Biotechnol Lett (2010) 32:1333–1337
DOI 10.1007/s10529-010-0287-8
ORIGINAL RESEARCH PAPER
Immunophilin FK506 loaded in chitosan guide promotes
peripheral nerve regeneration
Xiaoxia Li • Wei Wang • Guoqiang Wei •
Guanxiong Wang • Weiguo Zhang • Xiaojun Ma
Received: 23 December 2009 / Revised: 12 April 2010 / Accepted: 13 April 2010 / Published online: 5 May 2010
Ó Springer Science+Business Media B.V. 2010
Abstract Immunophilin ligand FK506 has been
treated as adjunct therapy for nerve repair due to its
potent neurotrophic and neuroprotective actions. It
was hypothesized that FK506 releasing from biodegradable chitosan guide provided better nerve
regenerative response than the guide with no FK506.
The drug was entrapped in the semi-permeable wall of
chitosan guide with the drug-loading of 647 lg/g. Rat
sciatic nerve defect model treated with FK506-releasing chitosan guide showed more mature appearance of
myelinated fibers 8 weeks after surgery; furthermore,
the motor functional reinnervation occurred, the
amplitude and velocity of compound muscle action
potentials reached 60% and 73% with respect to the
normal. Thus, FK506-releasing chitosan guide should
be acted as a long-lasting delivery device of immunosuppressive and neuroregenerative agent for peripheral nerve repair.
Keywords Artificial nerve Chitosan FK506 Peripheral nerve regeneration
Introduction
Electronic supplementary material The online version of
this article (doi:10.1007/s10529-010-0287-8) contains
supplementary material, which is available to authorized users.
X. Li W. Wang X. Ma (&)
Lab of Biomedical Material Engineering, Dalian Institute
of Chemical Physics, Chinese Academy of Sciences,
457 Zhongshan Road, Dalian 116023, China
e-mail: [email protected]
X. Li W. Wang X. Ma
Graduate School of the Chinese Academy of Sciences,
19 Yuquan Road, Beijing 100049, China
G. Wei W. Zhang
Department of Orthopedics, The First Affiliated Hospital
of Dalian Medical University, Dalian 116011, China
G. Wang
School of Chemical Engineering, Beijing University
of Chemical Technology, Beijing 100029, China
The repair of peripheral nerve lesions is one of the
most challenging tasks in neurosurgery. Although
nerve autograft remains the gold standard, there are
several drawbacks such as sacrifice of functioning
nerves, loss of sensation and mismatch between nerve
and graft. Artificial nerve guides have shown promising results in directing axons from the proximal to
the distal stump and creating local microenvironment
for neurotrophic substances or cells (Ciardelli and
Chiono 2006). A variety of degradable biopolymers
have been used to bridge nerve-gap defects (Alluin
et al. 2009; Yang et al. 2007). Chitosan, the deacetylated derivative of chitin, is a copolymer of
D-glucosamine and N-acetyl-D-glucosamine. With
the benefit of its neural cell compatibility (Cheng
et al. 2003), biodegradation (Freier et al. 2005), and
neuroprotective effect of the biodegradation products
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(Jiang et al. 2009), chitosan has been used as a
candidate material for peripheral nerve regeneration
(Chavez-Delgado et al. 2005).
FK506 is an FDA-approved immunosuppressant
used primarily for the prevention of allograft rejection
after transplantation. Recently, most studies are focus
on its role as a neuroprotectant and neurotrophic agent
(Gold et al. 1995; Sosa et al. 2005). Administration
treatment of FK506 in graft therapy or tube repairing
enhances the rate of nerve regeneration and the degree
of neurological recovery (Chen et al. 2009; Navarro
et al. 2001; Udina et al. 2004). However, considerable
systemic adverse effects, such as nephrotoxicity,
hypertension, hyperesthesia, muscular weakness and
gastrointestinal symptoms, can be expected because
of the non-selective immunosuppressive mechanism.
Both the potential side effects and the immunosuppressive properties may induce marginal effect or
even preclude nerve regeneration (Chunasuwankul
et al. 2002; Navarro et al. 2001). Thus, a localized,
effective and sustained delivery is crucial for a
potentially broader use of FK506.
Because of the prospective beneficial effects of
biodegradable chitosan guide and the neuroregenerative and immunosuppressive effects of FK506, the
objective of this work was to design FK506-loaded
chitosan guide used as in situ delivery vehicles of
FK506 in bridging the defects in rat sciatic nerves.
Materials and methods
Guide fabrication and characterization
Chitosan (degree of deacetylation = 93%, Mg =
230 kDa, purchased from Zhejiang Yuhuan Co.,
China), 0.64 g, was dispersed with 18 mg FK506
(Fujisawa Pharmaceuticals Inc., Japan), and dissolved
in 8 ml acetic acid (2 %, v/v) to obtain a homogeneous
solution. It was then cast in a mould with an inner
stainless steel and an outer tube, and soaked in 5%
(w/v) NaOH solution to obtain a tubular gel. Finally,
the tubes were rinsed with distilled water and airdried.
The amount of FK506 loaded in the tube wall was
analyzed by an automated microparticle enzyme
immunoassay performed on the IMx analyzer (Abbott
Labs.) after dissolving the chitosan guide with 2%
(v/v) acetic acid. The microstructure of the guide wall
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Biotechnol Lett (2010) 32:1333–1337
was examined by a JEOL/JSM-5600 scanning electron microscope, and analyzed with Image J. Chitosan and FK506-loaded chitosan tubes were grounded
into powder and scanned with a Bruker vector 22
Fourier transformed infrared spectrophotometer.
Animal model and surgical procedures
Male Wistar rats (200–220 g) were randomly divided
into four groups: (1) 13 received silica guides; (2) 13
received empty chitosan guides; (3) 13 received
FK506-releasing chitosan guides; and (4) three were
used as control in electrophysiological test. To avoid
the influence of FK506 on the contralateral sciatic
nerve, both sciatic nerves of each rat were operated.
The 7 mm guide was placed in a gap of 3 mm
according to standard procedures.
Electrophysiological studies and histological
techniques
In the first few weeks, rats implanted with tubes were
euthanized to examine the morphology of the graft
and the connection of the nerve fibers. After 6 and
8 weeks, electrophysiological evaluations were preformed on 3 or 4 rats. The sciatic nerve trunk was
stimulated with a needle electrode at its proximal and
distal portion respectively, while the receiving electrode was placed near the gastrocnemius belly to
record the compound muscle action potentials
(CMAPs). The evoked potentials were amplified to
measure the amplitude and the latency from stimulus
to the onset. The conduction velocity of motor fibers
(MNCV) between the two stimulation points was
calculated.
Eight weeks after surgery, the regenerated nerves
compounded with the graft were harvested, postfixed, and embedded in Eponresin. Transverse semithin sections (0.5 lm) were stained with Toluidine
Blue and examined by light microscopy. To estimate
the number of myelinated fibers and the diameter of
the axons, photographs of each nerve section were
taken from random fields on different rats and
analysis was then accomplished.
Statistical analysis
All parameters were expressed as mean values ±
standard deviation. Possible differences between the
Biotechnol Lett (2010) 32:1333–1337
groups were evaluated using one-way analysis of
variance, and the statistical significance was set at
P \ 0.05.
Results and discussion
FK506 was dispersed in chitosan guide wall during
fabrication, the drug-loading and entrapment efficiency were 647 lg/g and 2.3% respectively. The
drug entrapment was rather low, but FK506 was
potential in stimulating neuron outgrowth and as little
as 1 pM produced detectable augmentation (Lyons
et al. 1994). From FTIR spectrum analysis shown in
the Supplementary Figure, the macrocyclic lactone
FK506 revealed complicated structure, but due to its
low loading, the spectrum of the drug-loaded guide
was almost the same as that of chitosan, suggesting
that the introduction of FK506 did not influence the
properties of chitosan.
The morphology of FK506-loaded chitosan tube
was the same as the empty chitosan guide. SEM
photograph of the tube wall (Fig. 1c) showed that the
wall was hollow and asymmetric, the outer wall was
much compacter and thicker than the lumen. The
semi-permeable structure should impede the axon
extension out, and make FK506 located in the tube
wall releasing into the lumen. Furthermore, the
biodegradation of the chitosan graft makes room for
the growing nerve fibers as well as controlling the
Fig. 1 a FK506-loaded chitosan tube gel before drying; the
wet gel was smooth and transparent. b Air-dried FK506-loaded
chitosan guide; it became rigid with much shrinkage after
drying, and turned slight yellow. c SEM of cross section of the
guide; semi-permeable wall was noted, with the wall thickness
of 65 ± 3 lm, the compact lateral layer of 15 ± 1 lm and
medial surface of only 7 ± 2 lm
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drug release (Freier et al. 2005). The in situ
delivery was more effective and favorable for
the growing nerve fibers (Chavez-Delgado et al.
2005).
The rat sciatic nerve models treated with silica
guides exhibited strong inflammatory response in the
first few weeks, and most of the dissections showed
thick fibrous capsule with much of pus cells in the
tube (Fig. 2a). Chitosan guide group elicited mild
foreign body reaction. The tube became softer and
more translucid after 6 weeks, and the sharp and
defined edge appeared smooth, indicating the degradation of the biopolymer (Fig. 2b). Compared with
the empty chitosan guide group, the rats treated with
FK506-loaded guides did not show apparent secondary effect, and the fibrous around the tube was
thinner. The caliber of regenerated nerve fibers inside
the chamber was thicker than the empty chitosan
group, although remain thinner than the normal nerve
(date not shown). These results indicated that trace
FK506 delivered from chitosan guide decreased
inflammatory reaction, and may even promote the
growth of injured nerves.
To gain greater insight into the differences
observed among the groups, we therefore relied on
the histological result. Higher density of more mature
regenerated fibers with various sizes, shapes and
myelin-sheath thickness was observed in the FK506loaded guide group 8 weeks after surgery (Fig. 3a),
while the empty chitosan and silica groups showed
delayed regeneration with few myelinated nerve fibers
(Fig. 3b, c). Further details of the axonal diameter
distribution were shown in Fig. 4. For the silica guide
group, the proportion of myelinated fibers was converged below 2 lm and the mean value was 1.79 lm.
The histogram distribution was skewed to 2 lm in the
chitosan guide group, indicating that chitosan guide
served as a better substrate for axonal growth than the
non-biodegradable silica. On the other hand, the
FK506-loaded chitosan group had more myelinated
fibers, most of the fibers were between 2 and 3 lm,
with the mean diameter 2.77 lm. This result demonstrated that FK506 incorporated in the tube wall could
release into the lumen and further promoted fast
reinnervation of the nerve sheath besides suppressing
inflammation.
CMAPs are directly proportional to the number of
nerve fibers innervating the target muscle and allows
the conduction velocity of motor nerves. Though the
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Biotechnol Lett (2010) 32:1333–1337
Fig. 2 Gross observation of the guides after 6 weeks’
implantation. a Silica tube; pus was in the lumen and thick
fibrous capsule was noted in two rats, inflammatory cells were
found around the injury. b Chitosan tube; a thin capsule of
fibrous tissue grew and covered the intact tubes, the defined
edge became smoother. c FK506-loaded chitosan guide; inflammatory infiltration was slight around the wound, thin capsule of
fibrous tissue was around the tube and thicker nerve fibers were
clear in the tube. All of the guides were 7 mm long
Fig. 3 Light micrographs of Toluidine Blue stain of the transverse sections from the mid region in the graft 8 weeks after surgery.
a The silica guide group. b Chitosan guide group. c FK506-loaded chitosan guide group. Bar represents 10 lm
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Silica guide
Chitosan guide
FK506 loaded chitosan guide
Number of fibers
100
80
60
40
20
0
0
1
2
3
4
5
6
7
Fiber diameter (µm)
Fig. 4 Fiber diameter distributions of regenerated nerves
8 weeks after surgery; the axon densities within both empty
and FK506-loaded chitosan guide groups were significantly
higher than those in the silica groups
amplitude and conduction velocity in the guide
groups were lower than normal (Fig. 5), the two
indexes in FK506-releasing chitosan guide group
progressively reached 59.5% and 73% respectively
with respect to preoperative values, indicating the
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occurrence of motor functional reinnervation. The
result demonstrated that treatment with FK506-loaded
chitosan guide significantly enhanced the muscle
activity, which was accompanied by the increase of
gastrocnemius mass (date not shown), consistent with
the results (Chunasuwankul et al. 2002).
Though chitosan is biocompatible, the impurities in the material would evoke inflammation infiltration, influence the biocompatibility of graft and
even hinder successful nerve regeneration. Sustained
release of FK506 from the biodegradable polymer
was highly effective in suppressing inflammation
around the injured areas (Sakurai et al. 2003). Also,
sustained release of FK506 resulted in earlier onset of
reinnervation and improvement of neuroregenerative
actions.
The present results demonstrate that, incorporating
FK506 into the semi-permeable chitosan guides
during fabrication was effective and favorable for
minimal inflammatory response and quicker onset of
target reinnervation, and the regenerated myelinated
fibers showed a more mature morphometric profile at
8 weeks. However, further studies are needed to
Biotechnol Lett (2010) 32:1333–1337
35
60
Silica guide
Empty chitosan guide
FK506-loaded chitosan guide
30
50
25
MNCV (m/s)
CMAP amplitude (mV)
Fig. 5 Compound motor
action potentials amplitude
and motor nerve conduction
velocity (MNCV)
6 and 8 weeks after
transplantation; Amplitude
and MNCV showed
remarkable restoration in all
the three groups after
8 weeks, and the FK506releasing chitosan guide
group showed satisfactory
recovery compared with
silica and chitosan groups
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20
15
40
30
20
10
10
5
0
0
Normal
6 weeks
investigate the long term of target reinnervation and
the proper concentration of FK506 in the guides as
well as long gap repairing.
Acknowledgments We are grateful to Aihua Xu for assistance
in manuscript preparation and Fujisawa Pharmaceuticals, Inc.
(Osaka, Japan) for the generous gift of FK506. This study was
supported by Knowledge Innovation Project of The Chinese
Academy of Sciences (KJCX2-YW-210-02) and the National
Natural Science Foundation of China (Grant No. 20736006).
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