Preponderance of Sensory Versus Sympathetic Nerve Fibers

and Increased Cellularity in the Infrapatellar Fat Pad in

Anterior Knee Pain Patients after Primary Arthroplasty
Birgit Lehner,2 Franz X. Koeck,1 Silvia Capellino,2 Thomas E.O. Schubert,3 Raphael Hofbauer,1 Rainer H. Straub2
1
Department of Orthopedic Surgery, University Hospital Regensburg, Regensburg, Germany
2
Laboratory of Exp. Rheumatology and Neuroendocrino-Immunology, Department of Internal Medicine I,
University Hospital Regensburg, 93042 Regensburg, Germany
3
Department of Pathology, University Hospital Regensburg, Regensburg, Germany

Received 17 January 2007; accepted 4 July 2007

Published online 27 September 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20498

ABSTRACT: Sensory nerve fibers transmit pain perception and secrete pro-inflammatory substance
P (SP). Sympathetic nerve fibers secrete anti-inflammatory norepinephrine and endogenous opioids,
which inhibit pain perception in a bidirectional crosstalk with sensory fibers. In patients with
anterior knee pain after primary arthroplasty of the knee (AKP), this study investigated in parallel
the innervation of the infrapatellar fat pad by sensory and sympathetic nerve fibers. A total of 32
patients with osteoarthritis (OA) of the knee (n ¼ 10), AKP after primary knee joint replacement
(n ¼ 7), and OA of the hip (n ¼ 15) were included. Sensory nerve fibers were semiquantitatively
detected by immunohistochemistry against SP, and sympathetic nerve fibers were stained with an
antibody against tyrosine hydroxylase. Cellular density of the tissue was investigated by counting
cell nuclei. The density of sympathetic nerve fibers in the fat tissue was similar in knee OA as
compared to AKP. In the fat tissue, density of sensory substance P–positive nerve fibers was higher
in AKP than in knee OA, which was not observed in the fibrosis capsule of the fat pad. The
preponderance of sensory over sympathetic nerve fibers was accompanied by an increased cellular
density in fat tissue in patients with AKP compared to knee OA. A positive correlation existed
between cellularity and sensory nerve fiber density in fat tissue. This study revealed a
preponderance of sensory over sympathetic innervation in the infrapatellar fat pad in AKP after
primary arthroplasty of the knee, which possibly leads to aggravation and continuation of AKP and
local inflammation. ß 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
J Orthop Res 26:342–350, 2008
Keywords: anterior knee pain; sympathetic nerve fibers; sensory nerve fibers;
substance P; osteoarthritis

INTRODUCTION It has been demonstrated that neuronal dys-

The vast majority of cases of anterior knee pain
(AKP) are caused by trauma superimposed on
predisposing anatomical malalignment.1 In approxi-
mately 5% of patients with AKP,1 the pain is
referred from a source outside the knee, for
example, the infrapatellar fat pad (Hoffa’s fat
pad2). These extra-articular reasons for AKP can
include aspects of the nervous system, which is
subject of this present study.
Birgit Lehner and Franz X. Koeck contributed equally to
this work.
Correspondence to: Rainer H. Straub (Telephone: 49-941-
944-7120; Fax: 49-941-944-7121;
E-mail: rainer.straub@klinik.uni-regensburg.de)
ß 2007 Orthopaedic Research Society. Published by Wiley Periodicals,
Inc.
function with reduced coordination of motor units
between the medial and lateral vastus muscles play
a role in AKP.3 Knee joint position sense is reduced
in AKP,4 and saline injection into the infrapatellar
fat pad caused AKP in healthy subjects.5 Peripa-
tellar soft tissues are in close contact with the
patellofemoral joint and are perfectly innervated.6
The occurrence and distribution of sensory sub-
stance P (SP)–positive nerve fibers inside the
infrapatellar fat pad suggest a nociceptive function
in AKP.7 Witonski and colleagues found high
numbers of SP-positive nerve fibers in patients
with chronic AKP in the infrapatellar fat pad.8 One
study demonstrated that patients with AKP benefit
from electroacupuncture treatment, which indi-
cates that central pain inhibition by afferent
stimulation is a plausible explanation.9 In 1998,

342 JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2008

 INCREASED SENSORY OVER SYMPATHETIC NERVES IN AKP
Scott Dye performed arthroscopy of his own knee
without intra-articular anesthesia and reported
the infrapatellar fat pad to be a highly sensitive
structure.10
The main neuropeptide of sensory afferents in
the infrapatellar fat pad is SP, which is a pro-
inflammatory neuropeptide. For example, SP stimu-
lates interleukin(IL)-1,11 tumor necrosis factor
(TNF),11 prostaglandin E2,12 NF-kB,13 and super-
oxide anion production14 from various cell types. Its
pro-inflammatory role in acute arthritis has been
demonstrated earlier.15 In addition, SP has a
strong effect on fibroblast activation and extrac-
ellular matrix production leading to increased
fibrogenesis.16–20 We recently demonstrated
sprouting of SP-positive nerve fibers in two dis-
eases with exaggerated fibrogenesis, Achilles ten-
dinosis and Dupuytren’s contracture.21,22 Thus, SP
might be an important factor for the aggravation
and continuation of AKP.
In contrast to SP, neurotransmitters of the sym-
pathetic nerve endings can have anti-inflammatory
effects at high neurotransmitter concentrations.
Norepinephrine binds preferentially a-adrenoceptors
(at high physiological concentrations it also
binds b-adrenoceptors). Adenosine preferentially
binds adenosine 1 (A1) receptors (at high physio-
logical concentrations it also binds adenosine
2 (A2) receptors; reviewed in Ref.23). Ligation of b-
adrenoceptors or A2 adenosine receptors increase
intracellular cAMP levels and ligation of a2-
adrenoceptors or A1 adenosine receptors decrease
intracellular cAMP levels (reviewed in Ref. 23).
Generally, an elevated sympathetic tone due to
increased firing rates at sympathetic nerves results
in increased levels of norepinephrine and adeno-
sine (after conversion from ATP) in the vicinity of
the nerve terminal. This leads to an increase of
intracellular cAMP in multiple peripheral target
cells. Elevation of cAMP by these mechanisms has
been repeatedly demonstrated to induce many
anti-inflammatory effects on target immune mech-
anisms such as secretion of TNF or interferon g
(reviewed in Ref. 23). Thus, presence of sympa-
thetic nerves at a high fiber density would yield a
high enough anti-inflammatory concentration of
norepinephrine and adenosine (reviewed in Ref.
23). In addition, sympathetic nerve terminals bear
vesicles with endogenous opioids that are able to
inhibit release of the pro-inflammatory SP from
afferent sensory nerve fibers.24 In a highly inflam-
matory disease such as rheumatoid arthritis, the
preponderance of SP-positive nerve fibers over
sympathetic nerve fibers is approximately 8:1,
whereas in healthy tissue, density of sensory
343
versus sympathetic nerve fibers is balanced at
1:1.25 Taken together, the presence of sympathetic
nerve fibers leading to high concentrations of
sympathetic neurotransmitters can inhibit pro-
inflammatory effects of SP, which might be also
relevant in AKP. However, the parallel investiga-
tion of sensory and sympathetic nerve fibers has
never been reported.
In patients with AKP after primary arthroplasty
of the knee and in patients with osteoarthritis of the
knee, it was the aim of this comparative study to
investigate the density of sensory and sympathetic
nerve fibers in the infrapatellar fat pad and the
surrounding connective tissue capsule. We com-
pared the density of these fibers with the synovial
tissue of patients with osteoarthritis of the hip
undergoing replacement surgery. In addition, we
studied the density of cells in the infrapatellar fat
pad and the surrounding connective tissue capsule
because presence of sensory SP-positive nerve
fibers is often linked to connective tissue disease
with increased fibrogenesis.21,22
SUBJECTS AND METHODS
Subjects
A total of 32 patients with osteoarthritis (OA) of the knee
(n ¼ 10), AKP after primary arthroplasty of the knee
(n ¼ 7), and OA of the hip (n ¼ 15) were included.
The characteristics of patients are given in Table 1.
We included a group of hip OA patients in order to get a
more complete picture of synovial innervation and
cellular density independent of the anatomical location.
We did not include other patients without arthroplasty
of the knee or hip (e.g., younger people undergoing
endoscopy), because the material needed has a size of
approximately 5 cm2, which can only be obtained during
replacement surgery. The three groups were not differ-
ent in age, sex, systemic inflammation measured by the
erythrocyte sedimentation rate, and weight (Table 1).
Patients with OA of the hip were taller as compared to
AKP patients (Table 1). The patient groups did not differ
in the WOMAC score, the Besette–Hunter score, the
femur axis, the patella tilt and shift, and the patella
height index (Table 1). As expected, patients with OA as
compared to AKP had a stronger aberration of the lower
limb mechanical axis (Table 1). All three groups had a
similar percentage of patients treated with nonsteroidal
anti-inflammatory drugs (NSAIDs; Table 1). In AKP
patients, between index surgery and revision (approx-
imately 2 years, see Table 1), postoperative/intermittent
physiotherapy and NSAIDs were the sole treatment
options.
Patients with knee OA underwent primary total knee
joint replacement surgery, patients with AKP had a
revision of the infrapatellar fat pad with partial resection
and surgical denervation (after primary arthroplasty of
the knee) due to AKP, and patients with hip OA
JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2008
344 LEHNER ET AL.

Table 1. Characteristics of Patients

Osteoarthritis Anterior Knee Pain after Osteoarthritis

Characteristic of the Knee Primary Arthroplasty of the Hip
n 10 7 15
Age (years) 68.6  2.2 66.9  3.4 67.3  2.6
Sex (F/M), n (%) 6/4 (60/40) 6/1 (86/14) 8/7 (53/47)
Duration since primary arthroplasty (years) n.a. 2.0  0.6 [0.8–4.8] n.a.
Erythrocyte sedimentation rate, 1st h 9.4  3.2 16.7  9.5 10.8  2.2
Weight (kg) 92  6 80  8 81  5
Height (cm) 165  2.5 162  4.9 171  2.0*
WOMAC score (points) 84.4  10.1 74.3  2.7 n.d.
Besette–Hunter score (points) 50.8  9.4 50.4  5.6 n.d.
Deviation of lower limb mechanical axis (8) 6.7  1.2** 1.2  0.9 n.d.
Femur axis (8) 7.9  0.4 8.6  0.5 n.d.
Patella tilt (8) 3.5  1.1 7.9  2.2 n.d.
Patella shift (mm) 2.8  0.7 3.5  2.0 n.d.
Patella height index 0.95  0.05 1.01  0.07 n.d.
Treated with NSAID, n (%) 4 (40) 2 (29) 2 (13)

Abbreviations: n.a., not applicable; n.d., not determined; NSAID, nonsteroidal anti-inflammatory drugs.
Data are given as means  SEM, percentages in parentheses, and ranges in brackets.
*p < 0.05 and **p < 0.01 for the comparison versus anterior knee pain.

underwent total hip arthroplasty. All patients were
informed about the purpose of the study and gave written
consent. The study was approved by the Ethical Com-
mittee of the University of Regensburg.
Tissue Preparation and Histology
Tissue samples were obtained immediately after open-
ing the infrapatellar fat pad (OA of the knee, AKP) or the
hip joint capsule (OA of the hip). The preparation of the
tissue for histology was as described.25 Fat tissue, fat
capsule tissue, and synovial tissue were used for
histology. Samples intended for the determination of
cell density and detection of nerve fibers were fixed for
18 h in phosphate-buffered saline (PBS) containing 3.7%
formaldehyde and then incubated in PBS with 20%
sucrose for 18 h. Thereafter, tissue was embedded in
Tissue Tek (Tissue Tek, Sakura Finetek, Zoeterwoude,
The Netherlands) and quick-frozen. All tissue samples
were stored at
808C.
Histological evaluation has been described in an
earlier study.26 Briefly, the frozen tissue samples were
cut into 6- to 8-mm-thick sections and cell density was
evaluated using a standard hematoxylin and eosin (HE)
staining. Cellular density in the tissue was determined
by counting stained cells in 17 randomly selected high
power fields (400) and expressed per mm2. The deter-
mination of synovial innervation has been described
previously.8 Briefly, 6 to 8 cryosections (5–9 mm thick) of
the formaldehyde/sucrose–fixed tissue samples were
used for immunohistochemistry with a primary antibody
against tyrosine hydroxylase (THþ, the key enzyme for
NE production in sympathetic nerve endings; catalog no.
AB152, Chemicon, Temecula, CA) and against substance
P (SPþ, the key neurotransmitter of SPþ sensory nerve
fibers; catalog no. AB1566, Chemicon). An Alexa 546
conjugated secondary antibody (catalog no. A-11010
against mouse IgG; catalog no. A-121010 against rabbit
IgG; Molecular Probes, Leiden, The Netherlands) was
used to achieve immunofluorescent staining of sympa-
thetic and sensory SP-positive nerve fibers (Fig. 1A,C).
The numbers of THþ sympathetic and SPþ sensory nerve
fibers per square millimeter were determined by averag-
ing the number of stained nerve fibers (typical bead chain
structure with at least four separated vesicles along the
axon, minimum length 50 mm, determined by a micro-
meter eyepiece) in 17 randomly selected high power fields
of view (400). We controlled the positive nerve fiber
staining by incubating the tissue with polyclonal control
antibodies that always yielded a negative result.
Presentation of Data and Statistical Analysis
Mean values are given  SEM. Group medians were
compared by the nonparametric Mann–Whitney test,
correlations were calculated by Spearman rank correla-
tion analysis (SPSS/PC, ver. 12.0, SPSS Inc., Chicago,
IL). Frequencies in two different groups were compared
by the w-squared test using Yates continuity correction
or Fisher’s Exact test if possible. p < 0.05 was the level of
significance.
RESULTS
Nerve Fiber Density and Cellular Density in the
Fibrosis Capsule of the Infrapatellar Fat Pad
In the fibrosis capsule of the infrapatellar fat pad,
nerve fiber density of sympathetic nerves did not
differ between AKP after primary arthroplasty
and knee OA, and it was also similar in synovial
tissue of patients with hip OA (Fig. 1B). With

JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2008

 INCREASED SENSORY OVER SYMPATHETIC NERVES IN AKP 345

Figure 1. Sympathetic and sensory innervation in the infrapatellar fat pad. (A) Staining of
tyrosine hydroxylase in the fat pad (sympathetic nerve fiber). The typical bead chain–like structure
is detectable at a magnification of 400 (arrows). (B) Staining of substance P–positive sensory nerve
fibers in the fat pad. Original magnification, 400. A similar bead chain–like structure is visible
(arrows). (C, D) Tissue density of sympathetic tyrosine hydroxylase–positive (C) and sensory
substance P–positive (D) nerve fibers in the fibrosis capsule of the infrapatellar fat pad in knee
osteoarthritis (OA) and anterior knee pain (AKP), and, in addition, in synovial tissue in hip OA.
Density is given in nerve fibers/mm2.

JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2008

346 LEHNER ET AL.
respect to density of SP-positive nerve fibers, fat
pad capsule innervation was not different in
knee OA versus AKP after primary arthroplasty
(Fig. 1D). Sensory innervation tended to be higher
in the synovium of hip OA patients as compared to
the fibrosis capsule of the infrapatellar fad pad in
knee OA and AKP after primary arthroplasty
(Fig. 1D).
In addition, we studied the cellular density in the
fibrosis capsule and found an increased cellular
density in patients with AKP after primary
arthroplasty as compared to knee OA (fibrosis
capsule) and hip OA (synovial tissue; Fig. 2).
Nerve Fiber Density and Cellular Density in
Fat Tissue of the Infrapatellar Fat Pad
These elements were studied in patients with knee
OA and AKP after primary arthroplasty because a
similar fat pad does not exist at the hip joint. In the
fat tissue, the density of sympathetic nerve fibers
Figure 2. Cellular density in the fibrosis capsule of the infrapatellar fat pad [in knee osteoarthritis
(OA) and anterior knee pain (AKP)] and in the synovium of hip OA. Cellular density is given in
cells/mm2.
JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2008
was similar in knee OA as compared to AKP after
primary arthroplasty (Fig. 3A). However, density
of sensory substance P-positive nerve fibers was
higher in AKP after primary arthroplasty than in
knee OA (Fig. 3B). This was accompanied by an
increased cellular density in fat tissue in patients
with AKP after primary arthroplasty compared to
knee OA (Fig. 3C).
In order to study the direct interrelation
between cellular density and substance P-positive
nerve fiber density in fat tissue, a correlation
analysis was carried out (Fig. 4). It is obvious that
a positive correlation existed between cellularity
and nerve fiber density in fat tissue (Fig. 4).
DISCUSSION
This study in patients with AKP after primary
arthroplasty demonstrates that sensory innerva-
tion of the infrapatellar fat tissue was increased as
 INCREASED SENSORY OVER SYMPATHETIC NERVES IN AKP 347

Figure 3. Nerve fiber density (A, B)and cellular density (C) in fat tissue of the infrapatellar fat pad
in patients with knee osteoarthritis (OA) and anterior knee pain (AKP). The results for sympathetic
tyrosine hydroxylase (A) and sensory substance P–positive nerve fibers (B) are given. The density is
given as nerve fiber/mm2 (in A, B) or cells/mm2 (in C), respectively.

compared to the same tissue of patients with knee
OA. The elevated density of sensory nerve fibers
was positively related to an increased cellular
density in fat tissue. This was not accompanied by
a change in sympathetic innervation, which has
not been investigated so far in AKP. However, a
similar finding was reported in traumatic patellar
tendinopathy.27 We know that the rate of AKP due
to pain outside the knee is only approximately
5%,2 which is a relatively rare cause of a very
common clinical entity. Nevertheless, our data
might help to explain a piece of pathophysiology of
AKP under these particular conditions.
The sympathetic nervous system confers direct
anti-inflammatory and pro-apoptotic effects when
concentrations of neurotransmitters are in the
range of 10
6 to 10
5 M via the b-adrenergic
receptor on target cells.28–37 High concentrations
of norepinephrine can appear in the vicinity of
sympathetic nerve terminals.38 In addition to
norepinephrine, endogenous opioids such as methi-
onine enkephalin and leucine enkephalin are
released from sympathetic nerve terminals, and
these opioids block release of substance P from
sensory nerve fibers in the bidirectional cross-
talk.24 This local analgesic effect of sympathetically
released opioids has been demonstrated in inflam-
matory lesions.39 In normal connective tissue, a
balance between density of sympathetic and sen-
sory nerve fibers exists, which has been demon-
strated in the synovial tissue in patients with knee
trauma and in the ruptured Achilles tendon of
athletes.22,25 This study confirmed this particular
balance in the fibrosis capsule of the infrapatellar
fat pad in patients with AKP and knee OA and in
the synovial tissue of hip OA. Others have
demonstrated that an experimentally induced loss
of sympathetic innervation increases sensory path-
ways relevant for pain perception.27,40–42 Interest-
ingly and vice versa, sensory denervation increased
sympathetic innervation.43 We hypothesize that a
balance of sympathetic and sensory nerve fibers is

JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2008

348 LEHNER ET AL.
Figure 4. Interrelation between cellular and nerve fiber
density in fat tissue of the infrapatellar fat pad. Filled symbols
stand for patients with knee osteoarthritis and open symbols
represent patients with anterior knee pain. The linear regres-
sion line, the Spearman rank correlation coefficient, and its p-
value are given.
important to maintain normal pain perception and
tissue homeostasis including control of inflamma-
tion.
Substance P is a pro-inflammatory neuropeptide
of sensory nerve fibers and it can stimulate cyto-
kine release and fibrogenesis under different con-
ditions.11–14,16–20 A preponderance of substance
P over sympathetic neurotransmitters would be a
pro-inflammatory signal leading to increased pain.
This study demonstrated such a preponderance of
sensory over sympathetic nerve fibers in the fat
tissue of the infrapatellar fat pad in AKP. Because
all our patients with AKP had primary arthro-
plasty 2 years earlier, sensory hyperinnervation
might be related to prior surgery, which could be a
JOURNAL OF ORTHOPAEDIC RESEARCH MARCH 2008
stimulus for the sprouting response of these nerve
fibers. This was accompanied by an elevated
cellular density within fat tissue, which supports
the proproliferative role of this neuropeptide. A
very similar preponderance of substance P positive
over sympathetic nerve fibers was observed in
rheumatoid arthritis, Achilles tendinosis, and
Dupuytren’s contracture.21,22,44 These rheumatic
diseases and AKP are accompanied by increased
pain, and it is conceivable that a higher density of
sensory over sympathetic nerve fibers is respon-
sible for increased pain. Our study was not able to
demonstrate the hen or the egg in the pathophysio-
logic sequence of AKP; however, the following
scenario is plausible.
A traumatic event can trigger a local inflamma-
tory repair process with infiltrating leukocytes and
activation of local cells (adipocytes, fibroblasts,
myocytes, endothelial cells, etc.). Under inflamma-
tory conditions, infiltrating macrophages can
secrete factors, which repel sympathetic nerve
fibers (e.g., semaphorin 3C)45 and which stimulate
sprouting of sensory nerve fibers (nerve growth
factor, brain-derived neurotrophic factor).25,46
Sprouting of sensory nerve fibers in the infrapa-
tellar fat pad in AKP has been confirmed in this
study and in others.7,8 In the presence of sensory
nerve fiber sprouting and sympathetic nerve
repulsion (or growth inhibition), a preponderance
of sensory over sympathetic nerve fibers evolves
(see above).
Substance P from sensory nerve fibers is known
to stimulate IL-111 and TNF secretion11,12 and
consequently other painful factors such as prosta-
glandins and bradykinin.12,47 Thus, neuronally
released substance P is per se a pro-inflammatory
and painful stimulus, which contributes to pain
sensitization. In contrast, because norepinephrine
and sympathetic endogenous opioids at physiolog-
ically high concentrations inhibit secretion of TNF
and other pro-inflammatory factors (reviewed in
Ref. 23), norepinephrine and endogenous opioids
most probably counteract the pro-inflammatory
effects of substance P when neurotransmitter
concentrations are high enough. Thus, the balance
of the two systems is an important factor in pain
and inflammation control. After knee arthroplasty,
sensory hyperinnervation might be a long-standing
phenomenon leading to a reduction of pain thresh-
olds because the normally present sympathetic
inhibition of inflammatory pathways is reduced. In
addition, elevated concentrations of substance P
might lead to smoldering mild inflammation, which
leads to continuation of AKP after primary arthro-
plasty. Under these conditions, triggering factors
 INCREASED SENSORY OVER SYMPATHETIC NERVES IN AKP
such as small traumatic events might trigger or
aggravate AKP after primary arthroplasty.
In conclusion, the preponderance of sensory over
sympathetic innervation in the infrapatellar fat
pad in AKP after primary arthroplasty compared to
OA is probably an unfavorable factor, which
possibly leads to aggravation and continuation of
AKP in this group. It is presently not known
whether drugs that inhibit substance P signaling
or that support b-adrenergic signaling can alleviate
symptoms in patients with AKP after primary
arthroplasty.
ACKNOWLEDGMENTS
This study was supported by a grant from the Deutsche
Forschungsgemeinschaft (FOR696).
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