University of Groningen
Artificial nerve guides
Meek, Marcel Frans
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Meek, M. F. (2000). Artificial nerve guides: assessment of nerve function Groningen: s.n.
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p(DLLA-ε-CL) nerve guide filled with modified denatured muscle tissue
CHAPTER 5
Evaluation of functional nerve recovery after reconstruction
with a poly(DL-lactide-ε-caprolactone) nerve guide, filled with
modified denatured muscle tissue
MF Meek,1 WFA den Dunnen,2 JM Schakenraad,3 PH Robinson, FRCS1
1
Department of Plastic Surgery, University Hospital Groningen,
Groningen, The Netherlands
2
Department of Pathology, University Hospital Groningen,
Groningen, The Netherlands
3
KEMA Medical,
Arnhem, The Netherlands
Microsurgery 1996;17:555-561
69
Chapter 5
Abstract
The aim of this study was to compare the speed of functional nerve recovery after
reconstruction with a biodegradable p(DLLA-ε-CL) nerve guide, as filled with either modified denatured muscle tissue (MDMT) or phosphate-buffered saline (PBS).
To evaluate both motor and sensory nerve recovery, walking track analysis and
electrostimulation tests were carried out after implantation periods, ranging from
3-15 weeks. Functional nerve recovery after reconstruction of a 15 mm nerve gap,
with a biodegradable p(DLLA-ε-CL) nerve guide filled with modified denatured
muscle tissue, was slightly faster, compared with nerve reconstruction of a 10 mm
gap with a biodegradable p(DLLA-ε-CL) nerve guide filled with PBS. We conclude
that our experiments have demonstrated that the use of MDMT increases the speed
of recovery after reconstruction of a nerve gap with a p(DLLA-ε-CL) biodegradable
nerve guide. Furthermore, the use of MDMT might open perspectives for repair of
longer nerve gaps.
Introduction
The most widely-used technique for
bridging nerve gaps is the use of autologous nerve grafts. This technique, however, has some disadvantages: harvesting of the graft causes sensory deficit at
the donor site and the risk of neuroma
formation there. To eliminate these problems, many alternative techniques, such
as the use of biodurable nerve guides,
have been developed to bridge a nerve
gap.1-6 However, biodurable biomaterials
remain in situ as a foreign body, potentially causing a chronic foreign body
reaction with excessive scar tissue formation, resulting in constriction of the
regenerating nerve, and ultimately limiting recovery of nerve function (e.g., secondary nerve impairment).1,2,7
Biodegradable nerve guides provide a
successful alternative. The idea behind
the use of biodegradable nerve guides is,
that the nerve guide directs the outgrowing nerve fibers towards the distal nerve
stump, while preventing neuroma forma70
tion and ingrowth of fibrous tissue into
the nerve gap. After serving these functions, the nerve guide should gradually
degrade, without inducing scar tissue formation. The use of a biodegradable nerve
guide composed of an amorphous copolymer of DL-lactide and εcaprolactone [p(DLLA-ε-CL)] has
proven to be successful.8-11 This nerve
guide degrades quickly and completely
within 1 year,9 and nerve regeneration
across a 1 cm nerve gap was faster and
qualitatively better, compared with nerve
regeneration through an autologous nerve
graft.10 Moreover, reconstruction with
this nerve guide showed good functional
nerve recovery.11 However, in all these
previous experiments, nerve gaps of 1 cm
in the sciatic nerve of rats were bridged.
To bridge a nerve gap of several centimeters (a more realistic point of view in
the clinical situation), the addition of
nerve growth stimulation factors will be
necessary. The addition of one of these
p(DLLA-ε-CL) nerve guide filled with modified denatured muscle tissue
factors can be of special importance in
nerve regeneration through a nerve guide
of several centimeters, since the maximum gap length that can be successfully
reconstructed with a nerve guide in the
rat is 2 cm.12 Several researchers have already studied the influence of growth factors,13 extracellular matrix molecules,1416
and freeze-thawed muscle tissue on peripheral nerve regeneration. Most factors
positively influence peripheral nerve
regeneration.17 The use of denatured
muscle tissue inside a nerve guide is very
promising, since the longitudinally oriented basal lamina of the muscle tissue
will direct the outgrowing nerve fibers
towards the distal nerve stump by functioning as a scaffold for the outgrowing
nerves.18 In addition, the basal lamina
contains both collagen type IV and laminin, which are known to enhance the
outgrowth and regeneration of peripheral
nerve fibers.19 The nerve guide prevents
neuroma formation and the ingrowth of
fibrous tissue into the nerve gap. Due to
the presence of muscle tissue inside the
nerve guide, there is less tendency for the
nerve guide to collapse.20 Recently we
evaluated different preparation techniques of denatured muscle tissue,21 aiming at an open structure of the extracellular matrix (ECM) and an intact basement
membrane. The aim of this study was to
evaluate the speed of functional nerve recovery after reconstruction with a
biodegradable p(DLLA-ε-CL) nerve
guide, filled with modified denatured
muscle tissue (MDMT).
Materials and Methods
Preparation of Nerve Guides
The biodegradable nerve guides in this
study were composed of a copolymer of
50% DL-lactide and 50% ε-caprolactone.
The lactide component contained 85% Llactide (LLA) and 15% D-lactide (DLA).
The nerve guide was made by a dip-coating technique, which is described in detail by den Dunnen et al.8 After preparation, the nerve guides were stored in
100% ethanol at 4 oC. Before implantation, the nerve guides were first washed
in 0.1 M sterile phosphate-buffered saline (PBS) at room temperature, and then
filled with 0.1 M sterile PBS (group A),
or with modified denatured muscle tissue (group B).
Preparation of the Denatured Muscle
Tissue
Muscle tissue specimens were harvested
from the back of male Wistar rats, stored
at -20 oC for 24 hr, and then placed in
demineralized water at 4 oC for 24 hr.
These freeze-thawed (denatured) muscle
tissue specimens were then treated using
the following technique: the sample was
first placed in 16% acetic acid, at room
temperature (RT) for 30 min, then placed
under vacuum (10 mBar) (Brand RS-4,
Wertheim, Germany) for 1 hr, and finally
placed in 0.1 M phosphate buffer at RT
for 1 hr to clean the muscle tissue (Fig.
1). The preparation of modified denatured muscle tissue is described in more
detail by Meek et al.21 This MDMT was
then cut to the appropriate dimensions
and put into the nerve guide, with the
basal lamina oriented longitudinally,
leaving approximately 1.5 mm at both
nerve guide ends.
Surgical Procedures
Male Wistar rats (n = 30), weighing approximately 250 g, were premedicated
71
Chapter 5
A
Fig. 1. Illustration (A) and cryoscanning electron micrographs (cry-SEM) (B) showing preparation of
modified denatured muscle tissue obtained from the back of the rat. B1, B2: Untreated (normal)
muscle tissue. B3, B4: Denatured muscle tissue shows that muscle fibers are swollen. B5, B6: Modified denatured muscle tissue shows that the original muscle-fiber shape has disappeared and that
striations are no longer present. Furthermore, modified denatured muscle tissue shows a wider
extracellular matrix. E, extracellular matrix; MFI, muscle fiber; My, myofibril. Bars in B1, B3 and
B5, 4.5 µm. Bars in B2, B4 and B6, 0.3 µ m.
72
p(DLLA-ε-CL) nerve guide filled with modified denatured muscle tissue
B
73
Chapter 5
with atropine (0.25 mg/kg body weight)
and anesthetized with 1% isoflurane
(Forene®) and O2/N2O. The left sciatic
nerve was exposed through a superficial
gluteal muscle-splitting incision. In group
A, a 7 mm segment was then resected,
leaving a gap of about 10 mm due to
retraction of the nerve ends. Continuity
was reestablished using a 12 mm nerve
guide. Both the proximal and distal cut
ends of the sciatic nerve were telescoped
into the ends of the nerve guide and fixed
with a single 9-0 nylon epineural suture
[Auto Suture (ussc), MV 100-4 needle].
In group B, a 12 mm nerve segment was
resected, leaving a gap of about 15 mm
due to retraction of the nerve stumps.
Continuity was reestablished by interposing an 18 mm nerve guide, filled with
MDMT (Fig. 1A). Both the proximal and
distal cut ends of the sciatic nerve were
telescoped into the ends of the nerve
guide and fixed with a single 9-0 nylon
epineural suture [Auto Suture (ussc), MV
100-4 needle]. Surgical procedures were
performed under an operation microscope (Zeiss OPMI-6, Weesp, The Netherlands), and a sterile technique was
used throughout the procedure.
We emphasize that in this study, we intentionally chose to compare two different gap lengths. We chose to reconstruct
a 1.5 cm nerve gap with a nerve guide
filled with MDMT, in order to evaluate
whether a longer gap could be successfully bridged, and whether the results
obtained after reconstruction using this
technique are comparable to those after
reconstruction of the “standard gap
length” (i.e., 1 cm) without MDMT,
which was evaluated in all previous studies. 8-11,16,20,22-25
In our opinion, the addition of MDMT
74
inside a nerve guide (group B) would create a three-dimensional (3-D) structure,
providing the possibility to reconstruct a
longer nerve gap. Therefore, a 50%
longer nerve gap was chosen.
After surgery, the animals were housed
in a temperature- and humidity-controlled room with 12 hr light cycles and
had access to standard rat food and water ad libitum. Good laboratory practice
(GLP) was observed, according to the
National Guidelines for Animal Welfare,
comparable with the international rules
for animal experimentation (International
Guide on Animal Biomedical Research
and Ethical Code for Animal Experimentation of the Council for International Organization of Medical Sciences).
Walking Track Analysis
After 3, 5, 8, 12, and 15 weeks of implantation, walking track analysis was
carried out to evaluate motor-nerve
recovery, as described in detail by Meek
et al.11 In brief, the rats were first allowed
conditioning trials in a 8.2 x 42 cm walking track. Then photographic paper was
cut to the appropriate dimensions and
placed on the bottom of the track. The
rat’s hind feet were dipped in film developer slightly thickened with glycerol.
The rat was permitted to walk down the
track, leaving its hind feet prints on the
photographic paper. From the footprints,
several measurements were obtained:
distance from the heel to the third toe,
the print length (PL); distance from the
first to the fifth toe, the toe spread (TS);
and distance from the second to the
fourth toe, the intermediary toe spread
(ITS). All three measurements were
taken from the left operated foot as well
as the contralateral nonoperated foot. As
p(DLLA-ε-CL) nerve guide filled with modified denatured muscle tissue
a result, print length factor (PLF), toe
spread factor (TSF), and intermediary toe
spread factor (ITF) could be calculated.26,27 As a result, a sciatic function index (SFI) could be derived as follows:26,27
SFI = -38.3 x PLF + 109.5 x TSF + 13.3
x ITF -8.8
An SFI of 0 is normal. An SFI of -100
indicates total impairment. To obtain significant data, several prints were measured for each rat. Sometimes several
walks were required to obtain clear print
marks.
Electrostimulation Tests
After 3, 5, 8, 12, and 15 weeks of implantation, electrostimulation tests to
evaluate sensory-nerve recovery were
carried out at three different places on the
lateral side of the left (operated) foot-sole,
as described in detail by Meek et al.11 In
brief, an electrical stimulator with an adjustable current between 0-1.0 mA was
used for this purpose. A healthy rat will
immediately withdraw its foot and spread
its toes when stimulated. The threshold,
i.e., the lowest current causing this reflex, was evaluated. Furthermore, it
should be mentioned that with electrostimulation tests, not only recovery of
sensory-nerve function (e.g., afferent
nerve fibers) is tested, but actually the
reflex arc (e.g., afferent/sensory nerve
fibers + efferent/motor nerve fibers) of a
stimulus to the sensory receptor. Theoretically, it might be possible that sensory-nerve fibers are recovered while
motor-nerve fibers are not. No withdrawal reflex is then observed.
Statistical Analysis
All data were submitted to linear regression analysis.
Results
Walking Track Analysis
Reproducible walking track patterns
could be measured for all rats. Preoperative sciatic function indices for the test
group did not differ from the control
value (Fig. 2A). As a control, the SFI for
a perfect functioning hindpaw (SFI = 0)
was used. Group A: Nerve guides. The
first signs of motor-nerve recovery were
already observed after 5 weeks. After 12
weeks, approximately 60% of the motor-nerve function was recovered. Group
B: Nerve guides + muscle. The first signs
of motor-nerve recovery were observed
after 3 weeks. During the evaluation period, the SFI increased with time. After
12 weeks the SFI was -43.4 (almost 60%
recovery).
Electrostimulation Tests
The current (mA) necessary to cause a
withdrawal reflex of the foot is outlined
in Figure 2B. The threshold of the contralateral control-foot was 0.18 mA (= optimal value). Group A: Nerve guides. In
the first 3 weeks after implantation of the
nerve guide, the maximum current of 1.0
mA was not enough to cause the reflex.
After 5 weeks, the first signs of sensorynerve recovery could be observed. After
12 weeks, the threshold decreased to approximately 0.48 mA. Group B: Nerve
guides + muscle. After 3 weeks, the first
signs of sensory-nerve recovery could
already be observed and the threshold decreased sharply to 0.71 mA at 5 weeks.
After 12 weeks, the threshold decreased
to 0.37 mA.
Statistical Analysis
Linear regression analysis was per75
Chapter 5
Fig. 2. A: Graph showing change in average sciatic function index (SFI) with time. Note that an
SFI of 0 is normal, whereas an SFI of -100 indicates total impairment. An increase of SFI in group
A can be observed after 5 weeks, whereas in group
B, an increase of SFI can already be observed 3
weeks after implantation. B: Graph showing
change in current, which is necessary to cause
withdrawal reflex of the foot of the rat with time.
The nonoperated contralateral foot-sole served as
a control. A decrease of current in group A can be
observed after 5 weeks, whereas in group B, a
decrease of current can already be observed 3
weeks after implantation.
Group A, 10 mm nerve gap/12 mm nerve guide
filled with PBS; group B, 15 mm nerve gap/18
mm nerve guide filled with modified denatured
muscle tissue
formed, and showed that motor- and sensory-nerve recovery in both groups A and
B were highly significant: in both cases
P < 0.001.
76
Discussion
This study demonstrates that functional
nerve recovery after reconstruction of a
1.5 cm gap with a biodegradable
p(DLLA-ε-CL) nerve guide filled with
MDMT is slightly faster, compared with
the reconstruction of a 1 cm gap with a
biodegradable p(DLLA-ε-CL) nerve
guide filled with PBS only.
The first signs of functional (motor and
sensory) nerve recovery in group A
(nerve guide + PBS) could be observed
after 5 weeks of implantation, whereas
the first signs of functional nerve recovery in group B (nerve guide + MDMT)
were already observed after 3 weeks. The
exact moment at which the first sign of
functional nerve recovery of group B
started must have been before our first
evaluation period (at 3 weeks). Furthermore, it can be observed (Fig. 2) that the
motor- and sensory-nerve recovery in
group A was somewhat slower than in
group B, although a 50% longer nerve
gap was reconstructed in group B. Complete return of motor- and sensory-nerve
function in both groups was not reached
at the end of the evaluation period. However, since the return of nerve function
started somewhat earlier in group B, it
might be concluded that the addition of
MDMT inside the nerve guides in group
B enhanced the onset and speed of nerve
regeneration. The results obtained in this
study (recovery of functionality) can not
be directly related to speed and quality
of nerve regeneration. To evaluate this
relationship more deeply, light-microscopy (LM), transmission-electronmicroscopy (TEM), and morphometric
analysis of the axon regeneration process
will be necessary.
p(DLLA-ε-CL) nerve guide filled with modified denatured muscle tissue
The use of denatured muscle grafts for
peripheral nerve repair was studied by
Glasby et al.17 They reconstructed 3 cm
gaps in the ulnar nerve of primates. By 6
months, normal function of the hand had
returned. Norris et al. 28 studied the use
of denatured muscle grafts in a series of
human patients in order to span gaps in
digital nerves. Nerve gaps of 15-25 mm
were bridged. Evaluation of 3-11 months
after reconstruction showed almost complete sensory recovery in 7 out of 8 patients. However, Stirrat et al.29 showed
no motor recovery in 7 patients after reconstruction of major mixed nerves with
degenerated skeletal muscle. This may
be explained by the fact that by using only
freeze-thawed denatured autologous
muscle (without a nerve guide) to bridge
a nerve gap, there is an increased risk that
nerve fibers will grow out of the muscle
tissue, thereby forming a neuroma-incontinuity. In our opinion, the risks as
stated should not be taken, with regard
to clinical application of denatured
muscle tissue in patients with nerve injuries.
When autologous nerve grafts are used
for the reconstruction of nerve gaps, regeneration of axons through these grafts
is thought to be guided by endoneurial
tubes (e.g., Schwann cell basement membranes).30 Modified denatured muscle tissue contains basement membranes that
can function as a scaffold for regenerating axons. The major non-collagenous
component of basement membranes is
laminin, which has neurite-promoting
activity,31 and stimulates Schwann cell
mitosis.32,33 This might explain the better
functional nerve recovery in group B.
In a previous study,22 it was suggested
that the idea behind the use of a thin-
walled nerve guide (i.e., less biomaterial
and less swelling) was correct; however,
additional mechanical support is then
needed. The nerve guide and basement
membranes direct the outgrowing nerve
fibers towards the distal stump, prevent
the ingrowth of fibrous scar tissue, and
provide an optimal environment enriched
with factors to enhance and orient axonal
regeneration. Furthermore, the risk that
nerve fibers will grow out of the muscle
tissue, thereby forming a neuroma-incontinuity, is prevented.
LM, TEM, and morphometric analysis
of the axon regeneration process is now
being performed to evaluate whether the
addition of modified denatured muscle
tissue inside nerve guides enhances the
speed of nerve regeneration, as was suggested earlier. In the future, the effects
of other additives (NGF, B50) inside
biodegradable nerve guides on the speed
and quality of functional nerve recovery
and nerve regeneration will be evaluated.
Appropriate applications of additives inside biodegradable nerve guides will not
only improve peripheral nerve regeneration, but also allow a larger nerve gap to
be reconstructed by nerve entubulation.
The latter is of extreme importance with
regard to the clinical application of biodegradable nerve guides, because in the
clinical situation nerve gaps of several
centimeters have to be bridged.
Finally, we conclude that our experiments
have demonstrated that the use of MDMT
increases the speed of recovery after
reconstruction of a nerve gap with a
p(DLLA-ε-CL) biodegradable nerve
guide. Furthermore, the use of MDMT
might open perspectives for repair of
longer nerve gaps.
77
Chapter 5
ACKNOWLEDGEMENTS:
The assistance of H.L. Bartels with microsurgical techniques is greatly appreciated.
The authors gratefully acknowledge I. Stokroos for preparation of figures, P. van
der Sijde, D. Huizinga, and B. Hellinga for photography, and M.B.M. van Leeuwen
for assistance in histological techniques.
78
p(DLLA-ε-CL) nerve guide filled with modified denatured muscle tissue
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