Background
The use of “surgical glues” to facilitate efficient nerve repairs is an appealing
and popular concept. Autologous and commercially available fibrin sealants, such as
Tisseel (Baxter Healthcare Corporation, Westlake Village, CA), are the most commonly
used substances for this application. While this usage is supported by clinical and
laboratory data [[1 ],[2 ],[3 ],[4 ],[5 ],[6 ],[7 ],[8 ]] there are concerns regarding ultimate repair strength as well as scar generation
[[8 ],[9 ],[10 ],[11 ]]. Because of these concerns, we began investigating alternative “surgical glues”
including polyethylene glycol (PEG) hydrogel (marketed as DuraSeal, Confluent Surgical,
Inc., Waltham, MA).
Like fibrin glues, DuraSeal is applied as two separate components: one is a water-soluble
amine solution and the other is a multiarmed polyethylene glycol based solution. As
these combine, cross-linking results in the rapid formation of a strong adherent gel
like substance[[12 ]] which can be applied as a cocoon or cylinder around approximated nerve ends. Previously
published biomechanical data demonstrated that the holding strength of DuraSeal when
applied in this manner is equivalent to commercially available fibrin glues[[13 ]]. DuraSeal has proven to be safe and nontoxic when applied as a dural sealant following
cranial neurosurgery[[12 ]]. Possible advantages of using this or a similar PEG hydrogel as a “nerve glue”
include delayed breakdown when compared with fibrin glues and possible adhesion inhibiting
properties[[14 ],[15 ]] that could prevent peri-neural scarring. The purpose of this study is to investigate
this potential role of a polyethylene glycol hydrogel by directly comparing it to
a commercially available fibrin glue in a rat sciatic nerve repair model.
Methods
The left lower extremity of 30 immature female Sprague-Dawley rats (200 gm) were shaved,
prepped with beta-dine, and draped with sterile towels after induction of general
anesthesia. Anesthesia was induced and maintained using 2-5% isoflurane gas continuously
administered via a nose cone throughout the procedure. The sciatic nerve was exposed
(semi-tendinosis biceps femoris splitting approach), isolated, and transected midway
between the spine and the knee. Under operating microscope magnification, the cut
ends were co-apted with two 10-0 nylon epineural sutures placed 180 degrees apart.
A small piece of rubber background (5 × 10 mm) was placed behind the repair site to
assist in glue application. Half of the repairs were “glued” with Tisseel and the
remainder with DuraSeal ([Figure 1 ]). Both products were prepared as described in the manufacturer’s insert. The fibrin
glue (or PEG hydrogel) was applied circumferentially around the approximated nerve
ends. The piece of rubber background was wrapped around the congealing mixture to
encourage the formation of a cylinder of glue. This was held for 3 minutes before
carefully pealing away the piece of rubber while taking care to leave the cylinder
in place. The wound was closed with 4-0 nonabsorbable monofilament and the animals
were allowed to recover from anesthesia before being returned to their cages. They
were maintained on water and rat chow and monitored daily for signs of illness or
distress.
Figure 1 .
At 10 weeks post surgery, the rats underwent a second surgery in which muscle function
or contraction strength of both lower limbs was tested. After the induction of general
anesthesia, both the right and left sciatic nerves were exposed as before. The achilles
tendon was exposed. The gastrocnemious muscle was isolated and its portion of the
achilles tendon secured to a 4-0 silk suture. The leg being tested was fixed to a
testing table with intra-osseous pins through the femoral condyles and the distal
tibia. The suture through the achilles was coupled to a force transducer (ADInstruments,
Inc., Colorado Springs, CO). A supra maximal stimulus of 5 V was applied to the proximal
sciatic nerve for 25 ms. The force of the gastrocnemious contraction was measured
with the force transducer and recorded using the PowerLab data acquisition system
(ADInstruments, Inc., Colorado Springs, CO) and the Apple iBook lap top computer (Apple
Computer, Inc., Cupertino, CA). The data was analyzed as a percentage of the experimental
to control sides and paired student t-test statistical analysis was performed.
The final analysis of the repaired nerve was by histologic inspection to assess scar
formation. The nerves were harvested and fixed in 10% formalin solution. Longitudinal
sections were taken at the repair site and stained with Masson’s Trichrome. The 10×
images were digitalized and peri-neural scarring was evaluated ([Figures 2 ] and [3 ]). A ratio between the thickness of the scar and the nerve diameter was calculated
using longitudinal sections through the middle plane of the nerve at the level of
the repair. Final statistical analysis was performed between the two groups using
paired student t-test.
Figure 2 .
Figure 3 .
Results
Twenty-nine of the original 30 rats survived to complete the study. Fifteen were in
the hydrogel group and fourteen in the fibrin glue group. Maximal medial gastrocnemious
contractile force was measured in all 29 rats. The amplitude of the force transducer
waveform was recorded, and a ratio between the experimental and uninstrumented sides
for each animal was obtained. Muscle strength testing revealed the average ratio of
experimental to control sides for the fibrin sealant group was 0.75 (+/- 0.24)and
for the hydrogel group was 0.72 (+/- 0.26). No significant differences (using paired
Student’s t-test) were demonstrated between these groups. ([Figure 4 ])
Figure 4 .
Peri-neural scarring was evaluated histologically at 10 weeks in all 29 rats. A ratio
between the thickness of the scar (collagen stained with Masson’s Trichrome) and the
nerve diameter was calculated using longitudinal sections through the middle plane
of the nerve at the level of the repair. These measurements demonstrated no significant
difference in nerve diameter between the two groups. However, there was a significant
reduction in scar thickness in the hydrogel group (P < 0.01, Student’s t-test).
Discussion
The use of a substance to “glue” nerve endings together is appealing for several reasons.
Microsurgical suture neurorrhaphy, currently the gold standard, is technically demanding,
time consuming, and traumatizes the nerve ends. Therefore, repairing nerves without
or at least with fewer sutures should be theoretically easier, faster, and, if truly
less traumatizing, then better. Substances currently being used in this capacity have
partially achieved this potential. Autologous blood clot was first applied to approximated
nerve ends in the 1940’s[[16 ]] but the use of commercially available fibrin glues, such as Tisseel, has emerged
as the most common nonsuture primary or augmenting method of nerve repair. This observation
is supported by Tisseel gluing techniques being regularly included in nerve textbooks
and international instructional courses [[17 ],[18 ],[19 ]]. Its effectiveness and ease has been documented in several animal nerve repair
models [[3 ],[4 ],[5 ],[6 ],[7 ],[8 ]] and clinical outcomes reports[[1 ],[2 ]]. Concerns, however, have persisted regarding ultimate repair strengths and scar
tissue proclivity [[9 ],[10 ],[11 ]]. Additionally, in this author’s opinion, preparation, application, and set up of
Tisseel can be cumbersome and, still, relatively time consuming. Because of concerns
about repair strength, a few sutures are often still used in the repair, which partially
negates advantages of technical ease and decreased nerve trauma.
Polyethylene glycol hydrogel, marketed as DuraSeal, was brought to our attention as
a possible nerve glue by a neurosurgeon colleague currently using the product in its
FDA approved capacity (and as the name implies) as a dural sealant. DuraSeal sets
up in about 2 seconds and is easy to apply as a consistent adhesive cocoon around
approximated nerve ends. Clinical and laboratory data has already demonstrated a lack
of neurotoxicity[[15 ],[20 ]]. Since, holding strength is a key prerequisite for any “glue”, this was our initial
focus. In a biomechanical comparison of the effects of augmenting a two suture epineural
repair of a median sized nerve, DuraSeal demonstrated equivalent holding strength
to Tisseel, though it should be noted that while both substances resisted gapping
neither significantly increased the ultimate strength of the repair[[13 ]].
These results were encouraging enough to prompt further investigation with a direct
in vivo comparison between DuraSeal and Tisseel. For the purposes of this initial
study, a standard rodent sciatic nerve repair model focusing on functional motor recovery
and scar tissue formation was utilized. Despite the well-known disadvantages associated
with the superior regenerative powers of the rodent, (which should be kept in mind
while interpreting the data), similar rodent models are routinely used in similar
preliminary investigations [[4 ],[9 ],[10 ],[11 ],[21 ],[22 ],[23 ],[24 ],[25 ]]. The repair was performed with two epineural sutures before applying either nerve
glue which reflects the senior author’s current clinical practice. PEG Hydrogel, like
fibrin glue, does not add significant holding strength to the repair but does decrease
gapping and is being used here only to augment the suture repair[[13 ]]. Likewise, since the goal of this study was to compare a novel “nerve glue” to
the gold standard “nerve glue” no comparison to suture only repairs was performed.
Multiple studies have compared “glue” and sutures with variable results [[4 ],[5 ],[6 ],[7 ],[8 ],[9 ],[11 ]], leaving us to conclude that both techniques are effective. Muscle contraction
was the primary outcome parameter focused on and the recorded force measurements were
compared as direct ratios of the experimental limb to the contra-lateral normal “control”
limb. This data demonstrated no difference between the fibrin glue and PEG hydrogel
groups and indirectly implied that either modality could result in acceptable motor
recovery. Mean generated force with a supramaximal stimulation of the sciatic nerve
was around 75%, which is similar to other nerve repair techniques[[26 ]]. Though nerve conduction studies and axonal counts would have been more informative
and helpful in a more extensive comparison, the equivalency of muscle contractions
between the two groups suggests at least effective motor axon regeneration in both
groups.
Measurement of repair site nerve diameter and collagen thickness was performed to
assess scar tissue formation and potential nerve compression as described by similar
reports making observations on nerve fibrosis[[10 ],[21 ]]. Though the nerves were not stretched, slides were prepared as consistently as
possible between the two groups with regards to avoiding undulation and comparing
equivalent samples though perfect standardization could not be guaranteed. Our findings
that nerve diameter was the same between the two groups suggests that PEG hydrogel
swelling, which has been reported[[27 ]], was not a problem. Masson’s Trichrome staining of collagen, however, did demonstrate
significantly more scar tissue around the repair site repaired with fibrin glue. Past
experience in exposing peripheral nerve tissue to new surgical adhesives have resulted
in some unexpected and catastrophic results prompting our interest in perineural scarring.
Both cyanoacrylate glue[[21 ]] and BioGlue (CryoLife, Inc., Kennesaw, GA) (bovine albumin and glutaraldehyde)[[28 ]] have been associated with neurotoxicity and marked fibrotic response around exposed
nerves. Additionally, one criticism of the use of fibrin glues in general is that
they may generate excessive scar tissue. Herter found that multiple components found
in fibrin glue all actually induced fibrosis[[10 ]]. PEG hydrogel, on the other hand, has been shown to inhibit adhesion formation
in a rabbit pericardial abrasion model[[29 ]], a canine durotomy repair model[[15 ]], and a porcine intra-abdominal adhesion model[[14 ]]. This potential advantage of PEG hydrogel “nerve glue” did not translate to superior
results in our rodent model.
Conclusion
Our preliminary comparison of fibrin sealant with Polyethylene Glycol hydrogel when
applied as an augmenting nerve glue in a rodent sciatic nerve repair model suggests
an equivalency in motor recovery. Though less scar tissue formation was associated
with PEG hydrogel the significance of this is not known. A more extensive comparison
of the two substances may be necessary before any definitive conclusion of superiority
can be drawn.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JI was responsible for conception and design, was intimately involved in experimental
surgeries, data collection, interpretation of data, and writing the manuscript.
IK performed significant portions of experimental surgeries, data collection, histological
preparation and interpretation, and was involved in writing of the manuscript.
CM performed significant portions of data collection.
