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Brain-Derived Neurotrophic Factor–Enriched Collagen Tubule as a Substitute for Autologous Nerve Grafts
David J. Terris, MD;
Kenneth M. Toft, MD;
Melinda Moir, MD;
Joanne Lum, BS;
Michelle Wang, BS
Arch Otolaryngol Head Neck Surg. 2001;127:294-298.
ABSTRACT
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Background Autologous nerve interposition grafts are frequently harvested by head
and neck surgeons. The sacrifice of these donor nerves guarantees some degree
of morbidity, including sensory loss, additional incision sites with associated
potential complications, and prolonged operative time. An alternative to autologous
nerve grafting is, therefore, desirable.
Objective To determine if a collagen tubule (CT) filled with either a plain collagen
gel or a brain-derived neurotrophic factor (BDNF)–enriched collagen
gel could be used to achieve functional and histologic outcomes equivalent
to an autologous nerve graft in bridging a 15-mm nerve gap in the rabbit facial
nerve.
Design A prospective, randomized, blinded animal study with a control group.
Methods Thirty rabbit facial nerves were resected (15-mm segments) to create
nerve gaps. The gaps were bridged using 1 of 3 methods, assigned randomly:
a reversed facial nerve (control), a collagen gel–filled CT, or a BDNF-enriched
collagen gel–filled CT. The animals were evaluated after 6 weeks in
a blinded fashion for functional nerve recovery, axon count, and axonal diameter.
Results There were no significant differences between the autologous nerve graft
group, the collagen gel–filled CT group, or the BDNF-enriched collagen
gel–filled CT group (n = 10 for each group) for functional nerve recovery
(P = .94). The mean axon count and the mean axonal
diameter were highest in the BDNF-enriched collagen gel–filled CT group,
but these differences failed to reach statistical significance (P = .18 and .96, respectively).
Conclusions Collagen tubules filled with BDNF-enriched collagen gel appear to be
at least as good as autologous nerve grafts for bridging short facial nerve
gaps. Larger experimental studies are warranted to determine if clinical trials
are justified.
INTRODUCTION
AUTOLOGOUS NERVE interposition grafts are frequently harvested by head
and neck surgeons. The choices for donor nerve include the sural nerve, the
greater auricular nerve, and the medial antebrachial cutaneous nerve, among
others. The sacrifice of these nerves guarantees some degree of morbidity,
including sensory loss, additional incision sites with associated potential
complications, and prolonged operative time. Furthermore, autologous nerves
may have significant limitations, including caliber mismatch, inadequate length,
and susceptibility to crush injury during harvest. An alternative to autologous
nerve grafting is, therefore, desirable.
Bioengineered artificial conduits have shown promise as substitutes
for grafts to bridge short nerve gaps.1, 2
Outcomes have been enhanced by filling the conduits with various extracellular
matrix components or growth factors, which help to sustain the growing nerve.3, 4, 5, 6, 7, 8
Collagen matrix has been demonstrated to support neural growth in silicone
tubule and collagen tubule (CT) models. Similarly, brain-derived neurotrophic
factor (BDNF) has shown promise when applied after neurorrhaphy8, 9
and when used to sustain growth across nerve gaps.
The facial nerve is an important model for nerve grafting because it
is sometimes necessary to sacrifice the nerve for oncologic reasons, and because
of clinical limitations in available length. Anatomic constraints make it
difficult to mobilize the human facial nerve to overcome more than a 15-mm
defect; larger gaps usually require a nerve graft. Therefore, an artificial
conduit capable of bridging a 15-mm gap in a facial nerve could have immediate
clinical applicability.
We sought to determine if a CT filled with either a plain collagen gel
or a BDNF-enriched collagen gel could be used to achieve functional and histologic
outcomes equivalent to those obtained with an autologous nerve graft in bridging
a 15-mm gap in the rabbit facial nerve. The long-term objective of this investigation
is to develop a synthetic substitute for autologous nerve grafts that provides
better reliability and availability without donor site morbidity.
MATERIALS AND METHODS
ANIMALS
The left and right facial nerves of 15 male New Zealand white rabbits
weighing 2.6 to 3.0 kg were used for the investigation, yielding a total of
30 nerves. After a 15-mm segment of the buccal branch of the facial nerve
was resected, nerve reconstruction was undertaken using 1 of 3 methods, assigned
randomly: a reversed facial nerve (control), a collagen gel–filled CT
(CT-gel), or a BDNF-enriched collagen gel–filled CT (CT-BDNF). Animals
were observed daily to inspect the surgical incisions and to ensure proper
wound healing. The protocol was approved by the Stanford University Administrative
Panel on Laboratory Animal Care.
TUBULES
The tubules were synthesized from highly purified type I collagen (<0.2%
hexosamine and <0.1% trichloroacetic acid–insoluble residues, with  4
tyrosine residues per molecule), derived from bovine deep flexor tendon (Integra
LifeSciences, Plainsboro, NJ). The fibrillar structure of the collagen was
maintained throughout processing. The structural stability of the tubules
was increased by gaseous formaldehyde cross-linking, which also controls the
rate of in vivo resorption. Previous studies1
have shown that the tubules are freely permeable to macromolecules as large
as bovine serum albumin (molecular weight, 68 kd).
The tubules were cut to 20-mm lengths, each with an inner diameter of
2.5 mm. They were initially hydrated in lactated Ringer solution and then
filled with plain collagen gel or BDNF-enriched collagen gel. The collagen
gel used was composed of purified collagen dissolved in 0.012 N hydrochloric
acid for a final concentration of 3 mg/mL (Cell Prime; Collagen Biomaterials,
Palo Alto, Calif). The BDNF was provided by Regeneron Pharmaceuticals, Tarrytown,
NJ, and used at a final concentration of 300 µg/mL.
SURGICAL TECHNIQUE
The anesthesia consisted of a subcutaneous injection of atropine, 0.05
mg/kg, 15 minutes before the procedure, followed by an intramuscular injection
of ketamine hydrochloride, 35 mg/kg, and xylazine hydrochloride, 5 mg/kg.
The anesthetic depth was monitored by toe pinch and adjusted as needed. The
respiratory and heart rates were monitored every 15 minutes according to Stanford
University institutional guidelines.
Each rabbit underwent bilateral exposure and resection of a 15-mm segment
of the buccal branch of the facial nerve. The nerves were then reconstructed,
according to randomization, with 1 of the following: a reversed segment of
the facial nerve, CT-gel, or CT-BDNF. A single 10-0 monofilament nylon suture
(Ethicon, Inc, Somerville, NJ) was used to secure each end of the tubule,
and 4 to 5 epineurial sutures were used on each end of the nerve graft. The
surgeon was blinded to the type of tubule used. The wounds of the animals
in each of the 3 groups were closed with absorbable sutures.
Postoperative analgesia consisted of subcutaneous injections of buprenorphine
hydrochloride, 0.01 to 0.05 mg/kg, every 6 to 12 hours as needed. The animals
were monitored postoperatively for increased heart or respiratory rate as
an indicator of distress. Six weeks after nerve reconstruction, the animals
were killed by a lethal injection of pentobarbital sodium.
FUNCTIONAL ANALYSIS
Six weeks after surgery, functional assessment of the animals was undertaken,
as previously described.10 All rabbits were
observed for spontaneous movements of the upper lip and whiskers. Induced
movements of the upper lip and whiskers were then elicited by performing a
gentle midline nasal tap. Movement was scored on a 5-point scale (0-4). The
observer was blinded to the type of nerve reconstruction used.
HISTOLOGIC ANALYSIS
At the time of sacrifice, the nerve graft or tubule was harvested with
an additional length of nerve 5 mm proximal and distal to the nerve reconstruction
site. The specimen was fixed in buffered 10% formalin solution and then embedded
in paraffin and stained with Bielschowsky silver stain.
Morphometric measurements were made of the myelinated axon count and
mean axonal diameter for each distal nerve segment using a light microscope
(Nikon Alphashot 2YS2; Technical Instruments, San Francisco, Calif) equipped
with a single-chip color video camera (JEDMED CCD model 70-5110; JEDMED Instrument
Company, St Louis, Mo) projected onto a color monitor (NEC model PM-1971A;
Tokyo, Japan). The monitor was connected to a computer (Performa 6115 CD MacIntosh;
Apple, Cupertino, Calif) equipped with a Scion Frame Grabber Card and Scion
Image program (model LG3; Scion Corp, Bethesda, Md), which captures images
from the video screen and digitizes the analog signal for editing on the computer.
The nerve cross-sectional area (measured in micrometers squared) was
determined from the digitized image of the nerve (original magnification x40).
The number of axons was determined by manually counting the axons in 10 randomly
selected areas (1290 µm2 each) within each nerve section
(original magnification x400). A mean axon count per random 1290-µm2 area was calculated and multiplied by the nerve cross-sectional area
and then divided by 1290 µm2 to obtain the total number of
myelinated axons for that specific nerve segment.
The mean axonal diameters were derived from the digitized images of
the nerves at original magnification x100. Twenty axons were chosen
randomly and individually outlined on the computer screen. Using the Scion
Image program, the areas of the axons were measured, and the diameters were
calculated from the areas, assuming a circular geometric shape. The mean axonal
diameter of each nerve section was then calculated. The observer remained
blinded during the histologic and anatomic evaluation.
STATISTICAL ANALYSIS
Morphometric variables and functional recovery were calculated for each
group. These data were entered into a customized spreadsheet and analyzed.
Differences in means between the 3 groups were compared using an analysis
of variance. Mean differences between the experimental and control groups
were compared using the 2-tailed t test. Data are
given as mean ± SD unless otherwise indicated.
RESULTS
There were no surgical complications and no perioperative animal deaths.
However, 2 of the nerves failed to demonstrate any axonal growth through the
reconstruction (1 was in the CT-gel group and 1 was in the nerve graft group);
28 of 30 nerves, therefore, were included in the statistical analysis. An
example of the typical appearance of a nerve that was reconstructed with a
CT is provided in Figure 1.
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Figure 1. Typical appearance of a nerve
that was reconstructed with a collagen tubule. A single 10-0 monofilament
nylon suture is used to secure each end of the tubule.
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HISTOLOGIC ANALYSIS
The axon count was highest in the CT-BDNF group (7210 ± 7001)
compared with the CT-gel (3717 ± 1574) and nerve graft (3978 ±
1873) groups, but this difference failed to reach statistical significance
(P = .18) (Figure
2).
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Figure 2. Axon counts from the distal nerve
segments of rabbits whose facial nerves were reconstructed with a reversed
facial nerve graft (n = 9), a collagen gel–filled collagen tubule (CT-gel)
(n = 9), or a collagen tubule filled with brain-derived neurotrophic factor
(BDNF)–enriched gel (CT-BDNF) (n = 10). Data are given as mean ±
SD.
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The axonal diameter was similar among the 3 groups. It was highest in
the CT-BDNF group (2.99 ± 0.45 µm) compared with the CT-gel (2.95
± 0.36 µm) and nerve graft (2.93 ± 0.35 µm) groups,
but these differences were not statistically significant (P = .96) (Figure 3). A representative
histologic specimen is shown in Figure 4.
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Figure 3. Axonal diameters from the distal
nerve segments of rabbits whose facial nerves were reconstructed with a reversed
facial nerve graft (n = 9), a collagen gel–filled collagen tubule (CT-gel)
(n = 9), or a collagen tubule filled with brain-derived neurotrophic factor
(BDNF)–enriched gel (CT-BDNF) (n = 10). Data are given as mean ±
SD.
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Figure 4. A representative histologic specimen
is shown: this is a distal nerve segment from an animal in the group that
underwent reconstruction with a collagen tubule filled with brain-derived
neurotrophic factor–enriched gel (magnification x50).
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FUNCTIONAL ASSESSMENT
Comparison of the mean facial movements (on the 5-point scale) at the
time of nerve harvest for the 3 groups revealed no significant differences
(P = .94). The movement score was 3.3 ± 0.5
for the animals whose nerves were bridged with the CT-BDNF, 3.2 ± 0.6
for the animals whose nerves were bridged with the CT-gel, and 3.2 ±
0.6 for the animals whose nerves were bridged with a nerve graft (Figure 5).
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Figure 5. Facial movements of rabbits whose
facial nerves were reconstructed with a reversed facial nerve graft (n = 9),
a collagen gel–filled collagen tubule (CT-gel) (n = 9), or a collagen
tubule filled with brain-derived neurotrophic factor (BDNF)–enriched
gel (CT-BDNF) (n = 10). Data are given as mean ± SD.
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COMMENT
An artificial nerve graft is desirable because it would minimize patient
morbidity that accompanies autologous donor nerve harvest, including the need
for longer anesthesia time, postoperative pain, harvest site scar, and sensory
or motor deficit. If an artificial conduit can be achieved that is functionally
equivalent to the gold standard (an autologous nerve with epineurial repair),11 it would significantly improve patient care. The
evolution of artificial nerve grafts was predicated on an understanding of
the processes of exudation, cell proliferation, and collagen synthesis that
occur immediately after nerve transection. During nerve growth across a gap,
Schwann cells emerge and grow into the fibrin clot from the proximal and distal
nerve ends before the growth of axons. Subsequently, it was demonstrated that
local microstructures (fibrin strands, cell surfaces, and basal laminae) determine
axonal orientation.12 Since Schwann cells precede
and guide the growth of axons, the interaction of the Schwann cells with the
local environment was considered critical. Early attempts to cross long gaps
with silicone tubules were unsuccessful, likely because Schwann cells were
unable to adhere to silicone, so the space within the tubule was filled with
loose connective tissue or fat.13, 14, 15
The use of a porous collagen matrix overcomes this obstacle by promoting Schwann
cell–substrate interaction along the walls of the tubule, while allowing
local micromolecules to diffuse in and out of the lumen.
We demonstrated in this prospective, randomized, and blinded study that
collagen gel–filled CTs provide a satisfactory substitute to autologous
nerve grafts. The animals whose nerves were grafted with CTs with either collagen
gel or BDNF-enriched gel achieved functional and histologic recovery at least
as good as an autologous nerve graft when reconstructing a rabbit facial nerve
over a 15-mm gap. There are several distinct advantages to the use of CTs.
A CT is easier to place than a nerve graft and requires only a single suture
on each end, compared with 4 to 6 sutures for a nerve graft. Furthermore,
placement of the CT requires less manipulation of the nerve repair site, thereby
minimizing crush damage to the recipient nerve ends.
There are several reasons why BDNF was chosen as the neurotrophic factor
to be added to the CT in one group. Originally described in 1982 by Barde
et al,16 BDNF has been shown to undergo retrograde
transportation to the cell body of  motor neurons from skeletal muscle.
Brain-derived neurotrophic factor has subsequently been reported to rescue
rat motor neurons from naturally occurring cell death in ovo (in the ovum)17 and to prevent the death of cultured embryonic rat
spinal motor neurons in vitro.18, 19
In vivo, BDNF promotes survival of the facial and sciatic motor neurons after
axotomy in newborn rats and has been identified in sciatic nerve Schwann cells.20, 21, 22 Utley et al23 used a peripheral nerve injury model to demonstrate
that locally administered BDNF enhances the functional recovery of nerves
repaired by collagen tubulization. A combination of BDNF and ciliary neurotrophic
factor (a neurocytokine) increased the rate of nerve recovery compared with
BDNF alone in a similar experiment.9 Despite
these theoretic advantages, BDNF did not appear to foster regeneration any
better than plain collagen gel–filled CTs in the current model.
Nerve graft size in this experiment was not a significant consideration
since a reversed nerve was used as the control. Under clinical circumstances,
however, there may be a significant caliber mismatch between the recipient
nerve and the donor nerve. The CTs can be prepared in any size, and can be
constructed to contain branches of any diameter, allowing for customization
as required. Moreover, since it is synthetic, an unlimited amount of material
is available, unlike autologous nerve. The facial nerve is an ideal model
to introduce the artificial conduit into clinical practice as it is entirely
motor, is often sacrificed during a planned procedure (eg, radical parotidectomy),
and requires an autologous nerve graft for gaps as small as 15 mm. A gap of
this length can likely be consistently bridged with a porous CT, optimized
with carefully selected extracellular matrix components and neurotrophic growth
factors.
In conclusion, CTs filled with BDNF-enriched collagen gel appear to
be at least as good as autologous nerve grafts for bridging short facial nerve
gaps. Larger experimental studies are warranted to determine if clinical trials
are justified.
AUTHOR INFORMATION
Accepted for publication July 13, 2000.
From the Division of Otolaryngology/Head and Neck Surgery, Stanford
University Medical Center, Stanford, Calif.
Corresponding author and reprints: David J. Terris, MD, Division
of Otolaryngology/Head and Neck Surgery, Stanford University Medical Center,
Edwards Bldg, Room R135, Stanford, CA 94305-5328 (e-mail: dterris{at}stanford.edu).
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