC cultured on the substrates was assessed
31
using the 3-(4,5-dimethylthiazohl- 2-yl)- 2,5-diph enyltetrazolium
32
bromide (MTT; Sigma-Aldrich, USA) and the absorbance at a
33
wavelength of 490 nm was measured. 9
ACCEPTED MANUSCRIPT 1
2
2.8. The transwell assay The transwell assay was further performed to evaluate the effects of Ad-MSCs
paracrine
cytokines
on
the
Schwann
cell
line
4
(RT4-D6P2T) migration. The Ad-MSCs were firstly cultured on the glass
5
slides in 24-well plats for 3 days and 2,0000 of RT4-D6P2T were seeded
6
into the upper chamber with a pore size of 8 µm (Corning, USA) in
7
serum-free medium. After incubation for 24 hours, the remained cells in
8
the upper chamber were gently removed, and the migrated cells under the
9
membrane were fixed and stained with crystal violet solution. The images
10
of immigrated cells were captured with an inverted microscope and the
11
cell numbers of 5 random separate fields from each sample (n=3) were
12
counted.
13
2.9. Gene expression assay
M AN U
SC
RI PT
3
After cultured for 3 days, Quantitative real-time PCR (qRT-PCR) was
15
applied to assay the mRNA levels of related neur-protective genes and
16
the ELISA was performed to measure the concentration of FGF-2
17
(fibroblast growth factor 2), BDNF (Basic fibroblast growth factor),
18
SDF-1 (Stromal cell derived factor 1, CXCL12) that secreted by rat
19
Ad-MSC using ELISA kit (RayBiotech, Norcross, GA, USA). The
20
concentration of proteins was then normalized to the total amount of
21
protein. For the qRT-PCR, briefly, the total RNA was extracted and
22
first-strand cDNA was synthesized according to the protocol using
23
PrimeScript RT reagent Kit (Takara, China). qRT-PCR was performed
24
using a Bio-Rad CFX (Bio-Rad, USA) using SYBR Select Master Mix
25 26
Thermo scientific, Logan, USA . FGF-2, BDNF, NGF (Nerve growth factor) and NT-3 (Neurotrophin-3) were analyzed and
27
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as
28
housekeeping gene. Three independent experiments were performed for
29
each group. The primers used for real-time PCR experiments were listed
AC C
EP
TE D
14
10
ACCEPTED MANUSCRIPT 1
in Supplementary Table S1.
2
2.10. Statistical Analysis. The results are presented as the mean ± SD. One-way analysis of
4
variance (ANOVA) was applied for statistical comparisons among groups
5
more than two, and t-test was used for the statistical comparisons among
6
two groups. All statistical analysis was performed in the software SPSS
7
13.0 (SPSS, Chicago, USA). The values of p < 0.05 or 0.01 was
8
considered statistically significant.
9
3.Results
SC
M AN U
10
RI PT
3
3.1.Fabrication of nerve conduits
In this study, EPO-Chitosan nerve conduits were fabricated by
12
freeze-drying approach to explore their application as nerve guidance
13
substrates in vivo. The conduits were of about 3.8 mm in outer diameter
14
and about 1.2 mm in inner diameter (Figure 1A). The topographical
15
observation by SEM were demonstrated in Figure 1B. SEM of Conduit’ s
16
cross-section
17
interconnected porous structures in EPO-Chitosan conduit. The porosity
18
of the internal surface (Figure.1 B-c) was less than that of the
19
cross-section. In addition, only a few porous structures (Figure.1 B-d)
20
were observed on the external surface of the conduits, which might be
21
helpful for cytokine or implanted cell reservation in conduits.
showed
the
flaky
texture
and
EP
(Figure.1B-a&b)
AC C
22
TE D
11
In vitro release curve showed that the conduits could sustain the
23
release of loaded cytokine for upto 18 days (Figure.1C), suggesting the
24
feasibility to achieve a long-term action of healing response in vivo.
25
What's more, the swelling ration
26
(Figure.1E) measurement confirmed that the physical and mechanical
27
properties of the prepared conduits were not significantly affected by the
(Figure.1D) and elastic modulus
11
ACCEPTED MANUSCRIPT
adding of the EPO to the conduit.
2
3.2. In vivo transplantation and walking-foot-print
3
analysis
RI PT
1
The EPO-Chitosan nerve conduits with or without MSCs were
5
transplanted into the peripheral nerve injured animal model (Figure.
6
2A-a-e). Walking-foot-print analysis results showed that the value of SFI
7
in both Gel/MSC and Conduit group were significantly increased as
8
compared with control group 4 and 8 weeks after surgery (Figure. 2B &
9
C),
yet
by
the
combination
of
SC
4
Gel/MSC
and
conduit
in
Conduit+Gel/MSC, the SFI value was further significantly increased
11
(Figure. 2B & C). Similarly, both EPT and WRL results showed that the
12
rat (Figure. 2E & F) in Conduit+Gel/MSC treated group were observed
13
with significantly higher extensor force and WRL time as compared with
14
those in other groups. In addition, EPT and WRL demonstrated that the
15
motor performance of rats in both Gel/MSC and Conduit groups was
16
better than that in control group.
TE D
M AN U
10
The above beneficial effects of conduits-based therapy were further
18
confirmed by MNCV. As shown in Figure. 2D, MNCV values in both
19
Gel/MSC and Conduit groups were significantly higher (p<0.01) than
20
that in control group at 4 weeks and 8 weeks after surgery. Especially for
21
Conduit group at 8 weeks, the MNCV was even better than Gel/MSC
22
group (P<0.05). Similarly, the highest MNCV values (p<0.01) were
23
observed in Conduit+Gel/MSC group, at both 4 weeks and 8 weeks.
24
These results suggested that combination of Gel/MSC and Conduit could
25
significantly enhance the therapeutic potentials of single Gel/MSC or
26
Conduit for sciatic nerve injury.
27
3.3. Histological examination
28
AC C
EP
17
The survival of DiI-marked MSCs was analyzed in Gel/MSC and 12
ACCEPTED MANUSCRIPT 1
Conduit + Gel/MSC groups 4 weeks after surgery. As shown in Figure.
2
3A, much more MSCs were observed in Conduit + Gel/MSC group
3
compared with Gel/MSC group. Quantitative analysis (Figure. 3B)
4
showed that the number of survived MSC in Conduit + Gel/MSC group
5
was about 4~5 fold than that in Gel/MSC group. H&E staining was performed to observe the circular nerve bundles
7
morphology. As shown in Figure. 4A, lots of injured nerve bundles with
8
irregular or incomplete ring shape were observed in control group. In
9
comparison, the application of Conduit or Gel/MSC alone significantly
10
improved the morphology of injured nerve bundles and more importantly,
11
the improvement was more significant by combination of Conduit and
12
Gel/MSC, which resulted in the highest level of morphological
13
restoration and regularity of nerve bundles (Figure. 4A, p<0.05 or
14
p<0.01).
M AN U
SC
RI PT
6
PGP 9.5 immunohistochemical staining and its histomophormetry
16
analysis showed that the protein expression levels in Gel/MSC and
17
Conduit group were significantly higher than those in control group, and
18
the expression level was further increased in Conduit+Gel/MSC group
19
(Figure. 4 B & C, p<0.05 or p<0.01).
20
3.4.Axon number, diameter and myelin thickness
EP
TE D
15
In order to count the axon number, the Modified Bielschowsky silver
22
staining was performed 8 weeks after surgery. As shown in Figure.5 A &
23
C, the number of axon in the injured nerve bundles were increased
24
significantly (p<0.01) in both Gel/MSC and Conduit groups compared
25
with that in Control group, and it was further increased in
26
Conduit+Gel/MSC group (p<0.01). The axon diameter (Figure. 5 B & D)
27
and myelin thickness (Figure. 5 B & E) of the regenerated nerve bundles
28
were measured according to the TEM images (Figure. 5 B). Similarly,
29
axon diameter and myelin thickness in Gel/MSC and Conduit alone
30
groups were increased compared with control group, and the best results
31
were observed in the Conduit+Gel/MSC group, indicating the best
AC C
21
13
ACCEPTED MANUSCRIPT 1
performance of combining Gel/MSC and Conduit on repair of sciatic
2
nerve injury among the four groups.
3
3.5. Re-innervation of target gastrocnemius muscles According to the Masson trichrome staining in Figure. 6, much more
5
muscular cells with irregular shape (red color) and accumulation of the
6
collagen (blue color) were observed in control group than those in
7
Gel/MSC, Conduit and Conduit+Gel/MSC groups. In addition, the areas
8
of muscular cells in both Gel/MSC and Conduit were significantly larger
9
than that in control group (p<0.01), while the largest mean area of
SC
RI PT
4
muscular cells was observed in Conduit+Gel/MSC group.
11
3.6. The in vitro beneficial effects of EPO/Chi on Ad-MSC
12
and Schwann cells
M AN U
10
As shown in Figure.7C, the coating of EPO/Chi on the glass
14
significantly improved the cellular viability of MSCs compared with Chi
15
or Control group. Wound healing assay (Figure.7 A & B) indicated that
16
Chiostan-coated glass could improve the migration of Ad-MSCs, which
17
was further enhanced by adding of EPO compared with control (p<0.05 or
18
p<0.01). In order to investigate the neuro-protective effects of
19
EPO/Chi-regulated Ad-MSCs on nerve regeneration, the neurotrophins
20
gene analysis and transwell migration of Schwann cells assay were
21
performed. As shown in Figure.7 D-H, the Ad-MSC cultured on
22
EPO/Chi-coated glass showed higher levels of FGF-2 and BDNF gene
23
expression and proteins secretion, as well as SDF-1 concentration. The
24
exposure of Schwann cells to Ad-MSCs on EPO/Chi coated glass caused
25
more cells to migrate through the insert membrane (Figure. 7 I & J).
26
Interestingly, even without Ad-MSC, the EPO/Chi coated glass could also
27
result in certain numbers of Schwann cells, which was consistently with
28
previously reports that the EPO alone could also promote the cellular
AC C
EP
TE D
13
14
ACCEPTED MANUSCRIPT 1
migration [11, 20]. What’s more, unlike the randomly distributed cells in
2
other groups, the migrated Schwann cells in EPO/Chi group were more
3
likely to cluster together (Figure. 7J), which might be resulted from the
4
combination of MSCs and EPO/Chi. These results indicated that EPO/Chi-coated glass could promote the
6
migratory response of Ad-MSCs, and activate its neurotrophins protein
7
secretion as well as gene expression. The EPO/Chi could also promote the
8
migration of Schwann cells alone or through the mediation of Ad-MSCs,
9
which might eventually contribute to the good performance of nerve repair
11
4. Discussion
SC
with EPO-loaded conduit + Ad-MSC.
M AN U
10
RI PT
5
Transplantation of a nerve implant between two nerve stumps has
13
been proposed for aiding injured peripheral nerve regeneration and
14
functional restoration guidance. In the past, biomaterial based strategies
15
like nerve conduits, or cell-based therapy were applied to bridge the
16
nerve gap for nerve repair as well as protect the injured nerve from
17
surrounding tissues during the healing process[3]. Some have achieved
18
certain success, but many results were still in controversy[2, 22, 23].
TE D
12
In the present study, a combination strategy of EPO-loading nerve
20
conduit and MSCs was developed to repair sciatic nerve injury. The
21
functional gait movements, global and detail nerve tissue morphologies,
22
neural protein expression and reinnervation of target gastrocnemius
23
muscles were used to evaluate the performance of these strategies. The
24
results indicated that, although the single application of nerve conduits (in
25
Conduit group) or mesenchymal stem cells plus hydrogels (in Gel/MSC
26
group) could improve axon regeneration compared with control group,
27
combining MSC with EPO-loading chitosan nerve conduits resulted in
28
the most favorable outcomes for guidance of nerve regeneration. In vitro
29
cellular experimental results indicated that EPO/Chi could increase the
30
migratory response of Ad-MSCs, and increase its neurotrophins protein
31
secretion and gene expression. The MSCs + EPO/Chi could also promote
AC C
EP
19
15
ACCEPTED MANUSCRIPT 1
the migration as well as the clustering of Schwann cells. As is well known, the directly administration of exogenous cytokines
3
without protection would result in quickly degradation due to
4
deactivation by enzymes and other physical-chemical effectors in vivo
5
and in vitro. In recent years, we focus on the construction of localized and
6
sustained delivery system for controlled release of exogenous cytokines
7
to enhance its repair effects in vivo. Previously, we found that the
8
chitosan-based and NGF-loaded nerve conduits could be used for sciatic
9 10
nerve repair in rats which highlights good potential of Chitosan conduit for peripheral nerve treatments[8]. However, it was also demonstrated
11
that NGF might promote neural apoptosis after peripheral nerve injury
12
under certain circumstance[24], which indicated the bifacial effects of
13
NGF during nerve repair. The hematopoietic cytokine erythropoietin
14
(EPO) was also proved to be beneficial for the functional recovery of
15
injured peripheral nerve system via EPO receptor (EPOR) expressed by
16
Schwann cells [12, 13, 20]. Recently, we reported that the encapsulating
17
EPO in PLGA microspheres for localized and sustained delivery could
18
significantly improve the neural function of injured peripheral nerve,
19
which indicated its great potential for peripheral nerve treatments [16, 25].
20
What’s more, the results of some published papers also indicated that the
21
EPO is better than NGF to enhance nerve regeneration and promote
22
functional recovery after peripheral nerve injury in the rat[26]. In addition,
23
EPO was also confirmed to enhance the migration of MSCs, which might
24
be beneficial for the recruitment of endogenous MSCs and improve its
25
repair performance[11]. Therefore, the EPO was used as the
26
neuroprotective cytokine for preparation of “bio-active” chitosan nerve
27
conduit in the study.
28
AC C
EP
TE D
M AN U
SC
RI PT
2
To our knowledge, the present study was the first report to
29
encapsulate
EPO protein
in
the
30
transplantation of EPO-loading nerve conduit and MSCs for peripheral
31
nerve repair was also firstly evaluated. As expected, the survival of
32
transplanted MSCs was significantly enhanced in EPO-loaded conduit +
33
gel/MSCs group compared with that in Gel/MSC group. Further, the best 16
Chitosan
scaffold. Combined
ACCEPTED MANUSCRIPT
results of SFI and MNCV were detected in EPO-loaded conduit +
2
gel/MSCs group compared with all the other groups. The histological
3
examination and histomorphometry results indicated the highest level of
4
morphological restoration and regularity of nerve bundles. Similarly,
5
PGP 9.5 protein expression, axon diameter and myelin thicknessas well
6
as the number of axon were also significantly improved in
7
Conduit+Gel/MSC groups compared with the other groups.
RI PT
1
In order to further investigate the beneficial effects of EPO-loading
9
Chitosan scaffolds on Ad-MSC and Schwann cells, as well as the
10
potential underlying mechanism, in vitro cell experiments were
11
performed. The surface mobilization of Chitosan and EPO on glass was
12
used for in vitro evaluation of MSC performance. CCK-8 assay showed
13
that the EPO loaded Chitosan could enhance the growth of seeding MSCs.
14
The scratch wound healing assay was applied as well in vitro.
15
Consistently, the results proved that EPO-loaded Chitosan could promote
16
the migration of MSCs during the wound healing process.
M AN U
SC
8
In recent years, transplantation of MSCs for neuro-diseases therapy
18
has drawn more and more attention. Several beneficial effects were
19
contributed to the therapeutic results of MSC transplantation, such as its
20
immunomodulatory, chemotactic properties, and the secretion of some
21
beneficial growth factors and cytokines [9, 18]. In the present study, the
22
qRT-PCR of gene expression and ELISA assay indicated that the EPO
23
loaded Chitosan could also promote the secretion of neuro-protective
24
cytokines FGF2, BDNF and SDF-1. What’s more, the transwell results
25
showed that the EPO/Chi coated glass could increase the migratory
26
response of Schwann cells directly or through the mediation of Ad-MSCs.
27
Taken together, we speculated that EPO/Chi conduit alone might also
28
enhance the response of both Schwann cells and endogenous MSCs
29
during the wound healing process, while single injection of Gel/MSC
30
only resulted in small distributed number of the exogenousMSCs (Figure.
31
3), and these might lead to better repair results (such as MNCV in
32
Figure.2) of Conduit group than control group, or even better than
33
Gel/MSC.
AC C
EP
TE D
17
17
ACCEPTED MANUSCRIPT
What’s more, the MSCs cultured on EPO/Chi coated substrates could
2
also induce the clustering of migrated Schwann cells, which played vital
3
role in the Schwann cells’ mediated peripheral nerve regeneration [27].
4
These in vitro results indicated that the EPO-loaded Chitosan conduits
5
could not only protect the peripheral nerve via EPO proteins delivery, but
6
also be beneficial for the repair of injured peripheral nerve via MSC
7
regulation.
RI PT
1
Despite the encouraging preliminary results from our study, there
9
were still some limitations in the present study. Because the negative
10
outcomes were always associated with gaps greater than 5 mm for the
11
marketed bioartificial conduits, only 5 mm of gap distance was applied
12
[28]. Actually, treatment of larger nerve defects remains to be the big
13
challenge of the relative field. Ongoing studies are also directed toward to
14
apply the nerve conduits for long-distance regeneration of peripheral
15
nerves, which might be of great importance for peripheral nerve
16
reconstruction and recovery.
17
5. Conclusion:
TE D
M AN U
SC
8
In the present study, a new strategy of EPO-loaded Chitosan scaffold
19 20
and MSC transplantation for nerve regeneration was reported and the its repair performance for the sciatic nerve injury was evaluated in vivo and
21
in vitro. In vivo results showed that the combination of EPO-loaded
22
Chitosan scaffolds with MSCs eventually resulted in accelerated nerve
23
healing process. In vitro results indicated that the EPO-loaded Chitosan
24
could promote the MSC and Schwann cell repair performance, which
25
might contribute to the therapeutic outcome of the new strategy.
26
Therefore, the combined transplantation of the EPO-loaded Chitosan and
27
MSCs for nerve replacement represents a new generation of medical
28
device for peripheral nerve reconstruction.
29
Appendix A. Supplementary data
30
More information could be found in the “Supplementary data.DOC”.
AC C
EP
18
18
ACCEPTED MANUSCRIPT 1
References
2
[1] Intiso D, Grimaldi G, Russo M, Maruzzi G, Basciani M, Fiore P, et al. Functional outcome and
3
health status of injured patients with peripheral nerve lesions. Injury 2010;41:540-543.
4
[2] Gu X, Ding F, Williams DF. Neural tissue engineering options for peripheral nerve regeneration. Biomaterials 2014;35:6143-6156.
RI PT
5 6
[3] Daly W, Yao L, Zeugolis D, Windebank A, Pandit A. A biomaterials approach to peripheral
7
nerve regeneration: bridging the peripheral nerve gap and enhancing functional recovery.
8
Journal of the Royal Society, Interface / the Royal Society 2012;9:202-221.
[4] Quigley AF, Bulluss KJ, Kyratzis IL, Gilmore K, Mysore T, Schirmer KS, et al. Engineering a
10
multimodal nerve conduit for repair of injured peripheral nerve. Journal of neural engineering
11
2013;10:016008.
M AN U
SC
9
12
[5] Bozkurt A, Boecker A, Tank J, Altinova H, Deumens R, Dabhi C, et al. Efficient bridging of 20
13
mm rat sciatic nerve lesions with a longitudinally micro-structured collagen scaffold.
14
Biomaterials 2016;75:112-122.
[6] Meyer C, Stenberg L, Gonzalez-Perez F, Wrobel S, Ronchi G, Udina E, et al. Chitosan-film
16
enhanced chitosan nerve guides for long-distance regeneration of peripheral nerves.
17
Biomaterials 2016;76:33-51.
TE D
15
[7] Cirillo V, Clements BA, Guarino V, Bushman J, Kohn J, Ambrosio L. A comparison of the
19
performance of mono- and bi-component electrospun conduits in a rat sciatic model.
20
Biomaterials 2014;35:8970-8982.
21
[8] Wang H, Zhao Q, Zhao W, Liu Q, Gu X, Yang Y. Repairing rat sciatic nerve injury by a
22
nerve-growth-factor-loaded, chitosan-based nerve conduit. Biotechnology and applied
23
AC C
EP
18
biochemistry 2012;59:388-394.
24
[9] Keilhoff G, Goihl A, Stang F, Wolf G, Fansa H. Peripheral nerve tissue engineering: autologous
25
Schwann cells vs. transdifferentiated mesenchymal stem cells. Tissue engineering
26
2006;12:1451-1465.
27
[10] Hsueh YY, Chang YJ, Huang TC, Fan SC, Wang DH, Chen JJ, et al. Functional recoveries of
28
sciatic nerve regeneration by combining chitosan-coated conduit and neurosphere cells
29
induced from adipose-derived stem cells. Biomaterials 2014;35:2234-2244.
30 31
[11] Nair AM, Tsai YT, Shah KM, Shen J, Weng H, Zhou J, et al. The effect of erythropoietin on autologous stem cell-mediated bone regeneration. Biomaterials 2013;34:7364-7371.
19
ACCEPTED MANUSCRIPT 1
[12] Ning R, Xiong Y, Mahmood A, Zhang Y, Meng Y, Qu C, et al. Erythropoietin promotes
2
neurovascular remodeling and long-term functional recovery in rats following traumatic brain
3
injury. Brain Res 2011;1384:140-150. [13] Yin ZS, Zhang H, Bo W, Gao W. Erythropoietin promotes functional recovery and enhances
5
nerve regeneration after peripheral nerve injury in rats. AJNR American journal of
6
neuroradiology 2010;31:509-515.
RI PT
4
7
[14] Elfar JC, Jacobson JA, Puzas JE, Rosier RN, Zuscik MJ. Erythropoietin accelerates functional
8
recovery after peripheral nerve injury. The Journal of bone and joint surgery American volume
9
2008;90:1644-1653.
11
[15] Li X, Gonias SL, Campana WM. Schwann cells express erythropoietin receptor and represent
SC
10
a major target for Epo in peripheral nerve injury. Glia 2005;51:254-265.
[16] Zhang W, Gao Y, Zhou Y, Liu J, Zhang L, Long A, et al. Localized and sustained delivery of
13
erythropoietin from PLGA microspheres promotes functional recovery and nerve regeneration
14
in peripheral nerve injury. BioMed research international 2015;2015:478103.
15
[17] Wyse RD, Dunbar GL, Rossignol J. Use of genetically modified mesenchymal stem cells to
16
treat
17
2014;15:1719-1745.
19
neurodegenerative
diseases.
International
journal
of
molecular
sciences
[18] Hofer HR, Tuan RS. Secreted trophic factors of mesenchymal stem cells support
TE D
18
M AN U
12
neurovascular and musculoskeletal therapies. Stem cell research & therapy 2016;7:131. [19] Lee EJ, Xu L, Kim GH, Kang SK, Lee SW, Park SH, et al. Regeneration of peripheral nerves by
21
transplanted sphere of human mesenchymal stem cells derived from embryonic stem cells.
22
Biomaterials 2012;33:7039-7046.
EP
20
[20] Inoue G, Gaultier A, Li X, Mantuano E, Richardson G, Takahashi K, et al. Erythropoietin
24
promotes Schwann cell migration and assembly of the provisional extracellular matrix by
25 26 27 28 29 30 31
AC C
23
recruiting beta1 integrin to the cell surface. Glia 2010;58:399-409.
[21] Kim CL, Kim DE. Self-healing Characteristics of Collagen Coatings with Respect to Surface Abrasion. Scientific reports 2016;6:20563.
[22] Geuna S, Gnavi S, Perroteau I, Tos P, Battiston B. Tissue engineering and peripheral nerve reconstruction: an overview. International review of neurobiology 2013;108:35-57. [23] Chiono V, Tonda-Turo C. Trends in the design of nerve guidance channels in peripheral nerve tissue engineering. Progress in neurobiology 2015;131:87-104.
32
[24] Petratos S, Butzkueven H, Shipham K, Cooper H, Bucci T, Reid K, et al. Schwann cell apoptosis
33
in the postnatal axotomized sciatic nerve is mediated via NGF through the low-affinity 20
ACCEPTED MANUSCRIPT 1
neurotrophin
2
2003;62:398-411.
receptor.
Journal
of
Neuropathology
&
Experimental
Neurology
3
[25] Zhang W, Sun B, Yu Z, An J, Liu Q, Ren T. High dose erythropoietin promotes functional
4
recovery of rats following facial nerve crush. Journal of clinical neuroscience : official journal
5
of the Neurosurgical Society of Australasia 2009;16:554-556.
7
[26] Yin Z-S, Zhang H, Gao W. Erythropoietin promotes functional recovery and enhances nerve
RI PT
6
regeneration after peripheral nerve injury in rats. Am J Neuroradiol 2010;31:509-515.
8
[27] Parrinello S, Napoli I, Ribeiro S, Wingfield Digby P, Fedorova M, Parkinson DB, et al. EphB
9
signaling directs peripheral nerve regeneration through Sox2-dependent Schwann cell sorting. Cell 2010;143:145-155.
SC
10
[28] Weber RA, Breidenbach WC, Brown RE, Jabaley ME, Mass DP. A randomized prospective
12
study of polyglycolic acid conduits for digital nerve reconstruction in humans. Plastic and
13
reconstructive surgery 2000;106:1036-1045.
M AN U
11
14 15
17
TE D
16
Figures captions
19
Figure.1 Preparation and characterization of the EPO-loaded Chitosan scaffold. (A)
20
The schematic summary of the EPO-loaded Chitosan scaffold constructing process;
21
and (B) SEM observation of the scaffold; (C) in vitro release of EPO; (D) Swelling
22
ration and (E) elastic modulus of the Chi or EPO/Chi conduits.
AC C
23
EP
18
24
Figure.2 In vivo transplantation and functional evaluation. (A) The schematic
25
summaryof the implantation surgery process, (B) Representative images of
26
walking-foot-print analysis and its quantitative analysis results SFI (C);(D) MNCV of
27
the electrophysiological examination; and the motor performance evaluated by EPT
28
(E) and WRL (F). “*” for P<0.05, and “**” for P<0.01.
29
21
ACCEPTED MANUSCRIPT 1
Figure.3 In vivo observation of the DiI labeled MSCs (A) and quantitative analysis
2
results (B) after 4 weeks surgery. “*” for P<0.05, and “**” for P<0.01.
3
Figure.4 In vivo histological and immunohistological examination. (A) H&E staining
5
results of the injured nerve 8 weeks after surgery; immunohistochemical staining of
6
the PGP9.5 protein (B) and the quantitative analysis results (C). “*” for P<0.05, and
7
“**” for P<0.01.
RI PT
4
8
Figure.5 Axon number, diameter and myelin thickness of the regenerated nerve. (A)
10
Modified Bielschowsky silver staining for axon numbers calculation (C); the
11
represent images of atomic force microscope (AFM) results (B), and quantitative
12
analysis of the axons diameter (D) andmyelin sheaths thickness (E). “*” for P<0.05,
13
and “**” for P<0.01. Scale bar = 20µm.
M AN U
SC
9
14 15
Figure.6 Masson trichrome staining of the targeting gastrocnemius muscles and the
16
quantitative analysis. “*” for P<0.05, and “**” for P<0.01.
17
Figure.7 In vitro cell viability and wound-healing migration assay. (A)
19
Wound-healing migration assay of the MSCs on EPO/Chi coated glass and (B) the
20
quantitative analysis of migrated cells; (C) MTT results for the cell viability assay; (D)
21
qRT-PCR results for measurement of the neur-protective genes expression in MSCs;
22
(E) ELISA assay results for measurement of the SDF-1(E), FGF-2 (F), BDNF (G)
23
proteins concentration from MSCs; the quantitative analysis (H) of the migrated
24
Schwann cell(RT4-D6P2T) (I); “*” for P<0.05, and “**” for P<0.01.
26 27
EP
AC C
25
TE D
18
28 29 30 31 32 33 22
ACCEPTED MANUSCRIPT 1 2 3 4 5
RI PT
6 7 8 9
SC
10 11
M AN U
12 13 14 15 16
18
A
19
EP
20
TE D
17
21
AC C
22 23 24 25 26
B
C
23
ACCEPTED MANUSCRIPT EPO
100 80 60 40 20 0 0
1
D
E
15
18
Figure. 1
EP
A
AC C
9
TE D
8
15 16 17 18
24
B
0.5
/C
hi
0.0
EP O
7
14
12
1.0
hi
6
13
9
SC
Elastic Modulus (Mpa)
5
12
1.5
M AN U
4
11
6
Time (day)
3
10
3
C
2
RI PT
Cumulative Release (%)
120
21
24
ACCEPTED MANUSCRIPT 1 2
Control
Gel/MSC
Conduit
C
Conduit+Gel/MSC
D
**
3
**
**
**
**
**
**
** *
5 6
**
E
*
**
15
WRL Time (Sec)
100
9 EPT (%)
80
10
60 40 20
11
0
17 18 19
EP
16
A
AC C
14
TE D
Figure. 2
13
10
*
** **
5
0
Time = 8 weeks
12
F
M AN U
8
SC
7
15
*
RI PT
4
20 21 22
25
B
Time = 8 weeks
ACCEPTED MANUSCRIPT 1 2 3
Figure. 3
RI PT
4 5 6
SC
7
M AN U
8 9 10 11
13 14 15
A
AC C
16
EP
TE D
12
17 18 19 20 21
B
22
26
ACCEPTED MANUSCRIPT 1 2
**
C
**
3
RI PT
4 5 6
SC
7
M AN U
8
Figure. 4
9 10 11
15 16 17
EP
14
AC C
13
TE D
12
27
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
1
Figure. 5
2
6 7 8 9
EP
5
AC C
4
TE D
3
10 11 12 13
28
ACCEPTED MANUSCRIPT 1 2 3
RI PT
4 5 6
**
SC
**
7
M AN U
8 9 10 11
TE D
12
Figure. 6
13
EP
14 15
AC C
16 17 18 19
A
B
20 21 22 29
ACCEPTED MANUSCRIPT C
1 2 3
D
E
RI PT
4 5 6
SC
7
9
F
G
10 11
15 16 17 18
I
EP
14
AC C
13
H
TE D
12
M AN U
8
Figure. 7
30
S0142-9612(17)30423-4
DOI:
10.1016/j.biomaterials.2017.06.024
Reference:
JBMT 18144
To appear in:
Biomaterials
Received Date: 5 January 2017 Revised Date:
9 June 2017
Accepted Date: 19 June 2017
Please cite this article as: Zhang W, Zhang L, Liu J, Zhang L, Zhang J, Tang P, Repairing sciatic nerve injury with an EPO-loaded nerve conduit and sandwiched-in strategy of transplanting mesenchymal stem cells, Biomaterials (2017), doi: 10.1016/j.biomaterials.2017.06.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Repairing sciatic nerve injury with an EPO-loaded nerve conduit
2
and sandwiched-in strategy of transplanting mesenchymal stem
3
cells
RI PT
1
Wei Zhang 1, Lihai Zhang 1, Jianheng Liu 1, Licheng Zhang1, jian
5
Zhang 2, Peifu Tang 1*
6
1 Department of Orthopedics, General Hospital of Chinese PLA,
7
Beijing, 100853, China
8
2 Department of Orthopedics, General Hospital of Liaoyuan Mining
9
Group, Jilin Province, 136201, China
M AN U
SC
4
10
*Correspondence should be addressed to Peifu Tang;
11
pftang [email protected]
12 13
17 18 19 20 21 22 23 24
EP
16
AC C
15
TE D
14
25 26 27
1
ACCEPTED MANUSCRIPT
Abstract: Transplantation of a biosynthetic nerve conduit carrying
2
neuro-protective cytokines is promising for treating peripheral nerve
3
injury. Here we developed a novel strategy for repairing sciatic nerve
4
injury by EPO-loaded Chitosan nerve conduit (EPO/Chi) and
5
sandwiched-in transplantation of mesenchymal stem cells (MSCs). Then,
6
the beneficial effect of EPO/Chi+MSCs on nerve regeneration was also
7
further investigated by in vitro cellular experiments. In vivo experiment
8
showed that combination of EPO-loaded Chitosan nerve conduit with
9
MSCs could significantly accelerate nerve healing and improve
10
morphological repair. Furthermore, the in vitro cellular experiments
11
results demonstrated that the loaded EPO in nerve conduit could
12
significantly reinforce the repair performance of both MSCs and
13
Schwann cells, which may also contribute to the therapeutic outcome of
14
the EPO/Chi + MSCs strategy. Collectively, the EPO-loaded nerve
15
conduit and sandwiched-in transplantation of MSCs we reported in the
16
study may represent a new potential strategy for peripheral nerve
17
reconstruction.
18
Key words: Sciatic nerve injury; Mesenchymal Stem Cells (MSC);
19
Nerve regeneration or repair; Erythropoietin (EPO); Chitosan
AC C
EP
TE D
M AN U
SC
RI PT
1
2
ACCEPTED MANUSCRIPT 1
1.Introduction Despite the certain degree of self-regenerative capability, repair of
3
long-distance peripheral nerve transection injury was still a major
4
challenge in clinic and the therapeutic outcome was usually weak [1, 2].
5
Thus, tissue engineering-based efforts, such as biomaterials supplemented
6
with or without stem cells or neurotrophic factors, were proposed as
7
alternative promising ways to repair the injured peripheral nerve[3, 4].
8
Among
these
approaches,
the
RI PT
2
artificial
nerve
guidance
conduit-based surgery was considered as one of the most promising
10
strategies for treating peripheral nerve injury. These biodegradable
11
guidance conduits, with single/multi-channel hollow tubes [5] or more
12
complicated structure for bio-mimicking themacro- and micro-structures
13
of nerves [6, 7] demonstrated promising therapeutic benefits. A number
14
of materials, including the synthetic Poly-L-Lactide Acid (PLLA),
15
Polycaprolactone (PCL), and some natural biomaterials such as collagen
16
or fibrin, had been used for the guidance conduits development [2, 8, 9].
M AN U
SC
9
Recently, the natural derivative chitosan based porous and
18
biodegradable scaffolds were proved to be a potentially effective
19
candidate conduit material for nerve regeneration, especially for
20
promoting the long-distance regeneration of peripheral nerves [6, 10].
21
However, the biological response of single synthetic chitosan conduit
22
using is still underactive, especially for lacking of seeding cells for nerve
23
regeneration. In addition to single nerve conduit, supplemention of
24
growth-promoting and neuronal regulation cytokines, such as neurotropic
25
factors glial cell line derived neurotrophic factor (GDNF) and nerve
26
growth factor (NGF), were proved to further enhance the nerve
27
regeneration[2]. Recently, the normal known hematopoietic cytokine
28
erythropoietin (EPO) was demonstrated with neuro-protective on the
29
peripheral nerve system by targeting Schwann cells after nerve injury
30
[11-15]. Previously, we have demonstrated that sustained release of EPO
31
by poly (lactide-co-glycolide) (PLGA) microspheres could significantly
AC C
EP
TE D
17
3
ACCEPTED MANUSCRIPT 1
promote the recovery of injuried peripheral nerve in rats [16], and thus
2
we hypothesized in the study that the loading exogenous EPO in the
3
synthetic nerve conduits might increase their performance for peripheral
4
nerve repair. The application of stem cells therapy would provide not only large
6
numbers of exogenous seeding cells, but also adequate amounts of
7
paracrine secreted bioactive cytokines[17, 18]. However, traditional stem
8
cell transplantation was usually accompanied with excessive loss and
9
death of transplanted cells, which hampered the therapeutic efficacy for peripheral nerve injury.
SC
10
RI PT
5
The present study aimed to construct a simpler intraluminal modified
12
nerve guidance conduits and develop a sandwiched-in strategy for stem
13
cells transplantation. Based on the tissue engineering approach, the EPO
14
was loaded in the Chitosan scaffold for sustained release to further
15
enhance nerve regeneration and the functional repair of the sciatic nerve
16
injury. The immunohistochemical and histological monitoring methods
17
were performed to evaluate the performance. Finally, the in vitro cellular
18
experiments were performed to investigate beneficial effects of
19
EPO-loading Chitosan scaffolds on the repair performance of MSCs and
20
Schwann cells, as well as the potential underlying mechanism.
21
2. Materials and methods
22
2.1. Cell culture
TE D
EP
AC C
23
M AN U
11
Rat adipose-derived Mesenchymal Stem Cells (Ad-MSC or MSC)
24
were bought from Cyagen Co. (China) and cultured at 37 °C in a
25
humidified atmosphere containing 5% carbon dioxide. The culture
26
medium of Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen
27
Co., USA) with 10% (v/v) fetal bovine serum (FBS, Thermo scientific,
28
Logan, USA), 100 units/ml Penicillin and 100 µg/ml streptomycin was
29
used. 4
ACCEPTED MANUSCRIPT 1
2.2. Preparation of the EPO-Chitosan nerve guidance
2
conduit EPO-Chitosan nerve conduits were prepared as previously described
4
[8]. Briefly, 10 mg/mL (w/v) of chitosan in hydrochloric acid aqueous
5
solution (pH 5.8) with 1000 U/mL EPO were prepared and injected into a
6
casting mold with an outer sleeve (diameter = 3.8 mm) and an inner
7
sleeve (diameter = 1.2 mm) (Figure. 1 A). The solution was further
8
solidified under minus 20 oC overnight and dried by using a vacuum
9
freeze-drying machine with a FDP80 Oil Vacuum Pumps (Thermo
10
Scientific™, USA). The prepared nerve conduits were then washed with
11
phosphate-buffered saline (pH 7.4) and sterilized with 25 kGy Cobalt 60
12
prior to use. The Scanning Electron Microscope (SEM) was performed to
13
characterize the scaffold by using Mira3 LMH Schottky FE-SEM (Tescan,
14
Czech).
15
2.3.
16
guidance conduit
M AN U
SC
RI PT
3
of
TE D
Characterization
the
EPO-Chitosan
nerve
To characterize the in vitro release of the EPO from the conduit, 10
18
mg of EPO-Chitosan nerve conduits were emerged into 1mL 10 mM PBS and incubated at 37 °C with shaking at 110 rpm. The released supernatant with EPO at day 0.5, 1, 2, 4, 7, 10, 14, 18 and 21 were obtained via centrifugation at 3000 rpm for 2 min and replaced the same volume of PBS after collecting the samples. The concentration of EPO protein in the supernatant was measured using an EPO ELISA kit. The cumulative release (%) of EPO was calculated and 5 samples were included. To determine the effective swelling ratio, the dry weights (Wdry) of lyophilized Chitosan (Chi) and EPO loaded Chitosan (EPO/Chi) conduits were measured. After that, the conduits were immersed in 10 mM PBS
20 21 22 23 24 25 26 27 28
AC C
19
EP
17
5
ACCEPTED MANUSCRIPT
and incubated for 24 hours at 37 °C to reach equilibrium swelling state.
2
Five samples of each group were obtained and the weight of conduit after
3
swelling (Wwet) was measured. The swelling ratio (SR) was calculated
4
by the following equations: SR=( Wwet- Wdry)/ Wdry*100%. The
5
elastic modulus of conduits was measured using a sans CMT700 type
6
universal testing machine. The conduit were incubated in PBS for 24
7
hours at 37 °C and loaded on the machine. The elastic modulus of
8
conduits was at a stretching speed of 10 mm/min.
9
2.4.
injury
treatment.
animal
model
SC
nerve
and
M AN U
10
Peripheral
RI PT
1
The effect of EPO-loaded nerve conduit with MSCs on nerve
12
regeneration was examined in an experimental sciatic nerve defect injury
13
animal model employing specific pathogen-free (SPF) Sprague-Dawley
14
(SD) rats. The rats (about 220 g) were purchased from the Experimental
15
Animal Center, Academy of Military Medical Science (Beijing, China).
16
All animal experiments were approved by the local institution of Animal
17
Experimental Ethics Committee of Experimental Animal Center,
18
Academy of Military Medical Science (Beijing, China).
TE D
11
Animals were prepared and underwent surgery using the method as
20
described [7]. Briefly, zoletil (500 µg per animal) and rompun 2% (10 µL
21
per animal) of intramuscular injection were used to anesthetize the animal.
22
In the central area underneath the gluteus maximus, the sciatic nerve in
23
the right hind limb was exposed and transected by a scissor cut at a
24
constant point and a 5mm nerve defect was created. After that, four
25
different treatment approach were applied for the injury healing: (1) For
26
Control group, simple saline treatment was applied; (2) for Gel/MSC
27
group, 20uL Matrigel mixed with 2×105 MSC was injected to the defect
28 29
area (3) For Conduit group, only EPO- Chitosan conduit was applied for surgery transplantation; (4) for Conduit + Gel/MSC groups, 20uL
30
Matrigel mixed with 2×105 MSC was injected into the conduit after
AC C
EP
19
6
ACCEPTED MANUSCRIPT 1
Conduits implantation and then applied for the defect replacement. For
2
tracking of the exogenous MSC, the cells were labeled by DiI
3
(Invitrogen). There were 12 rats in every experimental group at each time
4
points.
5
2.5.
6
examination
analysis
and
electrophysiological
RI PT
Functional
Four and eight weeks after surgery, the walking track test for
8
foot-print analysis was applied for the all animals for functional recovery
9
of never injury as described [13, 19]. Briefly, a confined walking track
10
system (7 × 50 cm2) of an open and a darkened end corridor was used
11
respectively. The rats with black ink dyed paws were allowed to walk on
12
it to obtain the tractable paws prints. The footprint parameter sciatic
13
functional index (SFI) was calculated accordingly [13, 19] and an SFI
14
value (-100% - 0) stands for the incomplete nerve function recovery.
15
To further evaluate the motor performance of the rat, the extensor
16
postural thrust (EPT) and withdrawal reflex latency (WRL) were also
17
measure at the end of week 8. For EPT analysis, the entire body of rat
18
was wrapped in a surgical towel and held upright except for its one hind
19
limb. The foot extension of the rat was induced via lowering the rat
20
toward the surface a platform. The extension force to the platform of a
21
digital balance was recorded in grams. The EPT of normal hind limb
22
(EPT_nor) and experimental hind limb (EPT_exp) from every rat were
23
measured. The reduction in the extensor muscle force was set as the
24
indicative of a deficit in the motor function. The deficit percentage was
25
calculated with the formula EPT (%) = (EPT_nor – EPT_exp) / EPT_nor
26
*100%. For WRL measurement, the rat was fixed and the hind paw was
27
placed on a 56 oC platform. The WRL time was measured as the required
28
time for the rat to withdraw the paw. If the rat remain still for more than
29
12 seconds, removed the rat from the hot platform and the maximal WRL
30
of 12 seconds was assigned to the animal. Detailed descriptions of the
AC C
EP
TE D
M AN U
SC
7
7
ACCEPTED MANUSCRIPT 1
methods could be found in many relative published papers. The electrophysiological examination was further conducted to obtain
3
the Motor nerve conduction velocities (MNCV) of right sciatic nerve
4
using MEB-7102 instrument (Nihon Kohden,Osaka, Japan). The distance
5
between the stimulating and recording electrodes was then divided by the
6
latency of the distal and proximal to calculate the MNCV.
7
2.6.
8
examination.
histological
vivo
and
immunohistological
SC
In
RI PT
2
The sciatic nerves or targeting gastrocnemius muscles were harvest
10
from each group at relatively time points after surgery and fixed in 4%
11
paraformaldehyde for at least 24 hours. The tissue was embedded in
12
paraffin and 3-5 µm of paraffin sections were then prepared. After that,
13
hematoxylineosin (H&E) and Masson trichrome staining were applied for
14
the histological examination of nerve regeneration. An immunohisto-
15
chemical method was then performed to localize the expression of PGP
16
9.5 protein (protein gene product 9.5, 1:200, Abzoom, Wuppertal,
17
Germany). Modified Bielschowsky silver staining was also used and the
18
axon numbers were measured accordingly. In order to measure the
19
diameter of axons and thickness of myelin sheaths, the samples were also
20
observed by atomic force microscope (AFM) and calculated as previously
21
reported [13, 20]. For each section, at least 6 - 8 of random fields were
22
selected for the number counting and quantitative analysis.
23
2.7. In vitro cell viability and wound-healing migration
24
assay
25
2.7.1 Preparation of EPO/Chi coated glass substrates
AC C
EP
TE D
M AN U
9
26
In order to investigate the migratory response of MSCs to EPO/Chi,
27
the wound-healing migration assay was performed. For the in vitro 8
ACCEPTED MANUSCRIPT
wound-healing assay, glass slides with a size of 1 × 1 cm2 were used. The
2
glass slides substrates with different coating were prepared using a
3
slightly modified approach as reported previously [21]. Briefly, the glass
4
substrate was firstly ultrasonic cleaned with acetone for 15 minutes,
5
followed by 15 minutes of ethanol for two times. At last, the slides were
6
further ultrasonic treated with distilled water for 15 minutes. The cleaned
7
glass surface was then covered with a small amount of the chitosan
8
solution (10 mg/mL, w/v) with or without 1000 U/mL EPO and squeezed
9
a clean glass on the top to form a thin film. After solidification process,
10
the top glass was gently removed away and the thin film coated slide was
11
dried in a freeze-drying machine over night at a temperature of − 80 °C.
12 13
Before the cell seeding the coated slides were firstly incubated with culture medium to neutralize acidic film.
14
2.7.2 Cell migration (wound-healing) assay
M AN U
SC
RI PT
1
For wound-healing assay, the slides covered with chitosan (Chi) or
16
EPO loaded chitosan (EPO/Chi) were seeded with Ad-MSCs at the
17
density of 2×104/cm2, and the glass substrates without coating were used
18
as control. When the cells reach to 80–90% confluence, an artificial
19
wound was created by using a 10µL pipette tip by longitudinally straight
20
scratching the confluent cell monolayer. After washed two times with
21
PBS solution to removed the un-attached cells, the cells were then
22
incubated in DMEM containing 1% FBS for 12 hours. The cells at 0 h
23
and 12 h time points after the scratching were stained with fluorescein
24
diacetate (FDA) for observation and the images were obtained using an
25
inverted microscope (Olympus IX71, Japan). The total migrated cells
26
were then calculated by counting at least five random separate fields from
27
each sample (n=5) and then the cell numbers were normalized to control
28
groups.
29
2.7.3 Cell viability measurement
AC C
EP
TE D
15
30
The cell viability of Ad-MSC cultured on the substrates was assessed
31
using the 3-(4,5-dimethylthiazohl- 2-yl)- 2,5-diph enyltetrazolium
32
bromide (MTT; Sigma-Aldrich, USA) and the absorbance at a
33
wavelength of 490 nm was measured. 9
ACCEPTED MANUSCRIPT 1
2
2.8. The transwell assay The transwell assay was further performed to evaluate the effects of Ad-MSCs
paracrine
cytokines
on
the
Schwann
cell
line
4
(RT4-D6P2T) migration. The Ad-MSCs were firstly cultured on the glass
5
slides in 24-well plats for 3 days and 2,0000 of RT4-D6P2T were seeded
6
into the upper chamber with a pore size of 8 µm (Corning, USA) in
7
serum-free medium. After incubation for 24 hours, the remained cells in
8
the upper chamber were gently removed, and the migrated cells under the
9
membrane were fixed and stained with crystal violet solution. The images
10
of immigrated cells were captured with an inverted microscope and the
11
cell numbers of 5 random separate fields from each sample (n=3) were
12
counted.
13
2.9. Gene expression assay
M AN U
SC
RI PT
3
After cultured for 3 days, Quantitative real-time PCR (qRT-PCR) was
15
applied to assay the mRNA levels of related neur-protective genes and
16
the ELISA was performed to measure the concentration of FGF-2
17
(fibroblast growth factor 2), BDNF (Basic fibroblast growth factor),
18
SDF-1 (Stromal cell derived factor 1, CXCL12) that secreted by rat
19
Ad-MSC using ELISA kit (RayBiotech, Norcross, GA, USA). The
20
concentration of proteins was then normalized to the total amount of
21
protein. For the qRT-PCR, briefly, the total RNA was extracted and
22
first-strand cDNA was synthesized according to the protocol using
23
PrimeScript RT reagent Kit (Takara, China). qRT-PCR was performed
24
using a Bio-Rad CFX (Bio-Rad, USA) using SYBR Select Master Mix
25 26
Thermo scientific, Logan, USA . FGF-2, BDNF, NGF (Nerve growth factor) and NT-3 (Neurotrophin-3) were analyzed and
27
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as
28
housekeeping gene. Three independent experiments were performed for
29
each group. The primers used for real-time PCR experiments were listed
AC C
EP
TE D
14
10
ACCEPTED MANUSCRIPT 1
in Supplementary Table S1.
2
2.10. Statistical Analysis. The results are presented as the mean ± SD. One-way analysis of
4
variance (ANOVA) was applied for statistical comparisons among groups
5
more than two, and t-test was used for the statistical comparisons among
6
two groups. All statistical analysis was performed in the software SPSS
7
13.0 (SPSS, Chicago, USA). The values of p < 0.05 or 0.01 was
8
considered statistically significant.
9
3.Results
SC
M AN U
10
RI PT
3
3.1.Fabrication of nerve conduits
In this study, EPO-Chitosan nerve conduits were fabricated by
12
freeze-drying approach to explore their application as nerve guidance
13
substrates in vivo. The conduits were of about 3.8 mm in outer diameter
14
and about 1.2 mm in inner diameter (Figure 1A). The topographical
15
observation by SEM were demonstrated in Figure 1B. SEM of Conduit’ s
16
cross-section
17
interconnected porous structures in EPO-Chitosan conduit. The porosity
18
of the internal surface (Figure.1 B-c) was less than that of the
19
cross-section. In addition, only a few porous structures (Figure.1 B-d)
20
were observed on the external surface of the conduits, which might be
21
helpful for cytokine or implanted cell reservation in conduits.
showed
the
flaky
texture
and
EP
(Figure.1B-a&b)
AC C
22
TE D
11
In vitro release curve showed that the conduits could sustain the
23
release of loaded cytokine for upto 18 days (Figure.1C), suggesting the
24
feasibility to achieve a long-term action of healing response in vivo.
25
What's more, the swelling ration
26
(Figure.1E) measurement confirmed that the physical and mechanical
27
properties of the prepared conduits were not significantly affected by the
(Figure.1D) and elastic modulus
11
ACCEPTED MANUSCRIPT
adding of the EPO to the conduit.
2
3.2. In vivo transplantation and walking-foot-print
3
analysis
RI PT
1
The EPO-Chitosan nerve conduits with or without MSCs were
5
transplanted into the peripheral nerve injured animal model (Figure.
6
2A-a-e). Walking-foot-print analysis results showed that the value of SFI
7
in both Gel/MSC and Conduit group were significantly increased as
8
compared with control group 4 and 8 weeks after surgery (Figure. 2B &
9
C),
yet
by
the
combination
of
SC
4
Gel/MSC
and
conduit
in
Conduit+Gel/MSC, the SFI value was further significantly increased
11
(Figure. 2B & C). Similarly, both EPT and WRL results showed that the
12
rat (Figure. 2E & F) in Conduit+Gel/MSC treated group were observed
13
with significantly higher extensor force and WRL time as compared with
14
those in other groups. In addition, EPT and WRL demonstrated that the
15
motor performance of rats in both Gel/MSC and Conduit groups was
16
better than that in control group.
TE D
M AN U
10
The above beneficial effects of conduits-based therapy were further
18
confirmed by MNCV. As shown in Figure. 2D, MNCV values in both
19
Gel/MSC and Conduit groups were significantly higher (p<0.01) than
20
that in control group at 4 weeks and 8 weeks after surgery. Especially for
21
Conduit group at 8 weeks, the MNCV was even better than Gel/MSC
22
group (P<0.05). Similarly, the highest MNCV values (p<0.01) were
23
observed in Conduit+Gel/MSC group, at both 4 weeks and 8 weeks.
24
These results suggested that combination of Gel/MSC and Conduit could
25
significantly enhance the therapeutic potentials of single Gel/MSC or
26
Conduit for sciatic nerve injury.
27
3.3. Histological examination
28
AC C
EP
17
The survival of DiI-marked MSCs was analyzed in Gel/MSC and 12
ACCEPTED MANUSCRIPT 1
Conduit + Gel/MSC groups 4 weeks after surgery. As shown in Figure.
2
3A, much more MSCs were observed in Conduit + Gel/MSC group
3
compared with Gel/MSC group. Quantitative analysis (Figure. 3B)
4
showed that the number of survived MSC in Conduit + Gel/MSC group
5
was about 4~5 fold than that in Gel/MSC group. H&E staining was performed to observe the circular nerve bundles
7
morphology. As shown in Figure. 4A, lots of injured nerve bundles with
8
irregular or incomplete ring shape were observed in control group. In
9
comparison, the application of Conduit or Gel/MSC alone significantly
10
improved the morphology of injured nerve bundles and more importantly,
11
the improvement was more significant by combination of Conduit and
12
Gel/MSC, which resulted in the highest level of morphological
13
restoration and regularity of nerve bundles (Figure. 4A, p<0.05 or
14
p<0.01).
M AN U
SC
RI PT
6
PGP 9.5 immunohistochemical staining and its histomophormetry
16
analysis showed that the protein expression levels in Gel/MSC and
17
Conduit group were significantly higher than those in control group, and
18
the expression level was further increased in Conduit+Gel/MSC group
19
(Figure. 4 B & C, p<0.05 or p<0.01).
20
3.4.Axon number, diameter and myelin thickness
EP
TE D
15
In order to count the axon number, the Modified Bielschowsky silver
22
staining was performed 8 weeks after surgery. As shown in Figure.5 A &
23
C, the number of axon in the injured nerve bundles were increased
24
significantly (p<0.01) in both Gel/MSC and Conduit groups compared
25
with that in Control group, and it was further increased in
26
Conduit+Gel/MSC group (p<0.01). The axon diameter (Figure. 5 B & D)
27
and myelin thickness (Figure. 5 B & E) of the regenerated nerve bundles
28
were measured according to the TEM images (Figure. 5 B). Similarly,
29
axon diameter and myelin thickness in Gel/MSC and Conduit alone
30
groups were increased compared with control group, and the best results
31
were observed in the Conduit+Gel/MSC group, indicating the best
AC C
21
13
ACCEPTED MANUSCRIPT 1
performance of combining Gel/MSC and Conduit on repair of sciatic
2
nerve injury among the four groups.
3
3.5. Re-innervation of target gastrocnemius muscles According to the Masson trichrome staining in Figure. 6, much more
5
muscular cells with irregular shape (red color) and accumulation of the
6
collagen (blue color) were observed in control group than those in
7
Gel/MSC, Conduit and Conduit+Gel/MSC groups. In addition, the areas
8
of muscular cells in both Gel/MSC and Conduit were significantly larger
9
than that in control group (p<0.01), while the largest mean area of
SC
RI PT
4
muscular cells was observed in Conduit+Gel/MSC group.
11
3.6. The in vitro beneficial effects of EPO/Chi on Ad-MSC
12
and Schwann cells
M AN U
10
As shown in Figure.7C, the coating of EPO/Chi on the glass
14
significantly improved the cellular viability of MSCs compared with Chi
15
or Control group. Wound healing assay (Figure.7 A & B) indicated that
16
Chiostan-coated glass could improve the migration of Ad-MSCs, which
17
was further enhanced by adding of EPO compared with control (p<0.05 or
18
p<0.01). In order to investigate the neuro-protective effects of
19
EPO/Chi-regulated Ad-MSCs on nerve regeneration, the neurotrophins
20
gene analysis and transwell migration of Schwann cells assay were
21
performed. As shown in Figure.7 D-H, the Ad-MSC cultured on
22
EPO/Chi-coated glass showed higher levels of FGF-2 and BDNF gene
23
expression and proteins secretion, as well as SDF-1 concentration. The
24
exposure of Schwann cells to Ad-MSCs on EPO/Chi coated glass caused
25
more cells to migrate through the insert membrane (Figure. 7 I & J).
26
Interestingly, even without Ad-MSC, the EPO/Chi coated glass could also
27
result in certain numbers of Schwann cells, which was consistently with
28
previously reports that the EPO alone could also promote the cellular
AC C
EP
TE D
13
14
ACCEPTED MANUSCRIPT 1
migration [11, 20]. What’s more, unlike the randomly distributed cells in
2
other groups, the migrated Schwann cells in EPO/Chi group were more
3
likely to cluster together (Figure. 7J), which might be resulted from the
4
combination of MSCs and EPO/Chi. These results indicated that EPO/Chi-coated glass could promote the
6
migratory response of Ad-MSCs, and activate its neurotrophins protein
7
secretion as well as gene expression. The EPO/Chi could also promote the
8
migration of Schwann cells alone or through the mediation of Ad-MSCs,
9
which might eventually contribute to the good performance of nerve repair
11
4. Discussion
SC
with EPO-loaded conduit + Ad-MSC.
M AN U
10
RI PT
5
Transplantation of a nerve implant between two nerve stumps has
13
been proposed for aiding injured peripheral nerve regeneration and
14
functional restoration guidance. In the past, biomaterial based strategies
15
like nerve conduits, or cell-based therapy were applied to bridge the
16
nerve gap for nerve repair as well as protect the injured nerve from
17
surrounding tissues during the healing process[3]. Some have achieved
18
certain success, but many results were still in controversy[2, 22, 23].
TE D
12
In the present study, a combination strategy of EPO-loading nerve
20
conduit and MSCs was developed to repair sciatic nerve injury. The
21
functional gait movements, global and detail nerve tissue morphologies,
22
neural protein expression and reinnervation of target gastrocnemius
23
muscles were used to evaluate the performance of these strategies. The
24
results indicated that, although the single application of nerve conduits (in
25
Conduit group) or mesenchymal stem cells plus hydrogels (in Gel/MSC
26
group) could improve axon regeneration compared with control group,
27
combining MSC with EPO-loading chitosan nerve conduits resulted in
28
the most favorable outcomes for guidance of nerve regeneration. In vitro
29
cellular experimental results indicated that EPO/Chi could increase the
30
migratory response of Ad-MSCs, and increase its neurotrophins protein
31
secretion and gene expression. The MSCs + EPO/Chi could also promote
AC C
EP
19
15
ACCEPTED MANUSCRIPT 1
the migration as well as the clustering of Schwann cells. As is well known, the directly administration of exogenous cytokines
3
without protection would result in quickly degradation due to
4
deactivation by enzymes and other physical-chemical effectors in vivo
5
and in vitro. In recent years, we focus on the construction of localized and
6
sustained delivery system for controlled release of exogenous cytokines
7
to enhance its repair effects in vivo. Previously, we found that the
8
chitosan-based and NGF-loaded nerve conduits could be used for sciatic
9 10
nerve repair in rats which highlights good potential of Chitosan conduit for peripheral nerve treatments[8]. However, it was also demonstrated
11
that NGF might promote neural apoptosis after peripheral nerve injury
12
under certain circumstance[24], which indicated the bifacial effects of
13
NGF during nerve repair. The hematopoietic cytokine erythropoietin
14
(EPO) was also proved to be beneficial for the functional recovery of
15
injured peripheral nerve system via EPO receptor (EPOR) expressed by
16
Schwann cells [12, 13, 20]. Recently, we reported that the encapsulating
17
EPO in PLGA microspheres for localized and sustained delivery could
18
significantly improve the neural function of injured peripheral nerve,
19
which indicated its great potential for peripheral nerve treatments [16, 25].
20
What’s more, the results of some published papers also indicated that the
21
EPO is better than NGF to enhance nerve regeneration and promote
22
functional recovery after peripheral nerve injury in the rat[26]. In addition,
23
EPO was also confirmed to enhance the migration of MSCs, which might
24
be beneficial for the recruitment of endogenous MSCs and improve its
25
repair performance[11]. Therefore, the EPO was used as the
26
neuroprotective cytokine for preparation of “bio-active” chitosan nerve
27
conduit in the study.
28
AC C
EP
TE D
M AN U
SC
RI PT
2
To our knowledge, the present study was the first report to
29
encapsulate
EPO protein
in
the
30
transplantation of EPO-loading nerve conduit and MSCs for peripheral
31
nerve repair was also firstly evaluated. As expected, the survival of
32
transplanted MSCs was significantly enhanced in EPO-loaded conduit +
33
gel/MSCs group compared with that in Gel/MSC group. Further, the best 16
Chitosan
scaffold. Combined
ACCEPTED MANUSCRIPT
results of SFI and MNCV were detected in EPO-loaded conduit +
2
gel/MSCs group compared with all the other groups. The histological
3
examination and histomorphometry results indicated the highest level of
4
morphological restoration and regularity of nerve bundles. Similarly,
5
PGP 9.5 protein expression, axon diameter and myelin thicknessas well
6
as the number of axon were also significantly improved in
7
Conduit+Gel/MSC groups compared with the other groups.
RI PT
1
In order to further investigate the beneficial effects of EPO-loading
9
Chitosan scaffolds on Ad-MSC and Schwann cells, as well as the
10
potential underlying mechanism, in vitro cell experiments were
11
performed. The surface mobilization of Chitosan and EPO on glass was
12
used for in vitro evaluation of MSC performance. CCK-8 assay showed
13
that the EPO loaded Chitosan could enhance the growth of seeding MSCs.
14
The scratch wound healing assay was applied as well in vitro.
15
Consistently, the results proved that EPO-loaded Chitosan could promote
16
the migration of MSCs during the wound healing process.
M AN U
SC
8
In recent years, transplantation of MSCs for neuro-diseases therapy
18
has drawn more and more attention. Several beneficial effects were
19
contributed to the therapeutic results of MSC transplantation, such as its
20
immunomodulatory, chemotactic properties, and the secretion of some
21
beneficial growth factors and cytokines [9, 18]. In the present study, the
22
qRT-PCR of gene expression and ELISA assay indicated that the EPO
23
loaded Chitosan could also promote the secretion of neuro-protective
24
cytokines FGF2, BDNF and SDF-1. What’s more, the transwell results
25
showed that the EPO/Chi coated glass could increase the migratory
26
response of Schwann cells directly or through the mediation of Ad-MSCs.
27
Taken together, we speculated that EPO/Chi conduit alone might also
28
enhance the response of both Schwann cells and endogenous MSCs
29
during the wound healing process, while single injection of Gel/MSC
30
only resulted in small distributed number of the exogenousMSCs (Figure.
31
3), and these might lead to better repair results (such as MNCV in
32
Figure.2) of Conduit group than control group, or even better than
33
Gel/MSC.
AC C
EP
TE D
17
17
ACCEPTED MANUSCRIPT
What’s more, the MSCs cultured on EPO/Chi coated substrates could
2
also induce the clustering of migrated Schwann cells, which played vital
3
role in the Schwann cells’ mediated peripheral nerve regeneration [27].
4
These in vitro results indicated that the EPO-loaded Chitosan conduits
5
could not only protect the peripheral nerve via EPO proteins delivery, but
6
also be beneficial for the repair of injured peripheral nerve via MSC
7
regulation.
RI PT
1
Despite the encouraging preliminary results from our study, there
9
were still some limitations in the present study. Because the negative
10
outcomes were always associated with gaps greater than 5 mm for the
11
marketed bioartificial conduits, only 5 mm of gap distance was applied
12
[28]. Actually, treatment of larger nerve defects remains to be the big
13
challenge of the relative field. Ongoing studies are also directed toward to
14
apply the nerve conduits for long-distance regeneration of peripheral
15
nerves, which might be of great importance for peripheral nerve
16
reconstruction and recovery.
17
5. Conclusion:
TE D
M AN U
SC
8
In the present study, a new strategy of EPO-loaded Chitosan scaffold
19 20
and MSC transplantation for nerve regeneration was reported and the its repair performance for the sciatic nerve injury was evaluated in vivo and
21
in vitro. In vivo results showed that the combination of EPO-loaded
22
Chitosan scaffolds with MSCs eventually resulted in accelerated nerve
23
healing process. In vitro results indicated that the EPO-loaded Chitosan
24
could promote the MSC and Schwann cell repair performance, which
25
might contribute to the therapeutic outcome of the new strategy.
26
Therefore, the combined transplantation of the EPO-loaded Chitosan and
27
MSCs for nerve replacement represents a new generation of medical
28
device for peripheral nerve reconstruction.
29
Appendix A. Supplementary data
30
More information could be found in the “Supplementary data.DOC”.
AC C
EP
18
18
ACCEPTED MANUSCRIPT 1
References
2
[1] Intiso D, Grimaldi G, Russo M, Maruzzi G, Basciani M, Fiore P, et al. Functional outcome and
3
health status of injured patients with peripheral nerve lesions. Injury 2010;41:540-543.
4
[2] Gu X, Ding F, Williams DF. Neural tissue engineering options for peripheral nerve regeneration. Biomaterials 2014;35:6143-6156.
RI PT
5 6
[3] Daly W, Yao L, Zeugolis D, Windebank A, Pandit A. A biomaterials approach to peripheral
7
nerve regeneration: bridging the peripheral nerve gap and enhancing functional recovery.
8
Journal of the Royal Society, Interface / the Royal Society 2012;9:202-221.
[4] Quigley AF, Bulluss KJ, Kyratzis IL, Gilmore K, Mysore T, Schirmer KS, et al. Engineering a
10
multimodal nerve conduit for repair of injured peripheral nerve. Journal of neural engineering
11
2013;10:016008.
M AN U
SC
9
12
[5] Bozkurt A, Boecker A, Tank J, Altinova H, Deumens R, Dabhi C, et al. Efficient bridging of 20
13
mm rat sciatic nerve lesions with a longitudinally micro-structured collagen scaffold.
14
Biomaterials 2016;75:112-122.
[6] Meyer C, Stenberg L, Gonzalez-Perez F, Wrobel S, Ronchi G, Udina E, et al. Chitosan-film
16
enhanced chitosan nerve guides for long-distance regeneration of peripheral nerves.
17
Biomaterials 2016;76:33-51.
TE D
15
[7] Cirillo V, Clements BA, Guarino V, Bushman J, Kohn J, Ambrosio L. A comparison of the
19
performance of mono- and bi-component electrospun conduits in a rat sciatic model.
20
Biomaterials 2014;35:8970-8982.
21
[8] Wang H, Zhao Q, Zhao W, Liu Q, Gu X, Yang Y. Repairing rat sciatic nerve injury by a
22
nerve-growth-factor-loaded, chitosan-based nerve conduit. Biotechnology and applied
23
AC C
EP
18
biochemistry 2012;59:388-394.
24
[9] Keilhoff G, Goihl A, Stang F, Wolf G, Fansa H. Peripheral nerve tissue engineering: autologous
25
Schwann cells vs. transdifferentiated mesenchymal stem cells. Tissue engineering
26
2006;12:1451-1465.
27
[10] Hsueh YY, Chang YJ, Huang TC, Fan SC, Wang DH, Chen JJ, et al. Functional recoveries of
28
sciatic nerve regeneration by combining chitosan-coated conduit and neurosphere cells
29
induced from adipose-derived stem cells. Biomaterials 2014;35:2234-2244.
30 31
[11] Nair AM, Tsai YT, Shah KM, Shen J, Weng H, Zhou J, et al. The effect of erythropoietin on autologous stem cell-mediated bone regeneration. Biomaterials 2013;34:7364-7371.
19
ACCEPTED MANUSCRIPT 1
[12] Ning R, Xiong Y, Mahmood A, Zhang Y, Meng Y, Qu C, et al. Erythropoietin promotes
2
neurovascular remodeling and long-term functional recovery in rats following traumatic brain
3
injury. Brain Res 2011;1384:140-150. [13] Yin ZS, Zhang H, Bo W, Gao W. Erythropoietin promotes functional recovery and enhances
5
nerve regeneration after peripheral nerve injury in rats. AJNR American journal of
6
neuroradiology 2010;31:509-515.
RI PT
4
7
[14] Elfar JC, Jacobson JA, Puzas JE, Rosier RN, Zuscik MJ. Erythropoietin accelerates functional
8
recovery after peripheral nerve injury. The Journal of bone and joint surgery American volume
9
2008;90:1644-1653.
11
[15] Li X, Gonias SL, Campana WM. Schwann cells express erythropoietin receptor and represent
SC
10
a major target for Epo in peripheral nerve injury. Glia 2005;51:254-265.
[16] Zhang W, Gao Y, Zhou Y, Liu J, Zhang L, Long A, et al. Localized and sustained delivery of
13
erythropoietin from PLGA microspheres promotes functional recovery and nerve regeneration
14
in peripheral nerve injury. BioMed research international 2015;2015:478103.
15
[17] Wyse RD, Dunbar GL, Rossignol J. Use of genetically modified mesenchymal stem cells to
16
treat
17
2014;15:1719-1745.
19
neurodegenerative
diseases.
International
journal
of
molecular
sciences
[18] Hofer HR, Tuan RS. Secreted trophic factors of mesenchymal stem cells support
TE D
18
M AN U
12
neurovascular and musculoskeletal therapies. Stem cell research & therapy 2016;7:131. [19] Lee EJ, Xu L, Kim GH, Kang SK, Lee SW, Park SH, et al. Regeneration of peripheral nerves by
21
transplanted sphere of human mesenchymal stem cells derived from embryonic stem cells.
22
Biomaterials 2012;33:7039-7046.
EP
20
[20] Inoue G, Gaultier A, Li X, Mantuano E, Richardson G, Takahashi K, et al. Erythropoietin
24
promotes Schwann cell migration and assembly of the provisional extracellular matrix by
25 26 27 28 29 30 31
AC C
23
recruiting beta1 integrin to the cell surface. Glia 2010;58:399-409.
[21] Kim CL, Kim DE. Self-healing Characteristics of Collagen Coatings with Respect to Surface Abrasion. Scientific reports 2016;6:20563.
[22] Geuna S, Gnavi S, Perroteau I, Tos P, Battiston B. Tissue engineering and peripheral nerve reconstruction: an overview. International review of neurobiology 2013;108:35-57. [23] Chiono V, Tonda-Turo C. Trends in the design of nerve guidance channels in peripheral nerve tissue engineering. Progress in neurobiology 2015;131:87-104.
32
[24] Petratos S, Butzkueven H, Shipham K, Cooper H, Bucci T, Reid K, et al. Schwann cell apoptosis
33
in the postnatal axotomized sciatic nerve is mediated via NGF through the low-affinity 20
ACCEPTED MANUSCRIPT 1
neurotrophin
2
2003;62:398-411.
receptor.
Journal
of
Neuropathology
&
Experimental
Neurology
3
[25] Zhang W, Sun B, Yu Z, An J, Liu Q, Ren T. High dose erythropoietin promotes functional
4
recovery of rats following facial nerve crush. Journal of clinical neuroscience : official journal
5
of the Neurosurgical Society of Australasia 2009;16:554-556.
7
[26] Yin Z-S, Zhang H, Gao W. Erythropoietin promotes functional recovery and enhances nerve
RI PT
6
regeneration after peripheral nerve injury in rats. Am J Neuroradiol 2010;31:509-515.
8
[27] Parrinello S, Napoli I, Ribeiro S, Wingfield Digby P, Fedorova M, Parkinson DB, et al. EphB
9
signaling directs peripheral nerve regeneration through Sox2-dependent Schwann cell sorting. Cell 2010;143:145-155.
SC
10
[28] Weber RA, Breidenbach WC, Brown RE, Jabaley ME, Mass DP. A randomized prospective
12
study of polyglycolic acid conduits for digital nerve reconstruction in humans. Plastic and
13
reconstructive surgery 2000;106:1036-1045.
M AN U
11
14 15
17
TE D
16
Figures captions
19
Figure.1 Preparation and characterization of the EPO-loaded Chitosan scaffold. (A)
20
The schematic summary of the EPO-loaded Chitosan scaffold constructing process;
21
and (B) SEM observation of the scaffold; (C) in vitro release of EPO; (D) Swelling
22
ration and (E) elastic modulus of the Chi or EPO/Chi conduits.
AC C
23
EP
18
24
Figure.2 In vivo transplantation and functional evaluation. (A) The schematic
25
summaryof the implantation surgery process, (B) Representative images of
26
walking-foot-print analysis and its quantitative analysis results SFI (C);(D) MNCV of
27
the electrophysiological examination; and the motor performance evaluated by EPT
28
(E) and WRL (F). “*” for P<0.05, and “**” for P<0.01.
29
21
ACCEPTED MANUSCRIPT 1
Figure.3 In vivo observation of the DiI labeled MSCs (A) and quantitative analysis
2
results (B) after 4 weeks surgery. “*” for P<0.05, and “**” for P<0.01.
3
Figure.4 In vivo histological and immunohistological examination. (A) H&E staining
5
results of the injured nerve 8 weeks after surgery; immunohistochemical staining of
6
the PGP9.5 protein (B) and the quantitative analysis results (C). “*” for P<0.05, and
7
“**” for P<0.01.
RI PT
4
8
Figure.5 Axon number, diameter and myelin thickness of the regenerated nerve. (A)
10
Modified Bielschowsky silver staining for axon numbers calculation (C); the
11
represent images of atomic force microscope (AFM) results (B), and quantitative
12
analysis of the axons diameter (D) andmyelin sheaths thickness (E). “*” for P<0.05,
13
and “**” for P<0.01. Scale bar = 20µm.
M AN U
SC
9
14 15
Figure.6 Masson trichrome staining of the targeting gastrocnemius muscles and the
16
quantitative analysis. “*” for P<0.05, and “**” for P<0.01.
17
Figure.7 In vitro cell viability and wound-healing migration assay. (A)
19
Wound-healing migration assay of the MSCs on EPO/Chi coated glass and (B) the
20
quantitative analysis of migrated cells; (C) MTT results for the cell viability assay; (D)
21
qRT-PCR results for measurement of the neur-protective genes expression in MSCs;
22
(E) ELISA assay results for measurement of the SDF-1(E), FGF-2 (F), BDNF (G)
23
proteins concentration from MSCs; the quantitative analysis (H) of the migrated
24
Schwann cell(RT4-D6P2T) (I); “*” for P<0.05, and “**” for P<0.01.
26 27
EP
AC C
25
TE D
18
28 29 30 31 32 33 22
ACCEPTED MANUSCRIPT 1 2 3 4 5
RI PT
6 7 8 9
SC
10 11
M AN U
12 13 14 15 16
18
A
19
EP
20
TE D
17
21
AC C
22 23 24 25 26
B
C
23
ACCEPTED MANUSCRIPT EPO
100 80 60 40 20 0 0
1
D
E
15
18
Figure. 1
EP
A
AC C
9
TE D
8
15 16 17 18
24
B
0.5
/C
hi
0.0
EP O
7
14
12
1.0
hi
6
13
9
SC
Elastic Modulus (Mpa)
5
12
1.5
M AN U
4
11
6
Time (day)
3
10
3
C
2
RI PT
Cumulative Release (%)
120
21
24
ACCEPTED MANUSCRIPT 1 2
Control
Gel/MSC
Conduit
C
Conduit+Gel/MSC
D
**
3
**
**
**
**
**
**
** *
5 6
**
E
*
**
15
WRL Time (Sec)
100
9 EPT (%)
80
10
60 40 20
11
0
17 18 19
EP
16
A
AC C
14
TE D
Figure. 2
13
10
*
** **
5
0
Time = 8 weeks
12
F
M AN U
8
SC
7
15
*
RI PT
4
20 21 22
25
B
Time = 8 weeks
ACCEPTED MANUSCRIPT 1 2 3
Figure. 3
RI PT
4 5 6
SC
7
M AN U
8 9 10 11
13 14 15
A
AC C
16
EP
TE D
12
17 18 19 20 21
B
22
26
ACCEPTED MANUSCRIPT 1 2
**
C
**
3
RI PT
4 5 6
SC
7
M AN U
8
Figure. 4
9 10 11
15 16 17
EP
14
AC C
13
TE D
12
27
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
1
Figure. 5
2
6 7 8 9
EP
5
AC C
4
TE D
3
10 11 12 13
28
ACCEPTED MANUSCRIPT 1 2 3
RI PT
4 5 6
**
SC
**
7
M AN U
8 9 10 11
TE D
12
Figure. 6
13
EP
14 15
AC C
16 17 18 19
A
B
20 21 22 29
ACCEPTED MANUSCRIPT C
1 2 3
D
E
RI PT
4 5 6
SC
7
9
F
G
10 11
15 16 17 18
I
EP
14
AC C
13
H
TE D
12
M AN U
8
Figure. 7
30