Fracture Healing in a Denervation and/or Nerve Ending Interpositioning Model in the Rat

doi:10.4236/ijcm.2011.23051

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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.

Email: [email protected]

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)

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

0.53

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

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

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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