Neuropeptide Y innervation during fracture healing and remodeling. A study of angulated tibial fractures in the rat.

Abstract

Autonomic neuropeptide Y (NPY) is involved in local bone remodeling via the central nervous system. However, the role of peripheral neuronal NPY in fracture healing is not known. We investigated the relationship between bone healing and side-specific occurrence of NPY in angular and straight fractures.
Tibial fractures in Sprague-Dawley rats were fixed with intramedullary pins in straight alignment and anterior angulation. The samples were analyzed by radiography, histology, and immunohistochemistry (IHC) between 3 and 56 days postfracture.
In the angular fractures, radiography and histology showed a 3.5-fold increase in callus thickness on the concave side compared to the convex side at day 21, whereas a 0.2-fold reduction in callus thickness was seen on the convex side between days 21 and 56. IHC showed regenerating NPY fibers in the callus and woven bone in both fractures at day 7. In angular fractures, a 5-fold increase in NPY fibers was observed on the concave side compared to the convex side at 7 days, whereas a 6-fold increase in NPY fibers was seen on the convex side between 21 and 56 days; only a 0.1-fold increase in NPY fibers was seen on the concave side during the same time period. In straight fractures, similar bony and neuronal changes were observed on both sides.
The increase in NPY innervation on the convex side appears to correlate with the loss of callus thickness on the same side in angular fractures. Our results highlight the probable function of the peripheral NPY system in local bone remodeling.

Full text

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Acta Orthopaedica 2010; 81 (5): 639–646 639
Neuropeptide Y innervation during fracture healing and
remodeling
A study of angulated tibial fractures in the rat
Hua Long, Mahmood Ahmed, Paul Ackermann, André Stark, and Jian Li
Section of Orthopaedics, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
Corresponding author: jianli@ki.se
Submitted 09-02-20. Accepted 10-03-26
Open Access - This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use,
distribution, and reproduction in any medium, provided the source is credited.
DOI 10.3109/17453674.2010.504609
Background and purpose Autonomic neuropeptide Y (NPY) is
involved in local bone remodeling via the central nervous system.
However, the role of peripheral neuronal NPY in fracture healing
is not known. We investigated the relationship between bone heal-
ing and side-specific occurrence of NPY in angular and straight
fractures.
Methods Tibial fractures in Sprague-Dawley rats were fixed
with intramedullary pins in straight alignment and anterior
angulation. The samples were analyzed by radiography, histol-
ogy, and immunohistochemistry (IHC) between 3 and 56 days
postfracture.
Results In the angular fractures, radiography and histology
showed a 3.5-fold increase in callus thickness on the concave side
compared to the convex side at day 21, whereas a 0.2-fold reduc-
tion in callus thickness was seen on the convex side between days
21 and 56. IHC showed regenerating NPY fibers in the callus and
woven bone in both fractures at day 7. In angular fractures, a
5-fold increase in NPY fibers was observed on the concave side
compared to the convex side at 7 days, whereas a 6-fold increase
in NPY fibers was seen on the convex side between 21 and 56 days;
only a 0.1-fold increase in NPY fibers was seen on the concave side
during the same time period. In straight fractures, similar bony
and neuronal changes were observed on both sides.
Interpretation The increase in NPY innervation on the convex
side appears to correlate with the loss of callus thickness on the
same side in angular fractures. Our results highlight the probable
function of the peripheral NPY system in local bone remodeling.

The ability of bone to maintain its structures and proper-
ties or to remodel after trauma under different conditions is
well known, but the underlying neurobiological mechanisms
are poorly understood. The bone has a rich supply of nerve
fibers, which can be classified as belonging to the sensory,
autonomic, and opioid nervous systems according to the phe-
notype of the neurotransmitters (Bjurholm et al. 1988, Hill et
al. 1991). This strongly supports the peripheral role of neu-
ropeptides in bone physiology. Recently, the role of neural
signaling by the central nervous system in bone remodeling
through various pathways has been given some attention. One
such pathway is the neuropeptide Y (NPY) system. NPY, a
36 amino acid peptide, belongs to a class of peptides that
includes pancreatic polypeptide (PP) and peptide-YY (PYY),
which are expressed in the central and peripheral autonomic
nervous system. This system signals through 5 distinct recep-
tors (Y1, Y2, Y4, Y5, and Y6) expressed in various tissues.
NPY has several functions: among these are effects on energy
homeostasis, food intake, immunity, and the cardiovascular
system (Lin et al. 2004). Recently, an important role of NPY
in bone homeostasis has been identified through its effect
on Y2 and Y1 receptors: a study in mice with hypothalamic
deletion of Y2 receptors showed a generalized increase in
bone formation (Baldock et al. 2002). In contrast to this cen-
tral action of Y2 receptors, the presence of Y1 receptors on
osteoblasts suggests a direct local effect on these cells (Lun-
dberg et al. 2007). It has been shown that mice deficient in
Y1 receptors have a high bone mass (Baldock et al. 2007).
Whether it occurs through NPY-ergic pathways emanating
from the hypothalamus or through skeletal NPY innervation
has not been determined.
In the bone, NPY-immunoreactive fibers have been identi-
fied in areas of high osteogenic potential such as bone marrow
and the periosteum (Bjurholm et al. 1988, Hill et al. 1991).
These nerve fibers were found to be arranged as networks,
mainly around blood vessels. Experimental studies in vivo
have shown that NPY acts as a potent vasoconstrictor of blood
vessels in bone (Lindblad et al. 1994), but its role in local bone
turnover is not clear. Moreover, under conditions of trauma it
is not known whether NPY participates in different biological
events such as inflammation, cell proliferation, angiogenesis,
vasoregulation, and callus formation.

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Acta Orthopaedica 2010; 81 (5): 639–646
Recently, we reported a rat model of angulated tibial fracture
that showed side-specific changes in bone formation on the
concave side and bone resorption on the convex side of defor-
mities in the same bone (Li et al. 2004, 2007). The process
of bone formation/resorption in this animal model, leading to
bone healing and remodeling, might be affected by peripheral
expression of NPY because of its role in skeletal homeostasis.
We therefore assessed the temporal and side-specific expres-
sion of NPY at the fracture site during healing and remodeling
by morphological and semiquantitative analysis using immu-
nohistochemistry.
Materials and methods
44 male Sprague-Dawley rats with a mean weight of 200 g
were housed 3 per cage with free access to standard rat chow
and water under controlled temperature and a 12-hour light/
dark cycle. All animal experiments were performed with
approval from the Ethics Committee for Animal Research,
Stockholm North. The rats were allocated to 3 groups as
follows: 20 rats with a tibial fracture in anterior angulation
(group A); 20 rats with a tibial fracture in straight alignment
(group B); and 4 control rats with intact tibia (group C).
Surgery
The rats were anesthetized with an intraperitoneal (i.p.) injec-
tion of fentanyl-fluanisone (Hypnorm, 0.5 mL/kg). The right
tibia was first weakened at the mid-diaphysis by percutaneous
drilling with an 18-G needle. A small anteromedial skin inci-
sion was made proximal to the tibial tubercle and a 17-G can-
nula needle was inserted through the proximal metaphyseal
cortex into the medullary canal as described previously (Li
et al. 2004). The tibia was then fractured by manual 3-point
bending and fixed in either 40° anterior angulation or straight
alignment, which in both cases included the normal 13° ana-
tomical curve of the diaphysis.
Radiography
The progress of fracture healing was monitored on lateral
radiographs under Hypnorm anesthesia on day 0, 7, 21, 35,
and 56 after fracture (Figure 1A and B). The radiographic
set-up was adjusted to result in size enhancement of the image
by 33%. Callus size was then measured manually on a 300%
magnified printout. The total callus size was assessed by mea-
suring the transverse diameter (in mm) of the callus and the
thickness of cortical bone anterior and posterior to the nail at
the level of the apex of the angulated needle.
Sampling
The rats were anesthetized with sodium pentobarbitone (60
mg/kg, i.p.). 4 rats in each fracture group were killed on days
3, 7, 21, 35, and 56, while the 4 control rats with intact tibias
were killed on day 21. In vivo intra-aortic perfusion was car-
ried out with phosphate-buffered saline (PBS), followed by
fixation with Zamboni’s buffered 4% paraformaldehyde solu-
tion containing 0.2% picric acid in 0.2 M Sörensen phosphate
buffer, pH 7.2. The right tibia was dissected and then demin-
eralized at room temperature in a solution containing 7%
AlCl3, 5% formic acid, and 8.5% HCl for 12 h. The samples
were soaked in 20% sucrose for at least 2 days. Each demin-
eralized tibia was divided sagittally in two halves and 2-cm
long samples of the medial half of the tibial diaphysis, which
included the proximal, middle, and distal parts of the healing
fracture, were collected for analysis. Subsequently, the 2-cm
long samples were sectioned at a thickness of 15 µm using
a cryostat. Thus, the regions undergoing endochondral and
intramembranous ossification were all included in the same
section.
Immunohistochemistry
Serial sections were numbered consecutively from the middle
to the medial aspect. Two-interval sections, i.e. one close to
the middle part and another close to the medial part of the
tibia at each time point, were chosen for H & E staining and
for immunostaining according to the biotin-avidin system.
Thus, the sections were hydrated in 0.01 M PBS for 10 min
and then incubated with 10% normal goat serum at room tem-
perature for 30 min. Subsequently, the sections were incu-
bated at room temperature overnight in a humid atmosphere
with polyclonal antibody to NPY (1:5,000; Peninsula Labo-
ratories, San Carlos, CA). After rinsing in PBS (2 × 5 min),
Figure 1. Lateral view of tibia at day 0
and day 35 postfracture, fixed in straight
alignment (A) and anterior angulation
(B). The broken squares in the X-ray
images (panels A and B) depict the dis-
sected sample for microscopic analysis
of longitudinal sections shown in the
schematic illustration (C). The small
dotted squares in the drawing of the
dissected sample represent the 8 micro-
scopic fields studied in each tissue sec-
tion for morphological and semi-quanti-
tative analysis of NPY immunoreactivity.
D = day.

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Acta Orthopaedica 2010; 81 (5): 639–646 641
the sections were incubated with biotinylated goat anti-rabbit
antibody (1:250; Vector Laboratories, Burlingame, CA) for 40
min. Finally, fluorochrome Cy3-conjugated avidin (1:5,000;
Amersham) was used for visualization of the immune reac-
tion. Control staining was performed by omitting the primary
antibody. Addition of 50 µL of peptide to the correspond-
ing antiserum before application to tissue sections served as
another control.
For double staining, after completing the staining step for
NPY using fluorochrome Cy2-conjugated avidin for visualiza-
tion of the immunoreaction, the sections were incubated for 15
min with avidin-blocking solution followed by biotin-block-
ing solution. The sections were then incubated with goat anti-
mouse monoclonal antibody to GAP-43 (1:2,000), a marker
of axon growth, neuronal development, and nerve regenera-
tion, and mouse polyclonal antibody to PGP 9.5 (1:10,000;
both antibodies from Boehringer Mannheim Biochemicals,
Germany), the marker for mature nerve fibers. Then the sec-
tions were incubated with biotinylated anti-mouse antibodies
(1:250; Vector Laboratories) for 40 min. The fluorochrome
Cy2-conjugated avidin (1:1000; Amersham) was used for
visualization of the immunoreaction. An epifluorescence
microscope was used for analysis (20× objective).
Semiquantification
The nerve fiber density in and adjacent to the fracture area
during healing and modeling was quantified by computer-
ized image analysis as previously described (Li et al. 2007).
Briefly, 2 consecutive tissue sections per rat were included.
In each tissue section, 4 fields (20×) of each were selected in
a standardized manner from the concave and convex sides,
for comparison and quantification (Figure 1C). For quantifi-
cation by computerized image analysis, images of the tissue
sections were captured by a video camera attached to the
immunofluorescence microscope and then saved in a com-
puter. The fields selected were analyzed using Easy Analysis
software (Bergström Instruments, Stockholm, Sweden). A
standard lower and upper threshold of fluorescence intensity
was consistently applied for positively stained nerve fibers.
The results were expressed as the nerve fiber immunofluo-
rescent area in relation to the total area of each microscopic
field. Since there were 2 tissue sections in each rat, 8 micro-
scopic fields representing the convex side and 8 other fields
representing the concave side were analyzed. The mean value
(the percentage of neuronal immunofluorescenc area) based
on 8 microscopic fields was determined in 4 rats to obtain a
measure of fiber density for comparison of the concave and
convex sides of each tibia.
Statistics
For semiquantitative analysis of neuronal NPY-positive immu-
nofluorescence area, a non-parametric approach was used. In
each experimental group, the statistical analysis was based on
the mean of the values from 4 rats at each time point. Since our
data was skewed, the results are presented as mean, median,
and range (minimum and maximum). Mean values were used
to calculate the fold differences in NPY immunoreactivity
whereas median values were used to obtain the statistically
significant differences between the sides. To compare the dif-
ferences between the convex and concave sides within the
same group, the Wilcoxon test was used. A p-value of < 0.05
was considered statistically significant.
Results
Radiography
Radiographic analysis showed a statistically significant
increase in total callus formation from day 0 to day 21 post-
fracture (inflammatory and proliferative phases) in both frac-
ture groups. When compared to the bone thickness of each side
at day 0, the angular fracture exhibited a 3.5- fold increase in
callus thickness on the concave side, whereas only a 0.2-fold
increase in callus thickness was observed on the convex side at
day 21 (p = 0.03). During the remodeling phase, i.e. between
day 21 and day 56, a 0.2-fold reduction in callus thickness
was observed on the convex side (p = 0.05), while the callus
thickness remained almost the same on the concave side, as
compared to day 21. In the straight fracture, an almost equal
increase in callus thickness was observed on the concave side
(1.9-fold) and on the convex side (1.1-fold), reaching a peak
at day 21. It then decreased gradually, in a similar way on both
sides.
Histology
The longitudinal sections of tibia were stained with H & E at
7, 21, and 35 days postfracture in both fracture groups (Figure
2). In the straight fracture, a similar histological picture
depicting different phases of bone repair was seen on the two
sides of the fracture. Thus, there were equal amounts of new
woven bone and callus formation at day 7, complete thickened
cortical bridging at day 21, and reduction in cortical size with
normal bone architecture at day 35. However, in the angular
fracture a different histological picture was seen, with dispar-
ity in bone repair between the concave and the convex sides.
The increase in the size of new woven bone and callus forma-
tion was greater on the concave side than on the convex side
at day 7 (Figure 2A and B). At day 21, cortical bridging was
complete with periosteal hypertrophy and intense new bone
formation on the concave side, whereas these changes were
much less apparent on the convex side (Figure 2C and D). At
day 35, a further reduction in cortical thickness was observed
on the convex side whereas it remained the same on the con-
cave side, representing normal bone architecture (Figure 2E
and F). The morphological changes representing callus thick-
ness between the concave and convex sides were based on
observations in 4 rats at each time point (Figure 2A and F).

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Acta Orthopaedica 2010; 81 (5): 639–646
Distribution of NPY nerve fibers in fractures
NPY-positive nerve fibers were identified in the intact tibia
and at the fracture site in both straight and angular fractures at
all time points of the study.
In angulated fractures, ingrowth of nerve fibers containing
NPY was already observed at day 3 postfracture in the frac-
ture hematoma arranged as non-vascular nerve fibers (Figure
3A and B). At day 7, an increased number of NPY-positive
fibers was found on the concave side compared to the convex
side. These nerve fibers were seen as fibers sprouting into the
fibrocartilage callus from the deep layers of the periosteum.
Many fibers were seen close to the chondroid cells, and
Figure 2. Hematoxylin and eosin (H & E) staining (A-F) showing changes in bone thickness on the convex
(anterior) side (A, C, and E) and the concave (posterior) side (B, D, and F) of the angulated fracture at day 7
(A and B), 21 (C and D), and 35 (E and F) after fracture. The arrows depict the thickness of woven bone or
new bone, showing intensive bone formation on the concave side at early phases of fracture healing (days 7
and 21) and intense reduction of bone thickness on the convex side at a later phase of fracture healing (day
35). The small dotted square in each panel depicts the observed area of NPY immunostaining (20×) shown
specifically in Figures 4 and 5. 2× objective. Bar represents 1,000 μm. CA: cartilaginous callus; CO: cortical
bone; FG: fracture gap; NB: new bone; PE: periosteum; WB: woven bone.
Figure 3. Fluorescence photomicrographs showing site-specific occurrence of
NPY fibers on the convex (A and C) and concave (B and D) sides of angulation
fractures at days 3 (A and B) and 7 (C and D) after fracture. 20× objective. Bar
represents 50 μm. Arrows show NPY fibers. HE: hematoma; MU: muscle; PE:
periosteum; WB: woven bone.
Figure 4. Fluorescence photomicrographs showing site-specific occurrence of
NPY fibers on the convex (A and C) and concave (B and D) sides of angulation
fractures at days 21 (A and B) and 35 (C and D) after fracture. 20× objective.
Bar represents 50 μm. Arrows show NPY fibers. NB: new bone; MU: muscle;
PE: periosteum; VE: vessel; WB: woven bone.

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Acta Orthopaedica 2010; 81 (5): 639–646 643
they had entered the new-woven bone (Figure 3C and D).
Between days 21 and 35, NPY-positive fibers were mainly
observed as networks in and around the walls of blood ves-
sels, both in the deeper layers of hypotrophic periosteum and
in the new bone on the concave side, whereas few vascular
fibers entering into new bone were found on the convex side
(Figure 4A and B). At day 56 postfracture, during the remod-
eling, most of the NPY-positive fibers were seen retracted
from the fracture site and were present as vascular fibers in
the periosteum.
In straight fractures, the occurrence and distribution of
NPY-positive fibers over the time period was similar to that
seen in the angular fracture, with one notable difference. An
increased number of NPY-positive fibers with similar amount
were seen penetrating the woven bone on both sides of the
fracture at day 7. No side-specific differences in expression
were found over time.
Double-staining experiments showed that there was co-
expression of GAP 43, a marker of regenerating nerve fibers,
with NPY-positive fibers at the fracture site in both groups
at all time points. The co-localization of GAP 43 with NPY
was more intense between days 3 and 21 postfracture than that
at the later time periods (Figure 5A and C). Double-staining
studies of PGP 9.5, a neuronal marker of mature nerve fibers
and NPY showed a co-localization that was less obvious at
days 3–21, but which became more pronounced at days 35–56
postfracture (Figure 5B and D).
Semiquantitative analysis of NPY innervation
In general, there was a statistically significant increase in the
number of nerve fibers expressing NPY at the fracture site in
both straight and angular fractures at days 7, 21, and 35 post-
fracture as compared to intact tibia (Table).
In angular fractures, a 5-fold increase in NPY-immunore-
active fibers was seen on the concave side as compared to the
convex side when comparison was made with the innerva-
tion of intact bone at day 7. Notably, a statistically signifi-
cant (6-fold) increase in NPY-positive fibers was seen on the
convex side between days 21 and 56, whereas the correspond-
ing increase in NPY was only 0.1-fold on the concave side
during the same time period, when compared to day 7 (Table).
Interestingly, the peak in NPY-positive fibers was only seen
on the convex side in the angular fractures at days 21 and 35.
Although the amount of NPY-positive fibers was similar on
the concave and convex sides in the angular fractures between
days 21 and 56, an increase in NPY innervation on the convex
side was observed, but it was not statistically significant.
Figure 5. Fluorescence photomicrographs showing double staining of NPY and
GAP-43 (A and C) or PGP 9.5 (B and D) at the fracture site in angulation frac-
tures at days 7 (A and B) and 56 (C and D) after fracture. Co-localization of
NPY (red) with GAP-43 (green) and with PGP 9.5 (green), resulting in yellow
color, as seen in the woven bone and hypertrophic periosteum (A) and con-
nective tissue (B) at day 7 and vessels (C and D) at day 56. 20× objective. Bar
represents 50 μm. Arrows show co-localization of NPY with GAP-43 or with
PGP 9.5. CN: connective tissue; MU: muscle; PE: periosteum; VE: vessel; WB:
woven bone.
Changes of NPY immunoreactivity in straight and angulated fractures


Day
Straight fracture Angulated fracture
Posterior side
B
Anterior side
B
Posterior (concave) side
A B
Anterior (convex) side
A B A C A C D C C D
3
7
21
35
56
–0.07 –0.15 –0.28–0.29
0.45 0.39 0.09–0.92
0.44 0.44 –0.04–0.91
0.85 0.54 0.08–2.22
0.01 0.06 –0.40–0.34
–0.17 –0.26 –0.33–0.17
0.22 0.10 –0.16–0.85
0.60 0.45 0.17–1.33
0.70 0.39 0.07–1.97
–0.07 –0.21 –0.57–0.69
0.1
0.5
0.7
0.1
0.5
0.11 –0.16 –0.47–1.21
0.50 0.53 0.35–0.61
0.67 0.67 0.67–0.67
0.52 0.45 0.21–0.98
0.16 0.15 0.06–0.29
–0.18 –0.38 –0.60–0.70
–0.51 –0.53 –0.62 to –0.35
0.86 0.66 0.54–1.39
0.75 0.67 0.48–1.18
0.04 0.06 –0.06–0.10
0.4
0.04
0.9
0.8
0.1
The percentage changes of NPY immunoreactivity in both straight fracture and angulated fracture at each time point as compared to intact (control) bone.
A Mean
B Median
C Range
D P-value

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In the straight fractures, a statistically significant increase
in NPY-positive fibers was found both on the concave side
(by a factor of 0.6) and on the convex side (by a factor of
0.4) when compared to intact tibia at day 7. The maximum
increase in NPY-positive fibers (by a factor of 1) was observed
on both sides in the straight fractures at day 35, the phase of
bone remodeling. Later, the density of NPY fibers returned to
the normal (intact) level on both sides of the fracture (Table).
Discussion
Our study demonstrates temporal and side-specific changes in
NPY innervation during the early and the late phase of angular
fracture healing. The early increase in NPY innervation seen
on the bone-forming concave side during the inflammatory
phase may be a response to injury with the possible stimula-
tory effects on cell proliferation and angiogenesis leading to
enhanced bone formation. The increased expression of NPY
seen on the convex (bone resorption) side during the remod-
eling phase appears to coincide with the reduction in callus
thickness. The later increase in NPY expression on the convex
side may reflect the role of NPY in bone resorption, possibly
through the Y1 receptor. Given the proposed central role of
NPY in bone homeostasis, our results suggest that NPY may
have a peripheral neuronal function in local bone turnover,
especially during fracture healing and correction of angular
deformity.
The early inflammatory phase of fracture healing lays the
foundation for bone healing as inflammatory cells secrete a
number of proinflammatory cytokines with bone induction
properties. Accordingly, the cascade of reparative processes
such as matrix synthesis, cell proliferation, angiogenesis, and
neurogenesis is induced. Our results show an intense but simi-
lar pattern of nerve regeneration containing autonomic neu-
ropeptide NPY in the fracture callus and woven bone on the
two sides of a straight fracture (Figures 3 and 5). However,
in an angular fracture this phenomenon of nerve regenera-
tion was observed mainly on the concave side (Figure 3B and
D) (Table). The regenerative nature of NPY immunoreactive
fibers observed in the inflammatory phase of fracture healing
was confirmed by identification of co-expression of GAP-43,
a neuronal marker of nerve regeneration, in the NPY-positive
fibers (Figure 5). It is presumed that increased delivery of
NPY from the nerve endings during the inflammatory phase
of fracture healing would exert a beneficial effect on bone
repair by modulating various cellular events. A number of
studies have suggested that NPY exerts a strong angiogenic
effect, mainly on the Y2 receptor (Y2R) (Ekstrand et al. 2003,
Lee et al. 2003). In vitro studies have shown that NPY pro-
motes vessel sprouting and adhesion, migration, prolifera-
tion, and capillary tube formation in human endothelial cells
(Zukowska-Grojec et al. 1998, Movafagh et al. 2006). Fur-
thermore, it has been shown that vessel sprouting is reduced
to 50% after deleting Y2R or by using antagonists of Y2R
(Lee et al. 2003). In addition, a delay in skin wound healing
and reduced neovascularization was observed in mice lacking
Y2R (Ekstrand et al. 2003). It is known that early angiogen-
esis at the fracture site is one of the most important parameters
influencing the healing process (Keramaris et al. 2008). Our
study has shown early proliferation of nerve fibers containing
NPY at the fracture site where increased callus formation is
observed (concave side in angular fractures and on both sides
in straight fractures), which possibly contributes to the heal-
ing process by stimulating angiogenesis (Figure 3 and 5). In
addition to angiogenesis, recent studies have shown a possible
role of NPY in neurogenesis. A potential role of NPY in the
survival and the regenerative process of neurons was indicated
by the dramatic increase in NPY expression in dorsal root
ganglia after peripheral nerve injury (Wakisaka et al. 1991).
Moreover, a possible proliferative effect of NPY on neuro-
blasts in different areas of the neonatal and adult brain has
been shown to be mediated mainly through Y1R (Howell et al.
2003, 2005). In addition to the CNS, NPY has also been iden-
tified as a neurotrophic factor in the peripheral nervous system
by showing its regenerative effect on olfactory epithelium
(Hansel et al. 2001, Hökfelt et al. 2008). It has been shown
that NPY acting through Y1R stimulates the proliferation of
adult olfactory neuroblasts. Conversely, NPY-deficient mice
were found to show a significant reduction in olfactory neuro-
nal precursor cell proliferation (Hansel et al. 2001). Recently,
it has been shown that increased release of NPY promotes the
regeneration of damaged olfactory sensory neurons (Kanekar
et al. 2009). Altogether, the role of NPY implicated in neu-
roangiogenesis might set up the initial framework for a satis-
factory bone-healing process.
We have recently established a rat model of angular frac-
ture of the tibia, which covers not only the healing process
but also the subsequent bone remodeling (Li et al. 2004,
2007). In these studies, angulation gave a clear-cut difference
in local bone turnover between the concave and convex sides
of the fracture. We also found intense bone formation on the
concave side whereas bone resorption predominated on the
convex side, both sides thereby contributing to spontaneous
correction of the angular deformity (Figure 1 and 2). Notably,
our angular fracture model also permits sequential analysis
of local mediators involved in bone formation and resorp-
tion within well-defined areas of the same bone at different
stages of fracture healing. Thus, we have observed increased
expression of the sensory neuropeptide CGRP on the concave
side, where intense new bone formation occurred. On the
convex side where bone resorption predominated, there was
an increase in the occurrence of the bone resorption cytokine
interleukin-1 (Li et al. 2004, 2007), as confirmed by others
(Gordon et al. 2008).
In the present study a reduction in callus thickness during
the remodeling phase which coincided with a 6-fold increase
in NPY expression on the convex side, probably highlights

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Acta Orthopaedica 2010; 81 (5): 639–646 645
the role of NPY in bone resorption. Even though there is no
conclusive evidence to implicate NPY-mediated signaling
in bone homeostasis, a number of studies have indicated the
probable role of NPY in local bone turnover through the cen-
tral and peripheral nervous system (Baldock et al. 2002, 2007,
Lundberg et al. 2007). Recent studies analyzing germ line or
conditional knockout mice lacking Y2R have revealed that the
osteoblast activity is controlled by a central pathway through
hypothalamus. These studies have shown an increased rate of
bone mineralization and elevated bone mass due to increased
osteoblast activity in Y2R knockout mice (Lundberg et al.
2007). However, the presence of neuronal NPY in high osteo-
genic areas such as the periosteum, epiphysis growth plate,
and bone marrow may suggest a role of NPY in local bone
turnover by a peripheral pathway (Bjurholm et al. 1988, Hill
and Elde 1991). Recently, the Y1 receptor was reported to be
present in human osteoblastic and osteosarcoma-derived cell
lines (Lundberg et al. 2007), and treatment of wild-type mice
with NPY caused reduction in bone mass and volume (Ducy
et al. 2000). In addition, in vitro studies have demonstrated
that NPY can inhibit the osteoblastic parathyroid hormone
response through a receptor-receptor interaction (Bjurholm et
al. 1992). A more direct effect of NPY on bone cells involved
in bone remodeling has been reported. Not only the presence
of Y1R on osteoblasts but also a generalized increase in bone
formation on the cortical and cancellous surfaces of bone has
been shown in Y1R-deleted mice (Lundberg et al. 2007). It
can be assumed that the elevated levels of NPY seen in our
study caused an overstimulation of Y1R on osteoblasts, lead-
ing to bone resorption on the convex side of the fracture.
In summary, we have demonstrated upregulation in neu-
ronal expression of NPY during the early and late phases of
angular fracture healing, thus highlighting a probable role
of NPY in local bone remodeling. The findings of this study
combined with those in previous reports suggest that NPY
may play a role in the regulation of skeletal homeostasis,
probably through Y1R. Therapeutic application of the NPY
system has emerged as a promising area of research in clini-
cal orthopedics. A useful approach in this regard would be the
anti-ligand/anti-receptor strategy to prevent or minimize bone
loss in a number of skeletal disorders such as tumor, trauma,
and infection.
HL and JL contributed equally to the study (shared first authorship). They
performed the experiments, analyzed the data, and wrote the manuscript. MA,
PA, and AS participated in data analysis, discussion, and manuscript prepara-
tion. All five authors took part in interpretation of the results and in prepara-
tion of the final version of the paper.
This study was supported by grants from the AO/ASIF Foundation (99-K31)
and the Swedish Medical Research Council (13107).
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