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Nerve Regeneration by Using of Chitosan-Silicate Hybrid Porous Membranes
Yuki Shirosaki1,a, Satoshi Hayakawa1,b, Akiyoshi Osaka1,c José D. Santos2,d and Ana C. Maurício3,e
1 Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima, Kita-ku, Okayama, 700-8530, Japan
2 Faculdade de Engenharia da Universidade do Porto, Rua Dr. Roberto Frias, S/N, 4200-465, Porto,
Portugal 3 Centro de Estudos de Ciência Anomal / Institut de Ciências e Tecnologias Agrárias e
Agro-Alimentares, Universidade do Porto, Campus Agrario de Vairao, Rua Padre Armando Quintas, 4485-661, Vairão, Portugal
[email protected], [email protected], [email protected], [email protected], [email protected]
Keywords: nerve regeneration, inorganic-organic hybrid, chitosan, porous membrane
Abstract. The treatment of peripheral nerve injuries is still one of the most challenging tasks in
neurosurgery, as functional recovery is rarely satisfactory in these patients. The concept behind the
use of biodegradable nerve guides is that no foreign material should be left in place after the device
has fulfilled its task, so as to spare a second surgical intervention. In a previous study, flexible and
biodegradable chitosan-γ-glycidoxypropyltrimethoxysilane (GPTMS) hybrid membranes exhibited
better cytocompatibility in terms of osteoblastic cells than chitosan membrane. Porous chitosan
hybrid membranes, derived by freeze-drying the hybrid gels, showed that the cells were attached and
proliferated both on the surface and into pores. The aim of the present study was to evaluate the
influence of these chitosan hybrid membranes in terms of their inflammatory response and
remodeling of connective tissue during wound-healing processes before use as a periphery nerve
graft. The porous chitosan hybrid membranes showed good biocompatibility and improved
posttraumatic axonal regrowth and functional recovery.
Introduction
Peripheral nerve injuries have a high incidence in today’s society [1]. Despite recent progress in
peripheral nerve trauma management, recovery of functional parameters is usually far from normal
and thus much attention is being paid to nerve regeneration research. Over the last years, the research
on entubulation nerve repair has developed along two main lines: (1) testing different biomaterials for
fashioning of the conduit; (2) testing different additives for enriching the nerve guide. As regards the
first research line, many properties are desirable for a nerve scaffold. They included: (a) permeability
to prevent fibrous scar tissue invasion allowing nutrient and oxygen supply; (b) mechanical strength
to maintain a stable support structure for the nerve regeneration; (c) immunological inertness with
surrounding tissue; (d) biodegradability to prevent chronic inflammatory response and pain due to
nerve compression; (e) easy regulation of conduit diameter and wall thickness; (f) surgical
amenability. Among the various biomaterials proposed for the fashioning of nerve conduits, chitosan
has recently attracted much attention because of its biocompatibility, biodegradability, low toxicity,
low cost, enhancement of wound-healing, and antibacterial effects [2]. In a previous study [3-5],
flexible and biodegradable chitosan-γ-glycidoxypropyltrimethoxysilane (GPTMS) hybrid
membranes exhibited better cytocompatibility in terms of osteoblastic cells than chitosan membrane.
Porous chitosan hybrid membranes, derived by freeze-drying the hybrid gels, showed that the cells
were attached and proliferated both on the surface and into pores. In this study, new biodegradable
nerve guides from these chitosan hybrids were prepared and their biocompatibility was examined for
the nerve regeneration by using animal model.
Key Engineering Materials Vols. 529-530 (2013) pp 361-364Online available since 2012/Nov/29 at www.scientific.net© (2013) Trans Tech Publications, Switzerlanddoi:10.4028/www.scientific.net/KEM.529-530.361
All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of TTP,www.ttp.net. (ID: 130.15.241.167, Queen's University, Kingston, Canada-01/10/13,15:28:22)
Materials and Methods
Chitosan (high molecular weight, Aldrich®
, USA) was dissolved in 0.25M acetic acid aqueous
solution to a concentration of 2% (w/v). GPTMS (Aldrich®
, USA) was also added to the chitosan
solution and stirred at room temperature for 1h. The solutions for the solid membranes were then
poured into polypropylene containers with cover, and aged at 60°C for 2 days and then dried at 60°C
for 2 days. For the porous membranes, the solutions were frozen for 24h at -20°C and then
transferred to the freeze-dryer, where they were left 12h to complete dryness. The hybrid membranes
(both solid and porous types) were soaked in 0.25M sodium hydroxide aqueous solution to neutralize
remaining acetic acid, thoroughly washed with distilled water, and dried again at 60°C for 2 days or
freeze dried. All membranes were sterilized with ethylene oxide gas. Prior to their use in vivo,
membranes were kept 1 week at room temperature in order to clear any ethylene oxide gas remnants.
Poly (dl-lactide-co-glycolide) copolymers with ratio of 90PLA:10PLG were obtained from their
cyclic dimers, dl-lactide and glycolide. Non-woven constructs were used to prepare tube guides 16
mm long, internal diameter of 2.0 mm and thickness wall of 1.5mm. These fully synthetic non-woven
materials are extremely flexible, biologically safe and are able to sustain the compressive forces due
to body movement after implantation. They have also some degree of porosity to allow for influx of
low molecular nutrients required for nerve regeneration. The non-woven structure allowed the
tube-guide to hold suture without difficulties, however greater care had to be taken in order to ensure
its integrity. These tube-guides of PLGA are expected to degrade to lactic and glycolic acids through
hydrolysis of the ester bonds.
Prior to their use on crushed sciatic nerves, the biocompatibility of the chitosan hybrid membranes
was tested in vivo in four adult female Wistar rats. Under general anesthesia, three longitudinal 3cm
long dorsal incisions were made and 2 x 2 cm membranes were implanted. Animals were euthanized
on weeks 1, 2, 4, and 8 after implantation. The membranes remnants were collected together with
skin and subcutaneous tissues and fixed in a 10% formaldehyde solution for later histological
analysis. In vivo nerve regeneration, adult male Sasco Sprague-Dawley rats were used. The
standardized crush injury was carried out with the animals placed prone under sterile conditions and
the skin from the clipped lateral right thigh scrubbed in a routine fashion with antiseptic solution. The
right sciatic nerve was exposed through a skin incision extending from the greater trochanter to the
distal mid-half followed by a muscle splitting incision. After nerve mobilisation, a transection injury
was performed (neurotmesis) using straight microsurgical scissors. In Group 1 (End-to-EndCh),
immediate cooptation with 7/0 monofilament nylon sutures of the 2 injured nerve endings was
immediately performed under magnification and involved with a chitosan hybrid porous membrane.
In Group 2 (Graft180ºCh) the sciatic nerve was transected immediately above the terminal nerve
ramification and in a 10 mm distal point. The nerve graft obtained, with a length of 10 mm, was
inverted 180° and sutured with 7/0 monofilament nylon. The sutured graft was involved afterwards
with a chitosan hybrid porous membrane. In Group 3 (GapCh) the proximal and distal nerve stumps
were inserted 3 mm into the chitosan hybrid porous tube-guides and held in place, maintaining a
nerve gap of 10 mm, with two epineurial sutures using 7/0 monofilament nylon, respectively. These
groups were compared with three control groups, concerning the following experimental conditions:
in Group 4 (Graft180º) the sciatic nerve was transected immediately above the terminal nerve
ramification and in a 10 mm distal point. The nerve graft obtained, with a length of 10 mm, was
inverted 180° and sutured with 7/0 monofilament nylon; in Group 5 (PLGA) the proximal and distal
nerve stumps were inserted 3 mm into the PLGA tube-guides and held in place, maintaining a nerve
gap of 10 mm, with two epineurial sutures using 7/0 monofilament nylon, respectively; and in Group
6 (End-to-End) immediate cooptation with 7/0 monofilament nylon sutures of the 2 injured nerve
endings was immediately performed under magnification. Opposite leg and sciatic nerve were left
intact in all groups and served as control for normal nerves. All animals were tested preoperatively
(week 0), and weeks 1, 2 and then every two weeks until the end of the 20-week follow-up time.
Motor performance and nociceptive function were evaluated by measuring extensor postural thrust
(EPT) and withdrawal reflex latency (WRL), respectively. For EPT test, the affected and normal
limbs were tested three times, with an interval of 2 min between consecutive tests, and the three
362 Bioceramics 24
values were averaged to obtain a final result. The normal (unaffected limb) EPT (NEPT) and
experimental EPT (EEPT) values were incorporated into an equation 1 to derive the percentage of
functional deficit.
%Motor deficit = [(NEPT – EEPT) / NEPT] x 100 Eq. 1
The nociceptive withdrawal reflex (WRL) was adapted from the hotplate test developed by Masters et
al. [6] and described elsewhere. Normal rats withdraw their paws from the hotplate within 4s or less.
The cutoff time for heat stimulation was set at 12s to avoid skin damage to the foot.
At the end of the experiments, the rats were sacrificed under deep anaesthesia and, from two animals
from each experimental group, a 10-mm-long segment of the sciatic nerve distal to the site of lesion
was removed. The specimens were fixed by immediate immersion in 2.5% purified glutaraldehyde
and 0.5% sucrose in 0.1M Sorensen phosphate buffer for 6-8 hours. Nerves were then washed in a
solution containing 1.5% sucrose in 0.1M Sorensen phosphate buffer, post-fixed in 1%
osmiumtetroxide, dehydrated and embedded in resin. From each specimen, 2-µm thick series of
semi-thin transverse sections were cut, starting from the distal stump, using an Ultracut UCT
ultramicrotome (Leica Microsystems, Wetzlar, Germany). Finally, sections were stained using
Toluidine blue and analyzed with a DM4000B microscope equipped with a DFC320 digital camera
and an IM50 image manager system (Leica Microsystems, Wetzlar, Germany).
Results and Discussion
On a histological analysis, chitosan membranes elicited a chronic inflammatory reaction on the
implantation site, even on samples retrieved as early as 7
days. Whereas the solid hybrid membranes elicited mild
chronic inflammation, characterized by infiltration of
small numbers of macrophages, lymphocytes and plasma
cells, the porous hybrid membranes induced a strong
granulomatous reaction, with abundant multinucleated
giant cells. Mild peripheral fibrosis in the form of a
collagen capsule was observed with the solid hybrid
membranes, while the porous membranes induced
significant interstitial fibrosis. Although, the porous
hybrid membranes presented the stronger inflammatory
reaction on week-4, both with a strong cellular
component and a thick capsule, smaller values for
capsule thickness were obtained on week-8. None of the
tested membranes was rejected throughout the 60-day
healing period, nor elicited systemic or local clinical
signs of illness, infection or inflammation, with all animals remaining healthy throughout the study
period.
Figure 1 depicts the data for the WRL tests. In the first week post surgery all animals presented
severe loss of sensory function and all tests had to be interrupted at the selected cut off time of 12 s
that remained identical during week-2 except in Group 6. The withdrawal response recovered
progressively in the course of the 20 weeks but at different rates depending on treatment. As expected,
recovery of noxious thermal sensation was faster and more complete in Group 6 and Group 1 with no
differences between these two groups (p = 0.312). No differences in WRL data were observed for the
two groups treated with reversed sciatic nerve autografts covered (Group 2) or without a chitosan
membrane (Group 4) although the group with chitosan membrane (Group 2) tended to show a slower
rate of recovery (p = 0.056). WRL values were significant different between the groups treated with
nerve conduits fabricated with chitosan and PLGA (p<0.001). Significant differences were also
observed between the group treated with tube-guides and the reversed autograft group (p <0.001). No
differences existed between the later group and the one treated with PLGA-made nerve conduits.
Fig. 1 Withdrawal Reflex Latency (WRL)
test to evaluate the nociceptive function.
Key Engineering Materials Vols. 529-530 363
Since successful nerve regeneration should be paralleled by satisfactory functional recovery, we
assessed thermal nociceptive function and motor function using specific behavioral tests. Both these
functions showed good degree of recovery during the 20 weeks after the sciatic nerve transection in
all experimental groups. Acutely after sciatic nerve transection there was a complete loss of both
motor and thermal sensory function. Sensory and motor deficit then progressively decreased along
the post-operative. Overall inter-group statistical comparison showed that, in comparison to the gold
standard end-to-end direct suture, PLGA conduit group only presented worse recovery.
Axon regeneration occurred in all experimental groups with a good regeneration pattern with the
presence of microfascicles typical of regenerated nerves. As expected, when comparing nerve fiber
regeneration in the groups where a nerve gap was repaired with conduits, morphological analysis
revealed a better pattern of regeneration in Group 3 than in the Group 5, with the presence of larger
axons and with a better anatomical organization of the nerve fascicles. Higher resolution light
micrographs of nerve fiber regeneration along the chitosan hybrid tube guides disclosed the presence
of a rich perineurial connective architecture which contributes to nerve compartimentalization and
axonal fasciculation. The formation of an adequate stroma inside the nerve regenerated along a
non-nervous conduit is a positive predictor of successful recovery and is suitable to be attributed to
the interaction of the chitosan matrix with the regenerating autologous tissues, especially the
connective component. From a morphological point of view, our results showed that nerve
regeneration occurred in all experimental groups and that, at time of withdrawal, Wallerian
degeneration was almost completed and substituted by re-growing axons and the accompanying
Schwann cells. Qualitative comparison between the different experiment groups showed that the
axon regeneration pattern was similar in all groups with the exclusion of the PLGA where the
regeneration pattern was worse.
Summary
We have developed and tested in vivo, three types of nerve scaffolds made by the chitosan porous
hybrid membranes. Results showed that nerve regeneration was successful in all experimental and
control groups and that the chitosan porous hybrid tubulization induced a significantly better nerve
regeneration and functional recovery in comparison to PLGA tubulization control. Further
investigation is needed to explore the mechanisms at the basis of the positive effects of the chitosan
porous hybrid on axonal regeneration.
References
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364 Bioceramics 24
Bioceramics 24 10.4028/www.scientific.net/KEM.529-530 Nerve Regeneration by Using of Chitosan-Silicate Hybrid Porous Membranes 10.4028/www.scientific.net/KEM.529-530.361
