International Journal of Clinical Medicine, 2011, 2, 301-306
doi:10.4236/ijcm.2011.23051 Published Online July 2011 (http://www.SciRP.org/journal/ijcm)
301
Fracture Healing in a Denervation and/or Nerve
Ending Interpositioning Model in the Rat
Cagri Yegengil1, Mahmut Pekedis2, Hasan Yildiz2
1Bozyaka Training and Research Hospital, Orthopedics and Traumatology Clinic, Izmir, Turkey; 2Ege University, Engineering Fac-
ulty, Mechanical Engineering Department, Izmir, Turkey.
Received April 14th, 2011; revised May 17th, 2011; accepted July 3rd, 2011.
ABSTRACT
Background: In this experimental study, we aimed to determine the possib le changes in fracture healing du e to dener-
vation and/or nerve ending interpositioning. Methods: 50 Wistar Albino type male rats were divided into three study
groups. A standard transverse diaphysial fracture in the femurs of the same side of all subjects under anesthesia was
created and the fracture was fixed intramedullarily. Wh ile preserving the structural integ rity of the sciatic nerve in the
first group, neurectomy to the nerve in the second group was performed. In the third group, following the sciatic nerve
cut, the proximal end of the nerve were interposed the fracture line. After a 28-day observational period, the callus
formation in the subjects was examined radiologically, biomechanically and histopathologically. Results: Among all
groups, the third group subjects showed significant increase in radiological area measurements when they are com-
pared to the second g roup rats. Th ere was no sign ificant differen ce in biomechanical measuremen ts of fractu red femurs
of the three groups. In histopathological evaluations, it was observed that denervation had increased the thickness of
the cartilage an d the number of the chondrocytes and os teocla sts significantly but decreased the numb er of fibroblasts
compared to the control group. In addition to the denervation nerve ending interpositioning increased the bone thick-
ness and the number of the osteoblasts but decreased the number of the osteoclasts significantly. Conclusions: While
radiological observations exhibit that nerve ending interpositioning has resulted more hypertrophic callus formation,
histopathological evaluations led us to that denervation created partial (immature) callus formation and nerve ending
interpositioning demon strated larger but immature callus formation.
Keywords: Denervation, Nerve Ending Interposition, Fracture Healing, Callus Format ion
1. Introduction
Fracture healing is defined as a complex problem that
includes coordination of different processes in the lit-
erature [1]. Existence of neural formation in bone tissue
was first documented in 1545 [2]. Neural control of new
bone formation hypothesis extends to 1930 s [3]. The
neural tissue was thought only in periost, therefore neu-
ral tissue of bone is generally disregarded. Sudeck atro-
phia related to chronic regional pain syndrome, hetero-
typical ossification that is observed in patients with head
trauma and Charcot diabetic neuroarthropaty point out
that an interaction between nervous system and mus-
cle-skeleton system should exist [2]. Rapid joining with
plenty of callus formation in patients with neurological
damage such as head trauma, traumatic paraplegia etc.
and reduced ossification in some other neurological pa-
thologies such as Charcot disease were observed. Long
bones have a rich innervation by myelinated and non
myelinated nerve fibers. Functions and endings of these
nerve fibers were still not known, while blood flow
regulative roles of sympathetic nerves are known [4].
Denervation is one of the factors that effect fracture
healing. Concordantly, experimental studies related to
effects of denervation on fracture healing process exist
in the literature. One of these several denervation mod-
els that were used in these studies is neurectomy. Exis-
tence of neurological tissue and its effect on callus were
explained by methods devoted to immunohistochemical
analysis of some neuromarkers such as growth-associ-
ated protein 43 (GAP-43) and protein gene product 9.5
(PGP-9.5) that points existence of nerve tissue in exam-
ined fractural callus by forming denervation. A reinner-
vation without denervation after fracture which was
formed experimentally was given by Li et al. [5]. De-
spite the existence of sympathetic and sensorial nerve

Fracture Healing in a Denervation and/or Nerve Ending Interpositioning Model in the Rat
302
fibers in the periost and bone; and their specific recep-
tors, effect mechanism to callus formation and matura-
tion is not clear [4].
2. Materials and Methods
2.1. Study Groups
In the present study, 45 Wistar-Albino rats that were 12
- 14 weeks old with average weight of 299 grams were
used. Ethical Board approval was obtained from Ege
University Animal Ethics Board for this study. 45 rats
were divided into three groups. Standard transverse
fractures were created in right femur diaphyse that were
determined intramedullary for all groups. As difference,
in the first group integrity of nervous sciatica was pre-
served. 15 millimeters of resection was applied to nerv-
ous sciatica in the second group. In the third group, the
proximal part of the nerve was stabilized to fracture line
from free nerve end by applying sharp injury model with
a complete transvers cut to nervous sciatica in fracture
line level, while the distal part of the nerve was excised
widely (Figure 1).
2.2. Surgical Procedure
Anesthesia was applied to test subjects by administrating
Ketalar (Eczacıbaşı Warner Lambert) which includes
100 mg Ketamine Hydrochloride active substance as
100 mg/kg and Xylazine (Rompun Bayer İstanbul) as 10
mg/kg subcutaneously. A single dose of Ampisina 50
mg/kg was administrated as antibiotic prophylaxis. Long
axis of femur was entered by parallel skin incision from
external side of right thighbone of test subjects and
nervous sciatica was reached by cutting the gluteal mus-
cle parallel to the first incision. After that, femur
diaphysis was exposed by separating from adjacent
muscle tissues with dissection. Kirschner wire of 1 mm
thickness was sent from proximal of the femur intrame-
dullary via electric drill and the upper part of the bone
was cut such that 1 - 2 mm of wire came out of the bone.
Transverse diaphysis fracture was formed via a bone
forceps in femur diaphysis. Fascia and skin in all test
subjects were closed as 2 layers; in the first study group,
Figure 1. Study groups (nervous sciatica and femur are
shown with light and dark color respectively).
structural integrity of nervous sciatica was preserved. In
the second group, denervation was aimed by applying 15
mm of resection. In the third group, sharp injury model
was applied with #15 lancets from approximately 5 mm
distal part of the fracture line to prevent high tension on
nervous sciatica. After then, upper end of the nerve was
stabilized from epineureum with 10/0 nylon stitch mate-
rial (Figure 2). Lower part of the nerve was excised at
the level as the lowest level as possible.
Test subjects were allowed to move freely in wire
cages. Operated extremities of subjects were followed in
terms of sufficient stabilization and infection. It was
observed that test subjects used the other three extremi-
ties and protected the operated extremity as expected.
After four weeks, all test subjects were sacrified by ad-
ministrating high dose of Pentotal (Pantobarbutal 50
mg/kg) intraperitoneally. Both femurs of all test subjects
were removed by protecting the soft tissues at 1/3 mid-
dle part of femur diaphysis. Kirschner wires of all test
subjects were removed.
2.3. Statistical Evaluation
The statistical analysis of the results was done on “SPSS
15.0” statistics package program with a precision of
95%. During the statistic analysis of data, Wilcoxon
Signed Ranks, Mann-Whitney ‘U’, Spearman’s correla-
tion methods were used.
2.4. Radiological Evaluation
A-P direct pictures of all femur samples were taken on
the day of scarification. Imaging process was performed
with high resolution digital radiography system. The ob-
tained images were evaluated in “Image Viewer R 10.2”
software of PACS product of Philips Medical Systems
company as C: 8600 (contract), W: 9440 (brightness)
with their standard values digitally. During the evulation
of images, methaphisodiaphiser segment areas of the
Figure 2. Interposition of proximal end of nervous sciatica
to the fracture line (arrow).

Fracture Healing in a Denervation and/or Nerve Ending Interpositioning Model in the Rat 303
right femurs that callus formation was observed and the
left femurs that callus formation was not observed were
calculated. To determine the isolated diaphysis area of
the femur, vertical lines were drawn from trochanter mi-
nor level in upper end of femur and from joining level of
femur condyles with diaphysis to the longitudinal axis of
the femur and the area between these lines were calcu-
lated. With this technique, area increase that callus tissue
made to the diaphysis was observed (Figure 3).
2.5. Histopathological Evaluation
After the direct graphs were taken, the right femurs of
eight test subjects from the first and the third group and
seven test subjects from the second group were sepa-
rated for histopathological evaluation. Because two of
the samples from the first group were damaged during
the transportation, the histopathological evaluation of
this group was performed on six samples. Numbered
samples were fixed in the room temperature with 10%
formal solution. Following this process they were decal-
cified in the room temperature with 10% formic acid for
a week, with 48 - 72 hours intervals. After the decalcifi-
cation, 2 - 3 mm tissue sections were taken and blocked
into the paraffin. 6 mm longitudinal and transversal sec-
tions were taken via a microtome and coloration was
performed with HE (haematoxylon-eosin) and alkaline
blue. After this process, 6 to 8 sections were examined
in light microscope with 20×, 40×, 100× and 400×. Cell
count in per unit area from the most cellular regions of
samples (osteocyte, osteoblast, osteoclast, fibroblast,
chondrocyte) and bone and cartilage measurement of the
callus were performed (Figure 4). Obtained data were
evaluated for the groups with Mann-Whitney U test.
Figure 3. Isolated femur diaphysis areas of the number 16
number test subject of group 2.
(a) (b)
(c) (d)
Figure 4. Bone cells in the unit area (a) osteocytes (blue
arrows); (b) osteoblasts and osteoclasts (blue arrows show
osteoblast cell alignment, yellow arrows show multinuclear
osteclasts); (c) fibroblasts; (d) chondrocytes.
2.6. Biomechanical Evaluation
The right femurs where callus formation was observed
and opposite side healthy femurs of eight samples from
the first and the second group; seven samples from the
third group were separated for biomechanical evaluation.
Then, all femurs were embedded in to bone cement in
vertical direction via a goniometric ruler. In the study,
SHIMADZU Autograph AG-I 10kN was used as loading
device. The axial load is applied to the specimens at a
speed of 1 millimeter per minute (1 mm/min). The result
of measurements was recorded to a computer as force
(mN)/displacement (mm) data. Their hardness, elasticity
and energy storing capacities of specimens were com-
puted by using force-displecement diagrams.
3. Results
3.1. Radiological Evaluation
In the right femurs of all groups, callus tissue formation
was observed at the end of the fourth week. The bridg-
ing of fracture openings in all groups were observed
radiologically as periosteal bridging shaped (external
callus). While there was no meaningful difference be-
tween the first and the second groups, and the first and
the third groups during the comparison of isolated
diaphysis fields in the right femurs which showed callus
formation, there was a statistically meaningful differ-
ence between the second and the third groups (Figure
5).
3.2. Biomechanical Evaluation
There was no statistically meaningful difference be-
tween comparisons of fractured bones of three study

Fracture Healing in a Denervation and/or Nerve Ending Interpositioning Model in the Rat
304
Figure 5. Average areas of isolated femur diaphysis.
groups in terms of studied parameters: elasticity, hard-
ness and energy storing ability.
3.3. Histopathological Evaluation
While callus formation of different degrees and phases
was observed in all of the study groups, generally
smaller and more developed stage of callus formation
was observed in the first group (Figure 6), and more
hypertrophic and earlier staged callus formation was
observed in the second and the third groups.
In the group comparisons of callus bone and cartilage
parameters (osteocyte, osteoblast, osteoclast, fibroblast,
chondrocyte) with the method of cell count per millime-
ter square, it was observed that denervation increased
the cartilage thickness, the chondrocyte number and the
osteoclast number considerably (pct = 0.033, pcn =
0.018, pon = 0.024 respectively), and decreased the
number of fibroblast (p = 0.042) depending on the con-
trol group. In the denerved group, there was also an in-
crease in the number of osteocyte and bone thickness,
even though it was not considerably so (11.06% and
26.25% respectively). It was also observed that the nerve
ending interposition in the fractured callus considerably
increased the bone thickness compared to the first group,
Figure 6. Apparent trabecular bone formation regions in
group 1 (arrows).
0.3
0.32
0.39
0.4 0.4
0.53
0
0.5
Cartilage
thickness
(mm)
Bone
thickness
(mm)
Group 1Group 2Group 3
Figure 7. The bone-cement thickness comparisons.
and increased the number of osteoblast compared to the
first and the second group (p = 0.045, p = 0.034 and p =
0.004 respectively). It also decreased the number of os-
teoclast compared to the second group (p = 0.016).
Other histopathological parameters were similar to den-
ervation group (Figures 7 and 8).
4. Discussion
First of all, the formation of fracture on femur following
the nervous sciatica neurectomy leads us to believe that
the model can not denerve the bone completely. Fol-
lowing nervous sciatica neurectomy of rats, Frymoyer
and Pope and Hukkanen et al. formed fracture in fibula
and tibia respectively [6,7]. On the other hand Madsen et
al. created fracture in tibia after sciatic and femoral
nervous neurectomy [8]. As a quantifier indicator show-
ing how much neurectomy denerved the relative fracture,
Hukkanen et al. performed neuromarker analysis on
callus with immunohistochemical methods [7]. Owing to
26.5
25
10.5
46.83
25.83
29.43
22.14
19.29
37.29
42.57
25.63
34.38
7.88
39.25
35.63
0
10
20
30
40
50
Osteocyte
Osteoblast
Osteoclast
Fibroblast
Chondrocyte
Group 1Group 2Group 3
Figure 8. The number of cells comparisons.

Fracture Healing in a Denervation and/or Nerve Ending Interpositioning Model in the Rat 305
their findings, tibial fracture callus is not solely den-
erved by nervous sciatica, but it can also be innervated
by other sources. For this reason, in the following study,
Madsen et al. performed the neurectomy in both the
nervous sciatica and femoral nerve [8]. The analysis of
the callus with immunohistochemical methods in terms
of neural tissue may clarify (as in more or less) the den-
ervation amount in this study.
How do the denervation models in rats affect the
healing of fracture?
When the fracture healing is evaluated in radiography
and physical examination findings in clinics, quantifier
evaluation may be difficult [9]. It is observed that this
evaluation is done with radiographic, histopathologic,
densitometric and biomechanical measurements on rat
femurs [4,6-8,10,11]. Even though nervous sciatica den-
ervation was bigger, flat and calcific looking, Aro et al.
observed that in rat tibia fractures which are created as
intramedullary lead to a formation of callus which ex-
hibits more irregular density [4]. In the control groups,
callus was oval shaped and showed more regular density
distribution. Yüce et al. discovered radiologically that
the fibula fractured fragments of rats which had sciatic
denervation were combined on the 28th day and while a
minimal opening had found in the control group 10. In
their evaluation which was made with radiological scor-
ing system, Hukkanen et al. and Madsen et al. discov-
ered there was primer fracture healing in normal innerve
fractures, and there was secondary healing in denerve
cases which is characterized by large callus formation
[7,8]. In the first group, considering the comparisons of
fractured bones of the radiological findings at denerva-
tion, no change was discovered which is similar to the
literature.
During the healing of fibula fractures of fibula frac-
tures of rats whose nervous sciatica was denerved, Fry-
moyer snd Pope found an increase in biomechanical
properties such an elasticity, energy storing and hard-
ness on the 15th and 20th days [6]. During the earlier
stages of fracture healing (first 15 days), Aro et al.
found that the maximum fracture force increased in
denerve group 4. Hukkanen et al. and Madsen et al.
found that maximum bending moment, energy storing
capacity and hardness values in denerve group were
somehow lessened on the 35th day when compared to
control group [7,8]. In this study, the fact that no mean-
ingful difference was observed between fractured and
intact bone biomechanically resulting denervation and
nerve end interposition model are compatible with the
literature. This may be due to the fact that biomechanical
evaluation in this study is done with axial lading test as
opposed to fracturing from three points test in previous
studies.
The histopathological evaluation results of the dener-
vation group, considering the stages of the fracture
healing, point to the fact that denervation causes a delay
in callus formation process. In the control group, a callus
formation was observed and it is smaller than that of
denervation and nerve ending interposition groups, but it
was found to be is in more devoloped stage. In the heal-
ing of fibula fractures of rats whose nervous sciatica was
denerved, Frymoyer and Pope observed histological
increase [6] on the 15th and the 20th days (using a scoring
system which consists of hematom, callus, joining and
compact bone formation measurements). Yüce et al.
discovered lamellar bone formation and osteoclasts on
the 28th day histologically, and on control side, an earlier
stage of callus formation where the fibrosis and cartilage
tissues were dominant [10]. In their study concerning the
development of sensory innervations in rat tibias, Gajda
et al. observed an increase in osteoclastic activity in
denerved bones [12].
How does the performing of nervous sciatic denerva-
tion with nerve ending interposition in rats affect the
fracture healing in femur?
No experimental models concerning the interposition
of nerve end to fracture line were encountered in the
literature. The fact that a more hypertrophic callus for-
mation was observed in the nerve end interposition of
this study radiologically when compared to denervation
may be secondary to various physical and/or chemical
stimulus in the fracture line of the nerve. Since neither
immunohistochemical nor molecular analysis was done
about this subject, no further comments can be made. In
the histopathological evaluation, it can be observed that
nerve end interposition leads to a bigger but immature
callus formation when compared to denervation.
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