Nagai et al. Arthritis Research & Therapy 2014, 16:427
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RESEARCH ARTICLE

Open AccessBevacizumab, an anti-vascular endothelial growth
factor antibody, inhibits osteoarthritis
Toshihiro Nagai, Masato Sato*, Miyuki Kobayashi, Munetaka Yokoyama, Yoshiki Tani and Joji Mochida

Abstract
Introduction: Angiogenesis is an important factor in the development of osteoarthritis (OA). We investigated theefficacy of bevacizumab, an antibody against vascular endothelial growth factor and an inhibitor of angiogenesis, inthe treatment of OA using a rabbit model of anterior cruciate ligament transection.
Methods: First, we evaluated the response of gene expression and histology of the normal joint to bevacizumab
treatment. Next, in a rabbit model of OA induced by anterior cruciate ligament transection, we used macroscopic
and histological evaluations and real-time polymerase chain reaction (PCR) to examine the responses to intravenous(systemic) administration of bevacizumab (OAB IV group). We also investigated the efficacy of intra-articular (local)
administration of bevacizumab in OA-induced rabbits (OAB IA group).
Results: Histologically, bevacizumab had no negative effect in normal joints. Bevacizumab did not increase theexpression of genes for catabolic factors in the synovium, subchondral bone, or articular cartilage, but it increased
the expression of collagen type 2 in the articular cartilage. Macroscopically and histologically, the OAB IV groupexhibited a reduction in articular cartilage degeneration and less osteophyte formation and synovitis comparedwith the control group (no bevacizumab; OA group). Real-time PCR showed significantly lower expression of
catabolic factors in the synovium in the OAB IV group compared with the OA group. In articular cartilage,
expression levels of aggrecan, collagen type 2, and chondromodulin-1 were higher in the OAB IV group than in the
OA group. Histological evaluation and assessment of pain behaviour showed a superior effect in the OAB IA groupcompared with the OAB IV group 12 weeks after administration of bevacizumab, even though the total dosage
given to the OAB IA group was half that received by the OAB IV group.
Conclusions: Considering the dosage and potential adverse effects of bevacizumab, the local administration of
bevacizumab is a more advantageous approach than systemic administration. Our results suggest that intra-articularbevacizumab may offer a new therapeutic approach for patients with post-traumatic OA.

Introduction
Osteoarthritis (OA), the most common joint disease, isoften given less attention than other diseases, such as cancer, because it is not a disorder directly associated withthe sustainability of life. However, OA leads to severe joint
dysfunction and pain, and a decline in the patient’s qualityof life with an associated decrease in the ability to perform
activities of daily life. Patients with early to mid-stage OA
are given pharmacological treatment for pain relief, although the long-term benefits have not been shown convincingly. Patients with advanced OA are indicated for
total joint arthroplasty.
* Correspondence: sato-m@is.icc.u-tokai.ac.jp
Department of Orthopaedic Surgery, Surgical Science, Tokai University
School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan

Articular cartilage is an avascular tissue comprising asparse cell population with low mitotic activity, and its
capacity for self-repair is limited. Therefore, mature articular cartilage shows limited capacity for regeneration
after degeneration or injury. For this reason, various treatments have been developed with the aim of restoring tissue quality via regenerative methods. Techniques such as
microfracture [1], mosaicplasty [2], cell transplantation[3,4], and the implantation of tissue-engineered cartilage
with [5-7] or without [8-10] various scaffolding materials
have received increasing attention. However, the restorable areas are limited and tend to be replaced with boneor fibrocartilage tissue.
Previously, we investigated the use of an osteochondral
defect model to explore methods to repair cartilage defect© 2014 Nagai et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication
waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise
stated.

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sites. This was first accomplished by developing a threedimensional, scaffold-free, tissue-engineered cartilage [9]that was transplanted into osteochondral defects to initiate
cartilage differentiation [10]. This method achieved good
restorative effects in the long term, allowing us to confirm
that articular cartilage repair can be achieved during theearly stage of transplantation [10]. We noted that reparative cells from marrow had acquired anti-angiogenic properties, and we hypothesized that better cartilage repair
might be achieved by inhibiting the bioactivity of vascularendothelial growth factor (VEGF) in osteochondral defects.
We later reported that intravenous administration of an
antibody against VEGF contributed to articular cartilage
repair in an osteochondral defect model [11].
In OA, new blood vessels from the subchondral bonebreach the tidemark into cartilage [12], and it is thoughtthat these blood vessels contribute to articular cartilage ossification [13] and lead to osteophyte formation around the
cartilage [14]. Angiogenesis and inflammation are closely
integrated processes in the pathogenesis of OA, which is
associated with increased angiogenesis in the synovium[15]. Synovitis is also characteristic of rheumatoid arthritis(RA). Studies of angiogenesis that have compared thepathogenesis of RA and OA have concluded that angiogenesis correlates with the extent of synovial hyperplasia observed in these two diseases and that hyperplasia is most
severe in RA but is also present in OA-affected joints[16,17]. Angiogenesis also results in innervation of the articular cartilage [18], which may provide a source of pain
in OA patients. Thus, an angiogenesis inhibitor that could
suppress synovitis, osteophyte formation, and pain is an attractive candidate for the treatment of OA.
Although an anti-VEGF antibody is an attractive target
for the treatment of neovascular disease, several complications associated with its intravenous administration havebeen reported, including haemorrhage, thromboembolism,proteinuria, delayed wound healing, and hypertension [19].
In a recent study, we showed that the systemic intravenous
administration of bevacizumab improved articular cartilage
repair in an osteochondral defect model [11]. In thecurrent study, we first determined how normal joint tissue
responds to bevacizumab treatment. We then evaluatedthe effects of intravenous injection of bevacizumab in a
rabbit model of OA by comparing the restorative changes
in the cartilage of the synovium, subchondral bone, and articular joint in bevacizumab-treated and untreated rabbits.
Following this, we compared the effects on healing induced
by intra-articular administration with those induced byintravenous administration of bevacizumab (Figure 1).

accordance with the guidelines on animal use of Tokai
University (Authorization Number 122083). AdolescentJapanese white rabbits, aged 16 to 18 weeks and weighing about 2.5 kg, were used in this study. These rabbits
were purchased from a professional breeder (Tokyo Jiken
Dobutsu, Tokyo, Japan). The rabbits were anaesthetizedby exposure to sevoflurane and O2 gas. Under sterile conditions, a medial parapatellar approach was used to release
the joint capsule and to perform an anterior cruciate ligament transection (ACLT) [20]. After recovery from surgery, all animals were allowed to walk freely in their cages
without any splints. Animals were sacrificed after the experiments by an overdose of intravenous anaesthesia.

Methods

Experiment 2: OA versus OA intravenous bevacizumab

Animals and surgical procedures

Twenty rabbits underwent bilateral ACLT. Ten rabbits
were given an intravenous injection of 100 mg bevacizumab 1 week and 3 weeks after ACLT (OAB IV group).

Animal experiments were approved by the ethics review
board of Tokai University and were performed in

Bevacizumab dose
Intravenous (systemic) administration of bevacizumab

The half-life of bevacizumab in the normal blood circulation is reportedly 21.3 days [21]. In humans, the approved dose of bevacizumab is 5 mg/kg, with a clinicaladministration interval of more than 2 weeks [19]. Bevacizumab is cross-reactive with rabbit VEGF, but its affinity for rabbit VEGF is one-eighth that for human VEGF
[22]. Therefore, we investigated bevacizumab at a dose of
40 mg/kg administered on day 1 and again 2 weeks later.
Intra-articular (local) administration of bevacizumab

The local administration of bevacizumab is currently
performed in ophthalmology clinics, in which the drug
is injected into the vitreous body at a concentration of
25 mg/ml [23]. Intravitreal bevacizumab has been shown
to be non-toxic to the retina and optic nerve at the
2.5 mg dose tested in animals [24]. We thus chose to administer 25 mg/ml into the articular capsule of OA rabbits. A volume of 1 ml was deemed suitable because thisis typical for intra-articular injections in rabbits [25].
Experimental design
Experiment 1: normal versus normal bevacizumab

Twelve rabbits were used to investigate the effects ofbevacizumab treatment on normal articular cartilage,
synovium, and subchondral bone. Six rabbits were given
100 mg of bevacizumab intravenously on day 1 and thenagain 2 weeks later (normal bevacizumab group, n = 12
knees), and the other six rabbits were used as normal
controls (normal group, n = 12 knees). All rabbits weresacrificed after 3 weeks to examine gene expression(normal bevacizumab group, n = 6 knees; normal group,n = 6 knees) and histology (normal bevacizumab group,n = 6 knees; normal group, n = 6 knees).

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Figure 1 Study outline. This study comprised three experiments to assess the effects of bevacizumab in normal and rabbits with osteoarthritis(OA). Experiment 1: effects of bevacizumab treatment on normal articular cartilage, synovium, and subchondral bone were examined in 12
rabbits. Six normal (untreated) rabbits were given bevacizumab intravenously (iv) on day 1 and at 2 weeks (normal bevacizumab group, n = 12
knees), and the other six normal (untreated) rabbits were used as normal controls (normal group, n = 12 knees). All 12 rabbits were evaluatedafter 3 weeks to examine gene expression (normal bevacizumab group, n = 6 knees; normal group, n = 6 knees) and histology (normal
bevacizumab group, n = 6 knees; normal group, n = 6 knees). Experiment 2: 20 rabbits were given bilateral anterior cruciate ligament transection(ACLT): 10 rabbits were given bevacizumab intravenously 1 week and 3 weeks after ACLT (OAB IV group), and 10 rabbits served as untreated OA
controls (OA group). Rabbits were evaluated 4 weeks (n = 6 rabbits/group) and 12 weeks (n = 4 rabbits/group) after ACLT. Experiment 3: all
rabbits were given unilateral knee ACLT. Intra-articular bevacizumab (25 mg) was injected 4 weeks after ACLT, then weekly until 7 weeks (OAB IA
group). Intravenous bevacizumab (100 mg) was injected 4 and 6 weeks after ACLT (OAB IV group). Rabbits were evaluated 4, 6, and 12 weeks after
ACLT. As the comparison, untreated unilateral ACLT rabbits were examined at the same times (OA group).

The other 10 rabbits were used as the untreated control
group (OA group). It has been reported that, in unilateral and bilateral ACLT models, degenerative changes
appear in the articular cartilage 4 weeks after surgery[26-28]. At 4 weeks, six rabbits from each group weresacrificed for histology (n = 8 knees) and gene expression(n = 4 knees). The remaining four rabbits in each groupwere sacrificed 12 weeks after ACLT, and their tissues
were processed for histology (n = 8 knees).
Experiment 3: OA versus OA intravenous bevacizumab
versus OA intra-articular bevacizumab

The final experiment was designed to compare intravenous with intra-articular administration of bevacizumab.
In this experiment, ACLT was performed on 18 rabbits
on only one knee per rabbit to measure pain behaviour
in terms of weight-bearing asymmetry. Because ACLT
was performed unilaterally in experiment 3, the progressof OA was predicted to be slower than that in theexperiment involving bilateral ACLT. Therefore, we changed the timing of the administration of bevacizumab to
4 weeks after ACLT instead of 1 week. Six rabbits were
given 100 mg of bevacizumab by intravenous injection 4
and 6 weeks after ACLT (OAB IV group, n = 6 knees, total
dosage 200 mg), and six rabbits were given 25 mg bevacizumab by intra-articular injection 4, 5, 6, and 7 weeks after
ACLT (OAB IA group, n = 6 knees, total dosage 100 mg).
Six rabbits received no drug treatment (OA group, n = 6
knees). All animals were sacrificed 12 weeks after ACLT,
and the knee joints were evaluated.
Morphology of osteophyte formation

The femoral condyles were examined macroscopically.
Morphological changes were evaluated by two independent observers. The criteria for macroscopic gradingwere as follows: grade 0 (absent), grade 1 (mild osteophyte formation), grade 2 (moderate osteophyte formation), and grade 3 (severe osteophyte formation), asNagai et al. Arthritis Research & Therapy 2014, 16:427
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described previously [29]. We included both osteophytes
and chondro-osteophytes in our evaluation of osteophyte
formation because both are regarded as neoplastic tissuecaused by endochondral ossification as a result of angiogenesis in the articular margin associated with OA. The
formation of chondro-osteophytes by hypertrophic chondrocytes reflects the process of endochondral ossificationin the growth of osteophytes. The condyles were preparedfor histological evaluation and gene expression analysis
after morphological grading.

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cycles: 2 minutes at 50°C, 10 minutes at 95°C, 15 s at
95°C, 1 minute at 60°C, and then 15 s at 95°C, 30 s at
60°C, and 15 s at 95°C (dissociation step). Normal
articular cartilage, synovium, or subchondral bone wasused as the reference for comparisons of gene expression between samples in experiments 1 and 2. The relative expression of the target mRNA was standardized
to glyceraldehyde phosphate dehydrogenase, and theexpression level was calculated using the 2−ΔΔCT values.
Pain evaluation

Histological examination

The distal parts of the femur were excised and fixed in
4% paraformaldehyde for 7 days. Each specimen was
decalcified in a solution of 10% ethylenediaminetetraacetic acid in distilled water (pH 7.4) for 2 to 3 weeks, embedded in paraffin wax, and cut along the sagittal plane.
Each section was stained with Safranin O. We evaluated
OA repair sites at the medial femoral condyle semiquantitatively using a grading and staging system (Osteoarthritis Research Society International (OARSI) modified
Mankin score [30]). This system includes six histologicalgrades and four histological stages. The total score (gradescore multiplied by stage score) ranges from 1 point (normal articular cartilage) to 24 points (no repair). The sections were examined blindly by two observers, and thescores were averaged to minimize observer bias. The
synovium was sampled from the infrapatellar fat padregion. The synovial membrane was fixed in 4% paraformaldehyde for 7 days and subsequently embedded inparaffin. Each section was stained with H&E or Masson
trichrome stain.
Gene analysis by real-time polymerase chain reaction (PCR)

The synovium was obtained from the infrapatellar fat
pad region, articular cartilage was sampled from the femoral condyle, and subchondral bone was obtained fromthe femoral condyle with completely removed cartilage.
Each construct was homogenized using a Cryo-Press(Microtec Nition, Chiba, Japan) in liquid nitrogen. Total
RNA was isolated using the SV Total RNA Isolation System (Promega Corp., Madison, WI, USA), following the
manufacturer’s instructions. RNA quantification and quality were determined using the 260/280 nm ratio. EachRNA sample was then reverse-transcribed to cDNA using
TaqMan Reverse Transcription reagents (Applied Biosystems, Foster City, CA, USA) in a thermocycler set at 42°C
for 60 minutes and at 95°C for 5 minutes. The primer sequences used in this study are listed in Additional file 1:
Table S1. Real-time PCR was performed in an ABI SDS
7300 real-time PCR system (Applied Biosystems) using
SYBR Green Master Mix (Applied Biosystems). cDNA
(2 μl) was added to bring the final volume of the real-time
PCR sample to 25 μl. We then ran 35 to 45 amplification

An incapacitance test meter (Linton Instrumentation,
Norfolk, England) was used to identify and compare
trends in the weight distribution ratio of the undamaged
versus damaged limbs. The measurements were obtained
from rabbits after they were transferred into the rabbit
holder. The weight distribution of both hind legs wasmeasured 10 times, and the following formula was used
to calculate the damaged limb weight distribution ratios(%) obtained by loading the left and right limbs:
Damaged limb weight distribution ratio ð%Þ ¼
fDamaged limb load ðgÞ = ðUndamaged limb loadðgÞ þ Damaged limb load ðg ÞÞg  100:

Statistical analysis

All data are expressed as the mean ± SD. The nonparametric Mann-Whitney U-test was used to compare gene
expression, histology, and gross morphology between
two groups (experiments 1 and 2). The Kruskal-Wallis
test followed by post hoc comparisons (Mann-Whitney
U-test) was used to compare the pain evaluation test results and histological data between three groups (experiment 3). Differences were considered significant whenP-values were <0.05 for comparisons between two groups
or <0.017 for comparisons between three groups.

Results
Experiment 1: Effect of bevacizumab in normal tissues

Acute joint damage that occurs at the time of an injury
initiates a sequence of events that can lead to progressive articular surface damage. Therefore, we sought tocompare the gene expression and histological changes inthe synovium, subchondral bone, and articular cartilageof knees between normal untreated (normal group) andbevacizumab-treated normal rabbits (normal bevacizumab group) (Figure 2). We administered bevacizumab
intravenously and examined changes in the gene expression levels of various catabolic factors, including matrix
metalloproteinase-3 (MMP3), MMP13, ADAMTS5 (adisintegrin and metalloproteinase with thrombospondin
motifs-5), and interleukin-1β (IL-1β). In the synovium,

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Figure 2 Comparison of gene expression and histology between the normal bevacizumab and normal groups (experiment 1).
(A) Synovial gene expression. (B) Subchondral bone gene expression. (C) Articular cartilage gene expression. Gene expression data are expressed
as mean ± SD (n = 6 knees). The y-axis is defined as follows: 2–ΔCT normal B target gene/2–ΔCT normal target gene. *P <0.05 by the Mann-Whitney
U-test. (D) Representative articular cartilage histology for the normal group (left side) and normal bevacizumab group (right side) stained with
Safranin O. (E) Representative synovial histology for the normal group (left side) and normal bevacizumab group (right side) stained with H&E.
(F) Higher magnifications of the image in (E) stained with H&E. Histology data were obtained from six knees per group. Bars = 1 mm (E);bars = 300 μm (D, F).

the expression of most genes tended to be lower in thenormal bevacizumab group compared with the normalgroup; MMP3 and Runx2 expression levels were significantly lower in the normal bevacizumab group (Figure 2A).
In subchondral bone, IL1B and Runx2 expression levels
were significantly lower in the normal bevacizumab groupthan in the normal group (Figure 2B). In articular cartilage,
the expression levels of MMP3, MMP13, and ADAMTS5
were significantly lower in the normal bevacizumab
group compared with the normal group (Figure 2C). In
the articular cartilage, the expression level of collagentype 2, the major collagen in cartilage, was elevated, butthat of collagen type 1, the major collagen in bone andtendon, was lower in the normal bevacizumab groupcompared with the normal group. The expression levelsof chondromodulin-1 (ChM-1), an endogenous antiangiogenic factor, and aggrecan, the major structuralprotein in cartilage, were also lower in the normal bevacizumab group compared with the normal group. Intriguingly,
VEGF mRNA expression was unaffected by intravenous administration of bevacizumab (Figure 2A-C).
Joints from the normal bevacizumab group showedsmooth and uniform articular surfaces and Safranin O
staining throughout the articular cartilage, similar to theappearance in the normal group (Figure 2D). In the
synovium in the normal bevacizumab group, there
was no synoviocyte hyperplasia or proliferation, or
lymphoplasmacytic infiltration. One layer of synovial
membrane with underlying adipose cells was apparent(Figure 2E, F).

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Experiment 2: Comparison between OA and OA
intravenous bevacizumab

We next studied the effects of bevacizumab in a rabbit
model of OA, in which OA was induced by ACLT. Bevacizumab was administered 1 and 3 weeks after inductionof OA into 10 rabbits (OAB IV group); 10 untreated OAonly rabbits served as controls (OA group). All rabbits
were assessed for morphological differences and histological changes at 4 and 12 weeks, and gene expression
changes were assessed at 4 weeks.
Morphology of osteophyte formation

Four weeks after ACLT, macroscopic evaluation of joints
from both groups identified osteophyte formation but
nearly completely smooth joint surfaces of the articularcartilage (Figure 3A). The osteophyte formation score

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did not differ between the OA group and the OAB IV
group (Figure 3C). Twelve weeks after ACLT, the joint
surfaces showed a marked progression of arthritis andosteophyte formation in the OA group. The joints fromthe OAB IV group retained smooth joint surfaces in mostregions of the articular cartilage and had significantly lessosteophyte formation (Figure 3B, C).
Histological evaluation of the articular cartilage

We evaluated the medial femoral condyle at the femoraltibial (FT) site in the articular cartilage area. Four weeks
after ACLT, histological assessment showed a loss of
Safranin O-positive staining in the OA group, whereas
staining was retained in the OAB IV group (Figure 3D).
By 12 weeks after ACLT, the joints in the OA group
showed delamination of the superficial layer, erosion of

Figure 3 Comparison of macroscopic and histological evaluations between the osteoarthritis (OA) and intravenous administration of
bevacizumab in OA (OAB IV) rabbits (experiment 2). (A) Macroscopic articular cartilage appearance 4 weeks after anterior cruciate ligamenttransection (ACLT). (B) Macroscopic appearance 12 weeks after ACLT. Circles indicate osteophyte formation. (C) Osteophyte formation scores 4
and 12 weeks after ACLT. Data are expressed as mean ± SD (n = 8 knees). Safranin O staining of hyaline cartilage 4 weeks (D) and 12 weeks(E) after ACLT. (F) Osteoarthritis Research Society International histological score 4 and 12 weeks after ACLT. (G) Macroscopic synovia appearance
4 weeks after ACLT. (H) Representative synovial histology for OA group (left side) and OAB IV group (right side) stained with H&E. Higher
magnifications of the image shown in (H) stained with HE (I) and Masson trichrome (J). Data are expressed as mean ± SD (n = 8 knees). *P <0.05
by the Mann-Whitney U-test. Bars = 1 mm (D, E, H); bars = 300 μm (I, J).

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hyaline cartilage, and a lack of Safranin O staining. Joints
from the OAB IV group showed smooth and uniform articular surfaces and Safranin O staining throughout the articular cartilage (Figure 3E). The total OARSI histologicalscore did not differ between the OA and OAB IV groups
at 4 weeks but was significantly lower in the OAB IV
group than in the OA group at 12 weeks (Figure 3F).
Morphology and histology of the synovium

Four weeks after ACLT, the macroscopic appearanceof the synovium showed enlargement with redness andrecognized thick vascular invasion in the OA group. By
contrast, in the OAB IV group, the synovium was preserved and appeared pale yellow, although a thin vascular invasion was apparent (Figure 3G). Synovial sections
from the OA group showed severe synoviocyte hyperplasia with diffuse lymphoplasmacytic infiltration, bloodvessel proliferation, and severe fibroblast proliferation
with regression of adipose cells. These characteristics
were present in the OAB IV group, but they were more

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moderate (Figure 3H, I, J). These results indicate thatthe structural damage was less severe in the OAB IV
group than in the OA group.
Gene expression

We harvested the synovium, subchondral bone, and articular cartilage from the OA and the OAB IV groups 4 weeks
after ACLT. We used real time PCR to assess changes inthe expression of genes involved in anabolic and catabolic
factors, and we compared these changes between the OA
and OAB IV groups relative to the expression levels of thenormal tissues at the baseline. MMP13 gene expression in
the normal synovium was about 10-fold higher in the OA
group than in untreated animals. MMP13 gene expression
in the OAB IV group was one-tenth that in the OA group,a level that approximated the normal level. ADAMTS5 gene
expression did not increase in the OA group comparedwith the normal tissue but was significantly decreased byadministration of bevacizumab (Figure 4A). The effect ofbevacizumab on the OA synovial tissue differed from that

Figure 4 Comparison of relative gene expression levels between the osteoarthritis (OA) and intravenous administration of
bevacizumab in OA (OAB IV) groups (experiment 2). (A) Synovial gene expression. (B) Subchondral bone gene expression. (C) Articularcartilage gene expression. The y-axis is defined as follows: 2–ΔCT OA or OAB IV target gene/2–ΔCT normal target gene. The normal tissue isrepresented as 1 on the y-axis. Data are expressed as mean ± SD (n = 4 knees). *P <0.05 by the Mann-Whitney U-test.

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in the normal synovium (Figure 2A). In subchondral bone,
Runx2 expression was also significantly lower in the OAB
IV group compared with the OA group (Figure 4B). However, bevacizumab had a similar inhibitory effect on Runx2
expression as in normal subchondral bone (Figure 2B).
Articular cartilage from the OAB IV group showed significantly 3.5-fold higher collagen type 2 gene expression
compared with the OA group, and there was a tendency
toward lower collagen type 1 gene expression in the
OAB IV group compared with the OA group (Figure 4C).
The patterns of changes in the expression of types 1 and2 collagen genes were similar to those induced by bevacizumab treatment in normal articular cartilage (Figure 2C).
Unlike the effect of bevacizumab in normal tissue, aggrecan and ChM-1 gene expression levels were significantly
higher in the OAB IV group compared with the OA
group. By contrast, MMP13 and ADAMTS5 gene expression levels were about 2.0-fold higher and VEGF gene
expression level 1.4-fold higher in the OAB IV group thanin the OA group, but this increase was not significant.
MMP3 gene expression levels did not differ between the
OAB IV group and the OA group.
Experiment 3: differences between intravenous andintra-articular administration of bevacizumab in amodel of OA
Damaged limb weight distribution ratio (%)

A comparison of the weight distribution ratio between
the three groups (OA, OAB IV, and OAB IA) following
bevacizumab treatment is shown in Figure 5A. Up to
6 weeks, the rabbit weight distribution ratio did not differ
between the three groups. However, by 12 weeks, rabbits
in the OAB IA group showed a satisfactory improvementin the damaged limb weight distribution ratio, whereas
rabbits in the OA and OAB IV groups failed to show such
improvement. The OAB IA group showed significant improvement compared with the OA group (Figure 5A). Unfortunately, one rabbit from the OAB IV group developed
diarrhoea and lost weight, and this rabbit died from complications involving digestion at 8 weeks.
Histological evaluation

Because unilateral ACLT was performed, the OA change
was not as progressive at the FT site compared with thatobserved in rabbits that had received the bilateral ACLT
in experiment 2. Therefore, we divided the distal portionof the femur into the FT site, femoral-patellar (FP) site,and corner site, which was between FP and FT (Figure 5B).
In the OA group, diminished Safranin O staining andclear degeneration of the cartilage were observed at theFT site. Only minimal Safranin O staining was observed at
the corner and FP sites (Figure 5C). By contrast, in the
OAB IV group, matrix staining was strong at the FT sitethroughout the thickness of the cartilage and was stronger

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at the corner and FP sites compared with the OA group.
Some matrix staining depletion was observed within theupper third of the cartilage at the corner site and within
the full thickness of the cartilage within the FP site
(Figure 5D). In the OAB IA group, matrix staining depletion was minimal at all three sites, and staining wasobserved throughout the full thickness of the tissue(Figure 5E). As expected, the OARSI histological score
for Safranin O staining at the FT site did not differ significantly between the three groups (Figure 5F). However, atthe corner and FP sites, the histological score was significantly lower in the OAB IA group than in the OA group(Figure 5G, H). These results indicate that the OAB IA
group had less OA progression compared with the OAB
IV and OA groups.

Discussion
We previously demonstrated that the intravenous administration of bevacizumab repaired articular cartilage
in an osteochondral defect model [11]. Based on thoseresults, the present study was designed as three separate
experiments to (1) assess the effect of bevacizumab innormal joints; (2) clarify the therapeutic efficacy of theintravenous administration of bevacizumab in OA joints;and (3) evaluate the effects of a systemic versus local administration of bevacizumab in OA joints.
The intravenous administration of 100 mg of bevacizumab for a period of 2 weeks did not lead to inflammationor angiogenic effects in the three tissue types in thenormal joint as determined by histological and gene expression in experiment 1. The expression of the genes forsome anabolic and catabolic factors was suppressed by administration of bevacizumab in normal articular cartilage.
This environment may be the extremely stable hypometabolism, rather than the toxicity to cartilage with
bevacizumab.
Anderson et al. recently reported that early molecularinterventions can limit cartilage degradation by minimizing the extent of tissue damage and can help acceleratehealing by decreasing the inflammatory response [31]. In
the current study, early intervention by intravenous administration of bevacizumab appeared to contribute to a
reduction in articular cartilage degeneration, osteophyte
formation, and synovitis, as determined macroscopicallyand histologically in experiment 2.
We performed PCR analyses to assess whether bevacizumab has preventive effects on the whole joint (that is,by comparing the results of experiments 1 and 2). In the
synovium, MMP13 and ADAMTS5 expression levels weresignificantly lower in the OAB IV group than in the OA
group. Although bevacizumab did not decrease IL1B
expression significantly, we suggest that the decrease in
MMP13 and ADAMTS5 expression levels contributed tothe absence of synovitis.

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Figure 5 Comparison of pain and histological evaluations between osteoarthritis (OA), intravenous administration of bevacizumab in
OA (OAB IV), and intra-articular administration of bevacizumab in OA (OAB IA) groups (experiment 3). (A) An incapacitance test meter
was used to investigate the pain-ameliorating effects of treatment. The damaged limb weight distribution ratio (%) = {Damaged limb load(g)/(Undamaged limb load (g) + Damaged limb load (g))} × 100. The damaged limb weight distribution ratios 4, 6, and 12 weeks after surgery
are shown. Data are expressed as mean ± SD (4 and 6 weeks after surgery: for each group, n = 6 knees; 12 weeks after surgery: OA and OAB
IA groups, n = 6 knees, OAB IV group, n = 5 knees). *P <0.05 (Kruskal-Wallis post hoc test and Mann-Whitney U-test). (B) Distribution offemoral-patellar (FP), corner, and femoral-tibial (FT) sites. Safranin O-stained sections of articular cartilage. Bar = 1 mm. Representative SafraninO-stained sections for the (C) OA, (D) OAB IV, and (E) OAB IA groups. Top row, overview of the distal femur. Bar = 1 mm. Second, third, andfourth rows, enlarged images of the FT site, corner site, and FP site. Bar = 250 μm. Osteoarthritis Research Society International histological scoring
was performed at the (F) FT, (G) corner, and (H) FP sites. Data are mean ± SD (OA and OAB IA groups: n = 6 knees; OAB IV group: n = 5 knees).
*P <0.05 (Kruskal-Wallis post hoc test and Mann-Whitney U-test).

Higher VEGF mRNA and protein levels have been reported in the synovial tissue of patients with inflammatory joint disease who required synovectomy comparedwith healthy control subjects [32]. Acute joint damage
that occurs at the time of an injury initiates a sequenceof events that can lead to progressive articular surface
damage [31]. Subsequently, fragments of cartilage can
induce synovitis in these patients [33]. In this study, damage to the articular surface led to an elevation in VEGF expression in the articular cartilage. In the synovium, there
was a strong increase in the expression of MMP and IL1B,
which indicates inflammation, but VEGF expression, which
represents angiogenesis, was unchanged. This is probably

because our study examined the early stages of OA(4 weeks after ACLT). Therefore, it seems likely that VEGF
expression in the synovial tissue was not influenced by bevacizumab treatment.
Runx2 is the master gene of bone formation and contributes to sclerosis in subchondral bone and the formation of osteophytes [34]. From the early stages in ourmodel, we observed a significant decrease in Runx2 expression with bevacizumab treatment, suggesting that bevacizumab contributes to the inhibition of osteosclerosis in
the subchondral bone and inhibits osteophyte formation.
In this study, the expression of both VEGF and ChM-1
was examined in the articular cartilage. We consider that

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the balance between angiogenic and anti-angiogenic factors is important to articular chondrocyte survival andpreservation of the phenotype. The expression of ChM1, which was lower in the OA group than in the normalgroup, increased with bevacizumab treatment. ChM-1 isreported to be a strong inhibitor of angiogenesis involvedin the development of bone and regulation of vascular invasion during endochondral bone formation [35]. Articular cartilage contains considerable amounts of ChM-1,
which should induce the chondrocyte phenotype [36]. It
has been posited that the development of the OA phenotype might result from articular chondrocytes undergoinga differentiation route reminiscent of growth plate chondrocytes and expressing hypertrophy-like changes [37].
Thus, ChM-1 may act to prevent chondrocyte hypertrophy and to stabilize the articular chondrocyte phenotype [38].
VEGF is necessary for chondrocyte survival during
cartilage development [39]. In an osteochondral defectmodel, we showed previously that administration of
bevacizumab caused ChM-1 to accumulate in the interterritorial space of the repaired matrix surrounding thechondrocytes, which expressed VEGF [11]. In the articular cartilage, although VEGF expression was 2.4-foldhigher in the OA group than in normal tissue from untreated rabbits, bevacizumab did not significantly alter
VEGF expression.
The gene expression levels of the anabolic factors
aggrecan and collagen type 2 were significantly higher in
the OAB IV group than in the OA group. Interestingly,
the gene expression of the catabolic factors in the articular cartilage, MMP13 and ADAMTS5, were higher in
the OAB IV group than in the OA group, although thisdifference was not significant. Because this study was
performed in vivo, we consider that the expression of
the catabolic genes may have led to degradation of theexisting matrix and subsequent proliferation and synthesis of new matrix in the limited articular cartilage space.
The increase in ChM-1 expression in response to bevacizumab may have increased the anabolic and catabolic factors, leading to proliferation and synthesis of new matrix
in vivo. Interestingly, however, in normal articular cartilage, ChM-1 expression was decreased by administrationof bevacizumab (Figure 2C). Kitahara et al. suggested that
ChM-1 acts to inhibit vascular invasion in the immaturestate of articular cartilage and that ChM-1 levels decrease
gradually with age thereafter [40]. Such findings suggest aregulatory role of ChM-1 in vascular invasion; that is,
exposure of tissues to angiogenesis factors is reduced inmature articular cartilage concomitant with a reduction in
ChM-1 at both the protein and gene expression levels. In
other words, ChM-1 appears to decrease at the nonangiogenic sites. In the current study, it is possible thatangiogenic factors were reduced at the protein level after

Page 10 of 12

bevacizumab administration in the normal articular cartilage; if so, ChM-1 gene expression would have also decreased following the administration of bevacizumab innormal articular cartilage.
One limitation of this study is that we did not measure
VEGF protein levels in the tissue before and after
treatment with bevacizumab, which is important when
examining the treatment effects of bevacizumab. In thisexperiment, intermittent treatment with bevacizumab induced less OA progression, as shown by both histologicaland gene expression assays, through increases in the gene
expression of ChM-1 and VEGF in the articular cartilage
and decreases in the gene expression of MMP 13 and
ADAMTS5 in the synovium. Thus, although bevacizumabbound to VEGF protein, this did not prevent the expression of VEGF, which is needed for cartilage survival. In
addition, it is thought that bevacizumab functions tomodify the local environment by preventing angiogenesis
in articular cartilage and synovial inflammation. Further
research is required to understand better what determines
the magnitude of the increase in ChM-1 at both the protein and gene expression levels in response to bevacizumab in OA tissues. It is important to understand thepharmacokinetic profile of the drug if we are to optimize
dosing regimens for patients and to obtain a better assessment of its safety profile.
The second limitation of this study is that we did notmonitor the dose-response relationship for bevacizumab
because a sufficient preventive effect against OA progression occurred with the intravenous administration of
200 mg (2 × 100 mg) of bevacizumab and intra-articularadministration of 100 mg (4 × 25 mg) of bevacizumab.
In the absence of intravenous administration of bevacizumab, intra-articular administration may lead to bevacizumab being absorbed by the highly vascular synovial
membrane and then circulated throughout the wholebody. Therefore, some of the effects of the intra-articular
injection of bevacizumab may be attributable to its presence in the systemic circulation. Future studies should assess the effects of tapered dosages of bevacizumab and the
optimal systemic dosage.
We have shown here that intravenous administrationof bevacizumab is associated with reductions in synovitis, articular degeneration, and osteophyte formation in
an ACLT model of OA. Systemic bevacizumab-treatedjoints also generally showed greater aggrecan preservation, as indicated by the intensity of Safranin O stainingof the articular cartilage (Figure 3). These results arepromising for translating this study into the clinical setting. As described earlier, there are numerous potential
side effects of systemic administration of bevacizumab.
It is likely that the intra-articular administration of bevacizumab will decrease the risk of these adverse events.
In this study, there was no disadvantage associated with

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intra-articular administration of bevacizumab comparedwith intravenous administration. The total dose of the
drug given to the OAB IA group was half that given to
the OAB IV group. Moreover, the animals exhibited
significantly less pain in the OAB IA group. Bonnet andWalsh commented that angiogenesis may introduce sensory nerves into the aneural cartilage and that inflammation can sensitize nerves present in the joint [33]. It isbelieved that the intra-articular administration of bevacizumab inhibits angiogenesis and inflammation, thereby
reducing OA progression and providing pain relief. Because ACLT was performed unilaterally in experiment 3,
the progression of OA was slower than that in experiment2, which involved bilateral ACLT. Similar to experiment 2,
the differences in the histological score and pain behaviour between the OA and OAB IV groups in experiment 3
will seem to occur after 12 weeks. Intravenous administration of bevacizumab is a promising treatment option forpolyarthritis, whereas intra-articular administration might
be recommended for patients with single-site arthritis.

Conclusions
In conclusion, we show that bevacizumab reduced synovitis, osteophyte formation, and cartilage degradation ina rabbit model of OA. Importantly, intra-articular administration of bevacizumab also reduced pain behaviour. Because bevacizumab was administered before thedevelopment of structurally severe OA, its effects mightreflect changes in synovitis rather than direct effects on
cartilage pathogenesis. Our findings indicate that earlyintervention by the administration of bevacizumab following an ACL injury may be beneficial in retarding thedevelopment of post-traumatic OA.
Additional file
Additional file 1: List of primers used in real-time PCR.

Abbreviations
ACLT: anterior cruciate ligament transection; ADAMTS5: A disintegrin
and metalloproteinase with thrombospondin type 1 motif-5;
ChM-1: chondromodulin-1; ACL: anterior cruciate ligament; FP:
femoral-patellar; FT: femoral-tibia; H&E: haematoxylin and eosin;
IL-1β: interleukin-1β; MMP: matrix metalloproteinase; OA: osteoarthritis;
OAB IA: intra-articular administration of bevacizumab in OA; OAB
IV: intravenous administration of bevacizumab in OA; OARSI: Osteoarthritis
Research Society International; PCR: polymerase chain reaction;
RA: rheumatoid arthritis; Runx2: runt-related transcription factor-2;
VEGF: vascular endothelial growth factor.
Competing interests
The authors confirm that no competing interests of any kind exist for any of
the authors, not only financial conflicts of interest. The authors declare thatthey applied for a patent relating to the content of the manuscript in Japanbut did not receive any reimbursements, fees, funding, or salary from anorganization. The competitive companies developing or selling anti-VEGF
drugs (for example, Novartis, Wyeth, Bayer Schering) may keep them incheck.

Page 11 of 12

Authors’ contributions
TN carried out the animal experiment, performed the statistical analyses, hasbeen involved in drafting the manuscript and revising it critically forimportant intellectual content. MS has made substantial contributions toconception and design, interpretation of data, helped to draft themanuscript, and also agreed to be accountable for all aspects of the work inensuring that questions related to the accuracy or integrity of any part of
the work had been appropriately investigated and resolved. MK participatedin the animal experiment, performed the PCR analyses and has been
involved in drafting the manuscript. MY performed histological examinations
and participated in PCR analyses and has been involved in drafting themanuscript. YT performed histological examinations and participated in thestatistical analyses and has been involved in drafting the manuscript. JM
participated in the design and coordination of the study and agreed to beaccountable for all aspects of the work in ensuring that questions related tothe accuracy or integrity of any part of the work had been appropriately
investigated and resolved. All authors read and have given final approval ofthe version to be published.
Acknowledgements
This research was supported partly by Grants-In-Aid for Scientific Researchfrom the Ministry of Education, Culture, Sports, Science and Technology.
Received: 16 November 2013 Accepted: 13 August 2014

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2014 16:427.

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