Progression of Cartilage Degradation, Bone Resorption and Pain in Rat Temporomandibular Joint Osteoarthritis Induced by Injection of Iodoacetate
Bone Resorption and Pain in Rat
Temporomandibular Joint Osteoarthritis Induced by Injection of Iodoacetate. PLoS ONE 7(9): e45036. doi:10.1371/journal.pone.0045036
Progression of Cartilage Degradation, Bone Resorption and Pain in Rat Temporomandibular Joint Osteoarthritis Induced by Injection of Iodoacetate
Xue-Dong Wang 0
Xiao-Xing Kou 0
Dan-Qing He 0
Min-Min Zeng 0
Zhen Meng 0
Rui-Yun Bi 0
Yan Liu 0
Jie-Ni Zhang 0
Ye-Hua Gan 0
Yan-Heng Zhou 0
Andre Van Wijnen, University of Massachusetts Medical, United States of America
0 1 Department of Orthodontics, Peking University School and Hospital of Stomatology , Beijing , China , 2 Center for Temporomandibular Disorders and Orofacial Pain, Peking University School and Hospital of Stomatology , Beijing , China
Background: Osteoarthritis (OA) is an important subtype of temporomandibular disorders. A simple and reproducible animal model that mimics the histopathologic changes, both in the cartilage and subchondral bone, and clinical symptoms of temporomandibular joint osteoarthritis (TMJOA) would help in our understanding of its process and underlying mechanism. Objective: To explore whether injection of monosodium iodoacetate (MIA) into the upper compartment of rat TMJ could induce OA-like lesions. Methods: Female rats were injected with varied doses of MIA into the upper compartment and observed for up to 12 weeks. Histologic, radiographic, behavioral, and molecular changes in the TMJ were evaluated by light and electron microscopy, MicroCT scanning, head withdrawal threshold test, real-time PCR, immunohistochemistry, and TUNEL assay. Results: The intermediate zone of the disc loosened by 1 day post-MIA injection and thinned thereafter. Injection of an MIA dose of 0.5 mg or higher induced typical OA-like lesions in the TMJ within 4 weeks. Condylar destruction presented in a time-dependent manner, including chondrocyte apoptosis in the early stages, subsequent cartilage matrix disorganization and subchondral bone erosion, fibrosis, subchondral bone sclerosis, and osteophyte formation in the late stages. Nociceptive responses increased in the early stages, corresponding to severe synovitis. Furthermore, chondrocyte apoptosis and an imbalance between anabolism and catabolism of cartilage and subchondral bone might account for the condylar destruction. Conclusions: Multi-level data demonstrated a reliable and convenient rat model of TMJOA could be induced by MIA injection into the upper compartment. The model might facilitate TMJOA related researches.
Funding: This project is supported by the National Natural Science Foundation of China (Grant No. 81070849) and China International Science and Technology
Cooperation (Grant No. 2010DFB32980). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
Temporomandibular joint osteoarthritis (TMJOA) is an
important subtype of temporomandibular disorders (TMD) [1,2] and is
especially common in female patients with severe pain and
dysfunction of the temporomandibular joint (TMJ) [3,4]. OA is
characterized by a progressive degradation of cartilage,
subchondral bone remodeling, synovitis, and chronic pain [3,5,6].
However, the process of TMJOA remains obscure.
Chondrocyte death due to either apoptosis or necrosis is assumed to be a
central feature in the degeneration of osteoarthritic cartilage and
to contribute to the development of clinical or experimental OA
[7,8]. Resorption and abrasion of condylar subchondral bone are
unique in TMJOA, which usually shows no typical pannus in the
synovium whereas rheumatoid arthritis does . Therefore, a
proper animal model may provide a useful way to understand the
pathogenesis of TMJOA and to evaluate potential therapeutic
Thus far, several methods have attempted to create animal
models of TMJOA, including surgical , mechanical ,
druginducing [12,13], and spontaneously occurring methods . Due
to the limited availability of special animal species, slow
progression of the disease, and complicated operations, the use
of spontaneous or surgical-induced methods was limited [13,15].
In addition, the lack of progressive changes led to a number of
drug-induced models being merely models of cartilage damage
rather than OA . A simple and reproducible animal model of
TMJOA that mimics the histopathologic changes both in cartilage
and subchondral bone, as well as clinical symptoms, is still needed.
Intra-articular injection of monosodium iodoacetate (MIA) to
induce OA-like lesions is widely used to induce knee OA
[16,17,18,19,20,21]. MIA mainly inhibits the activity of
glyceraldehyde-3-phosphate dehydrogenase leading to apoptosis of
chondrocytes [18,20,22]. The MIA-induced OA model has the
great advantage of easy modulation of the progression and severity
of the articular lesions by modification of MIA concentration .
Although a few studies have attempted to induce OA-like lesions
in rabbit TMJ by MIA injection into the lower compartment with
or without surgical assistance [13,23,24], it is important to explore
whether MIA could induce OA-like lesions in the rat TMJ, since
rats are one of the most widely used species in experimental
research and drug toxicology testing . The TMJ is partitioned
by a disc, which forms a larger upper and smaller lower
compartment. Agent injection into the lower compartment is a
difficult procedure both in humans and animals because of its
limited space , whereas injection into the upper compartment,
even in rats, is technically and manually preferable, and has often
been used previously [26,27,28].
The question then arises as to whether MIA injection into the
upper compartment of the rat TMJ can be used to create a
comprehensive OA model. To address this question, investigations
at the histopathologic, radiographic, molecular, and noceiceptive
behavioral levels were performed in this study to examine whether
injection of MIA into the upper compartment of the rat TMJ
could induce OA-like lesions in the entire joint.
Materials and Methods
With the approval of the Peking University Institutional Animal
Care and Use Committee (NO: LA2012-59), all rats were housed
under controlled temperatures in a 12 h light/dark cycle with easy
access to food and water.
Induction of TMJOA
A total of 72 female Sprague-Dawley rats (180200 g) were
randomly assigned to either the experimental (n = 42) or control
(n = 30) groups. The experimental schedule is illustrated in Fig. 1A.
TMJOA was induced by injection of MIA (Sigma, Saint Louis,
USA) dissolved in 50 mL saline into the upper compartment of
bilateral TMJs using a 27-gauge 0.5-inch needle without surgical
assistance. We first confirmed the injection site by injection of
50 mL dye into the upper compartment (Fig. 1B).
Various doses of MIA (0.05, 0.1, 0.5, 1, or 2 mg) were injected
into the upper compartment of bilateral TMJs of rats in five
experimental groups (n = 3/group), while 50 mL saline was
injected into TMJs of rats in the control group (n = 3). All rats
were sacrificed on day 28 post-injection to validate the adequate
dose of MIA for further observations (Fig. 1A).
After the dose course test, 0.5 mg MIA (minimum effective
dose) or saline was injected into TMJs and rats were sacrificed on
days 1, 3, 7, 14, 28, 56, or 84 post-injection (n = 3/group)
To determine gene expression profiles during the induction of
TMJOA, an additional 12 rats, divided into two groups (n = 6/
group), were injected with 0.5 mg MIA or saline and sacrificed on
day 14 post-injection.
Head Withdrawal Threshold (HWT) Measurements
The nociceptive behavior of animals was assessed based on the
HWT as described previously . HWT measurements were
performed pre-MIA/saline injection and on days 1, 3, 7, 14, 21,
28, 35, and 42 post-injection (Fig. 1A). The HWT was calculated
as a mean value per joint of 3 rats/group.
All rats were sacrificed by pentobarbital overdose. For
histopathology, the TMJs of two rats in each group were removed
bilaterally en bloc, fixed in 4% paraformaldehyde, and
demineralized in 15% EDTA. For radiographic examination, the bilateral
condyles of one rat in each group were dissected.
For real-time PCR analysis, the condyle heads of six rats in each
group (0.5 mg MIA for 2 weeks or control) were dissected.
Bilateral condyle heads of each rat were pooled for sufficient RNA
extraction owing to the difficulty of isolating and acquiring enough
cartilage from the small condylar head of rat and the reason that
both the cartilage and subchondral bone were affected by MIA.
Scanning Electron Microscopy (SEM) and Transmission
Electron Microscopy (TEM)
TMJ discs and condylar cartilage were dissected from the rats
on day 1 post-injection (n = 3/group). SEM and TEM were
performed as described previously . Briefly, the samples were
fixed with 2.5% fluteraldehyde solution and 1% osmium tetroxide
(Sigma). For SEM, the disc section was produced by tearing
through the intermediate zone. For TEM, the intermediate zone
of the disc or condylar cartilage was embedded in epoxy resin.
Ultrathin sections (100 nm) were stained with lead citrate and
Paraffin-embedded TMJ blocs were sagittally cut in serial
sections at a 5-mm thickness. Sections were stained with
hematoxylin and eosin (HE) for routine histological evaluation.
Safranin O-fast green (S.O) and Toluidine blue (TB) stains were
used to evaluate proteoglycans in the cartilage matrix .
Radiographs of condyles were obtained with a high-resolution
MicroCT system (Inveon, Siemens, Germany). The specimens
were scanned at 60 kV, 300 mA, and 8.5 mm-effective pixel size.
The images were analyzed using software provided by the
manufacturer. All sagittal images were captured using the same
parameters: Ct = 2550; W = 550.
Total RNA was isolated from the condylar heads containing
cartilage and subchondral bone using TRIzol reagent (Invitrogen,
Carlsbad, USA) according to the manufacturer s instructions. The
condylar heads were ground into powder in liquid nitrogen using a
cryogenic grinder (6770 Freezer/Mill, SPEX SamplePrep, NJ,
USA). Reverse transcription were performed with an iScript
cDNA synthesis kit (Bio-Rad) in 20 ml reaction volume containing
1 mg of total RNA as described previously [27,30]. Real-time PCR
was performed with Power SYBR Green PCR Master Mix
(Applied Biosystems) using a 7500 real-time PCR System (Applied
Biosystems). The amplification specificity was confirmed by
melting curve. The sequences of primers for rat b-actin ,
Collagen I and Aggrecan , Collagen II , ADAMTS5
(aggrecanase-2) , Tissue Inhibitors of Metalloproteinase
(TIMP)2 , TNFa , Bax, Fas, FasL, Caspase2, Caspase3,
Figure 1. Outline of experimental design and confirmation of injection site into upper compartment of rat TMJ. A: Outline of
experimental design. B: Photograph of dye (fast green solution) injection into the upper compartment of the left TMJ. (a). Needle insertion was 5 mm
anterior to the external auditory canal. (b). Dissection showed that the needle was right under the root of the zygoma (dotted arrow), anterior of the
external auditory canal (arrow), stopped at the temporal fossa, and was located in the upper compartment (black circle). (c). 50 mL dye was injected.
(d). Opening the capsule revealed that the dye was restricted to the upper compartment of the TMJ (disc and condyle: hollow arrow).
Caspase8, and Caspase9 , and alfa-smooth muscle actin
(aSMA)  were all previously described and their efficiency was
confirmed by sequencing their conventional PCR products. The
primers for rat Matrix Metalloproteinase (MMP)3 (sense:
59GGTCTGTGGAGGACTTGTA-39), MMP13 (sense:
59CTGACCTGG- GATTTCCAAAA-39; anti-sense:
59ACACGTGGTTCCCTGAGAAG-39), TIMP1 (sense:
59-TTGATCTCATAACGC- TGGT-39), and Proliferating Cell Nuclear Antigen
(PCNA) (sense: 59-CCAGGG- CTCCATCCTGAA-39; anti-sense:
59-CCCAGCAGGCCTCATTGAT-39) were designed with
Primer Premier Version 5.0 software and their efficiency was confirmed
by sequencing their conventional PCR products.
Terminal Deoxynucleotidyl Transferase dUTP nick end
Labeling (TUNEL) Assay
Apoptosis was examined in situ using a TUNEL assay according
to the manufacturers instructions (Roche, Mannheim, Germany).
Briefly, sections were deparaffinized, rehydrated, pretreated with
protease K (10 mg/ml, Sigma) for 20 min, and blocked with 3%
bovine serum albumin for 20 min at room temperature. The
sections were incubated with TUNEL reaction mixture for 1 h at
37uC and covered with fluorescence mounting medium
(Zhongshan-Golden-Bridge-Biotechnology, Beijing). Confocal
microscopic images were acquired using a Zeiss laser-scanning microscope
Immunohistochemical (IHC) Staining
IHC staining was performed with a two-step detection kit
(Zhongshan-Golden-Bridge-Biotechnology) as described
previously . The primary antibodies were MMP3 (Abcam, 1:100
dilution), caspase3 (Cell Signaling Technology, 1:1000 dilution),
and a-SMA (Abcam 1:100 dilution).
Statistical analysis was performed using SPSS version 11.0 for
Windows. All data were presented as mean 6 SEM. Following
confirmation of normal data distribution, all data between the
experimental and control groups were analyzed using Students t
tests with P values ,0.05 considered to be statistically significant.
Confirmation of Injection into Upper Compartment
To confirm the injection site in the upper compartment of the
TMJ, one rat was preliminarily dissected after injection of fast
green solution into the upper compartment. The needle was
inserted right under the root of the zygoma, beneath the temporal
fossa into the upper compartment. The green stained region was
mainly limited to the upper compartment (Fig. 1B).
Ultrastructural Changes in Disc
To determine whether MIA injected into the upper
compartment of the TMJ could diffuse into the lower compartment, the
ultrastructure of the TMJ disc was evaluated by SEM and TEM 1
day after injection of MIA or saline (Fig. 2). SEM showed that the
surface of the disc in the control group was furrowed and covered
by an evenly distributed gelatinous layer, whereas the intermediate
zone of the disc in the MIA group lost these features and presented
a limited region with a thinner and smooth surface surrounded by
areas with a rough and uneven surface. From the section view of
the intermediate zone, disc cells in the control group inserted into
the collagen fibrils, whereas the disc cells in the MIA group were
crimpled and rounded, detached from the surrounding collagen
fibrils (Fig. 2A).
TEM showed that the disc cells of the intermediate zone in the
control group were surrounded by a dense, collagenous
extracellular matrix (ECM) and the cell junction was tight, with
ovoidshaped mitochondria around the nucleus. However, the disc cells
in the MIA group underwent morphological changes, including
cell shrinkage, condensation of the cytoplasm and nucleus, cell
membrane detachment from the surrounding collagen fibrils, and
loosened cell junctions accompanied by the disrupted ECM. Some
cells even presented features of apoptosis, such as chromatin
compaction, swelling mitochondria, and vacuolar degeneration
Dose-dependent Histopathologic Changes in TMJ
To understand the effects of MIA on the TMJ, the morphology
of the TMJ was examined for 4 weeks after injection with saline or
increasing doses of MIA (Fig. 3A). In the control TMJ, HE
staining showed that the condylar cartilage was a regular
alignment of multilayer chondrocytes. S.O and TB staining
showed that the hypertrophic layer was stained red and
metachromatically purple, respectively, indicating abundant
proteoglycans in the condylar cartilage. In the 0.05 mg MIA group,
HE staining showed that the condylar cartilage was slightly
decreased in cell number and thickness as compared with the
control. S.O and TB staining showed slight decreases in cartilage
proteoglycans. In the 0.1 mg MIA group, discontinuousness of the
hypertrophic layer with peripheral cartilage thickening was
observed. However, in the 0.5 mg group, HE staining showed
severe discontinuity of the four-layer cartilage, regional loss of
chondrocytes, peripheral proliferation and clustering of
chondrocytes, a disorganized matrix network, horizontal clefts, and
subchondral bone resorption with adjacent bone marrow filled
with fibroblast-like cells. TB and S.O staining showed severe loss
of staining in irregularly arranged chondrocytes and enhanced
staining at the periphery. In the 1 mg and 2 mg MIA groups,
complete loss of chondrocytes, severe thinning of cartilage, and
subchondral bone erosion were evident in the lesion, but without
peripheral clustering of chondrocytes and thickening of the
cartilage. Typical OA-like destruction of the cartilage and erosion
of the subchondral bone were observed in the 0.5 mg MIA group
. Therefore, 0.5 mg was defined as the minimum effective
dose of MIA for induction of typical OA-like lesions in the rat
Time-dependent Histopathologic Changes in TMJ
To further characterize the development of OA, the major
structures of the TMJ were evaluated after injection of 0.5 mg
MIA at different time points for up to 12 weeks (Fig. 3B, C).
With regard to the condyle, chondrocytes disappeared following
MIA induction and the matrix was less stained within the
proliferative zone in the anterior and central areas of the condyle
corresponding to the load-bearing region. Additionally, scattered
cells with nuclear condensation were evident after 3 days. Loss of
chondrocytes in all of the cartilage layers with no matrix staining
was observed by 1 week. In addition to the above features, regional
osteolysis and peripheral chondrocyte proliferation with deep
matrix staining were observed by 2 weeks. By 4 weeks, typical
OAlike lesions were observed, as described for the 0.5 mg MIA group
in the dose course. By 8 weeks, fibrosis in the lesions was evident
and the subchondral bone was developing sclerosis. By 12 weeks,
the condylar lesions were fully repaired by sclerotic subchondral
bone and thin cartilage with disorganized chondrocytes. These
changes over the 12-week period were not due to aging effects
when compared with the control group. (Fig. 3B).
Time-dependent changes, including synovitis, disc thinning, and
the destruction of temporal fossa cartilage following MIA
induction, are shown in Fig. 3C. Massive fibrin-like exudates
were observed in the upper compartments of TMJs in the
experimental group by 3 days to 1 week after MIA injection, but
not in the control group. Abundant proliferative villi consisting of
multi-layer synovial lining cells and apparent infiltrated
mononucleated cells were present in the upper compartment by 2 weeks.
The synovial villi decreased and became smaller by 4 weeks and
nearly disappeared by 12 weeks. Chondrocytes were almost lost in
the cartilage of the temporal fossa and intermediate zone of the
disc by 3 days after MIA injection. Until 2 weeks,there were almost
no further changes in the disc and temporal fossa. From 4 weeks to
12 weeks, the disc and the cartilage of the temporal fossa became
thinner, but the subchondral bone of the temporal fossa remained
intact and no disc perforation was observed.
Radiographic Changes in Subchondral Bone
To fully understand the changes in the subchondral bone after
MIA-injection (0.5 mg/joint), radiographic changes in the condyle
were evaluated by MicroCT scanning (Fig. 4A). On sagittal
images, the bone surface of the control condyle was smooth and
continuous, whereas the bone surface of the anterior and central
areas of the condyle was discontinuous by 1 week after MIA
injection. Multi-erosions, characterized by translucency disrupting
the bone surface of the load bearing areas, grew deeper and more
extensive with obvious defects from 2 to 4 weeks. By 8 weeks, the
Figure 2. Ultrastructural changes in disc 1 day after MIA injection into upper compartment of TMJ. A. SEM view of the disc. (a). The
furrowed surface (arrow) of the control joint. (b). The surface 1 day after MIA treatment, showing regional flattening in the intermediate zone (dotted
arrow) with residual gelatin condensed to a mass (hollow arrow), surrounded by an area with a rough and uneven surface (arrow). (c). Section view of
the control disc showed the disc cells studded in the collagen fibrils (white arrow). (d). Section view of the disc of the MIA-treated group showed that
disc cells were crimpled and detached from the collagen fibrils (white arrow). B. TEM view of the intermediate zone of the disc. (a). Cells in the control
disc were closely attached to the collagen fibrils (arrowhead) and cell junctions were tight (arrow). (b). The cells in the MIA-treated disc were shrunken
with condensed chromatin (white arrow), detached from the disrupted ECM (arrowhead), and had loose cell junctions (arrow). (c). Mitochondria were
regularly tubular-shaped (arrow) around the nuclei in the control disc. (d). Chromatin compaction (white arrow), swollen mitochondria (arrowhead),
and vacuolar degeneration (dotted arrow) were observed in the disc cells 1 day after MIA treatment. (CF: collagen fibers; N: nucleus; Bar = 50 mm in
Aa, b; Bar = 20 mm in A-c, d; Bar = 0.5 mm in B).
bone surrounding the lesion became sclerotic. By 12 weeks, the
lesion was replaced with smooth but sclerotic bone. Osteophytes
began to present from 4 weeks until 12 weeks after MIA injection.
Hyperalgesia of TMJ after Induction of TMJOA
To understand the relationship between the nociceptive
response and histopathological changes, the HWT was measured
at different time points after MIA injection (0.5 mg/joint).
(Fig. 4B). The HWT significantly decreased 24 h after MIA
injection (P,0.01), remained at a decreased level until 3 weeks
(P,0.05), but then gradually recovered to baseline by 4 weeks, as
compared with the control group.
Induction of Condylar Chondrocyte Apoptosis
To understand the mechanism underlying MIA-induced
chondrocyte loss in the condylar cartilage, TEM examinations
and a TUNEL assay were performed following MIA injection
(0.5 mg/joint). TEM showed that the chondrocytes in the control
group were polygonal with abundant mitochondria and
endoplasmic reticulum, whereas chondrocytes in the group treated with
MIA showed typical apoptotic features, including cell shrinkage,
nuclear condensation, vacuolar degeneration, and apoptotic
bodies, after 1 day (Fig. 5A). Three days after MIA injection,
TUNEL-positive chondrocytes were observed diffusely in the area
corresponding to the region with HE unstained nuclei, but not in
the control group. However, 1 week after MIA injection, the
TUNEL positive chondrocytes almost disappeared in the same
region due to the extreme loss of chondrocytes as shown by HE
staining (Fig. 5B).
Expression of Metabolism and Apoptosis Related Genes
of Condyle after MIA Injection
To further understand the molecular events underlying
condylar destruction following MIA induction (0.5 mg/joint), the
expressions of genes related to the metabolism of cartilage and
bone and apoptosis were examined from the condylar head
contain both cartilage and subchondral bone by real-time PCR
and IHC 2 weeks after MIA injection. As compared with the
control group, mRNA expression of main matrix components,
including aggrecan and collagen I and II, were significantly
downregulated. However, mRNA expression of the matrix
degrading proteases MMP3, MMP13, and ADAMTS5 were
significantly upregulated in the MIA group. In contrast, TIMP2,
but not TIMP1, was correspondingly downregulated (Fig. 6A).
MIA induction resulted in a significant increase in the expression
of the proapoptotic genes of the death receptor family, such as Fas,
FasL, caspase8, caspase3, and BAX, but not caspase2 and
caspase9 (Fig. 6B). PCNA and a-SMA, markers of proliferation
and fibrosis [40,41], respectively, were also upregulated in the
MIA group (Fig. 6B). Moreover, IHC showed that MMP3 was
mainly expressed in the hypertrophic layer in the control cartilage,
but diffuse staining of MMP3 was observed in the chondrocytes
adjacent to the lesion. Stronger staining of caspase3 was observed
diffusely in the proliferative and hypertrophic layers adjacent to
the lesion as compared with the control group. Expression of
aSMA was mainly in the hypertrophic chondrocytes in the control
group, whereas it was enhanced in the chondrocytes of the
proliferative and hypertrophic layers adjacent to the OA-like
lesion by 4 weeks after MIA injection (Fig. 6C).
In this study, we provided multi-level data to show that a
comprehensive rat model of TMJOA could be successfully
established through MIA injection into the upper compartment
of the TMJ. First, electron microscopy showed that the
intermediate zone of the disc loosened to facilitate the diffusion
of MIA into the lower compartment. Second, histopathologic
analysis illustrated that typical OA-like lesions in the TMJ,
including degenerative changes in the condyle, disc, and temporal
fossa, as well as synovitis, were induced by MIA in a dose- and
time-dependent manner. Third, subchondral bone destruction,
which is characteristic of the early stage of OA, and sclerosis,
which is seen during the later stage of OA, were observed by
MicroCT scanning. Fourth, the molecular analysis revealed that
chondrocytic apoptosis and the imbalance between the anabolism
and catabolism of cartilage and subchondral bone might account
for the advanced condylar destruction following MIA induction.
Fifth, nociceptive responses increased in the early stages
corresponding to the presence of synovitis. To the best of our
knowledge, this is the first report to demonstrate that MIA can
effectively induce typical OA-like lesions in the TMJ of a rodent
Figure 5. Apoptosis of chondrocytes in condyle after MIA treatment. A. TEM view of condylar chondrocytes. (a). The chondrocytes in the
control group were polygonal. (b). Chondrocytes treated by MIA were shrunken with vacuolar degeneration after 1 day (dotted arrow). (c). Magnified
photograph of the white frame in (a). Abundant mitochondria (arrowhead) and endoplasmic reticulum (hollow arrow) were observed around the
nuclei (N) in the control chondrocyte. (d). Magnified photograph of the white frame in (b). Apoptotic bodies (black arrow) were observed in the
chondrocyte following MIA induction. (Bar = 0.5 mm) B. Comparison of TUNEL assay and HE staining results. (a) There were few apoptotic
chondrocytes (arrow) in the control group and the corresponding HE staining shown in (b). (c). Diffuse apoptotic chondrocytes were observed in the
region corresponding to the lightly stained area with nuclear condensation of HE staining (black frame in d) at 3 days post-MIA injection. The TUNEL
positive chondrocytes almost disappeared (e) due to the extreme loss of chondrocytes as shown by HE staining (black frame in f) at 1 week.
(Bar = 80 mm).
The histopathologic features of MIA-induced lesions in the rat
TMJ were similar to that of TMJOA. The present study revealed a
typical time- and dose-dependent degeneration of TMJ tissues,
showing the progress of cartilage degradation, erosion, osteophyte
formation, and sclerosis in the subchondral bone, synovitis, and
thinning in the disc and temporal surface. The current results are
similar to the previous description of TMJOA . The lesions
were specifically limited to the load-bearing areas of the condyle.
Although MIA was injected into the upper compartment and
should have a more direct action on the surface of the temporal
fossa than on the condyle, the destruction of condylar cartilage and
subchondral bone was more severe than that of the temporal fossa.
This feature is similar to the clinical and experimental observations
that the condyle is active and undergoes greater destruction and
remodeling [42,43,44]. Interestingly, the disc did not prevent MIA
from penetrating into the lower compartment. In as little as 24 h
the disc cells underwent apoptotic changes, such as cell body
shrinkage and mitochondrial breakage, accompanied by disruptive
ECM and loosened junctions between cells and between cells and
the ECM. These changes facilitated the penetration of MIA
through the disc to the lower compartment after injection into the
The radiographic findings of MIA-induced lesions in the rat
TMJ were similar to that of TMJOA. The typical clinical
radiographic findings for the condyle are erosion, sclerosis, and
osteophytes . All of these features could also be observed with
MicroCT in our MIA-induced rat TMJOA model. Moreover, the
radiographic features of our TMJOA model corresponded well to
the histopathologic changes. Therefore, this MIA model provides
detailed histopathologic changes for the corresponding
radiographic changes. In addition, this model can also be used for in
vivo radiographic analysis of subchondral bone to understand the
pathogenesis of TMJOA, as it already known for knee OA .
Nociceptive responses of MIA-induced TMJOA corresponded
to the observed histopathologic changes. Pain is one of the
predominant clinical features of OA and it may arise from the soft
tissues around the joint or the subchondral bone undergoing
destruction . Therefore, a successful animal model of OA
should have appropriate nociceptive responses corresponding to its
histopathologic changes. The HWT is usually used evaluating
TMJ nociceptive responses and is inversely associated with TMJ
inflammation and pain . We observed that TMJ hyperalgesia
corresponded to the observed histological and radiographic
changes in the MIA-induced TMJOA. Specifically, the
hyperalgesia of TMJ in the first week after MIA injection could be mainly
inflammatory response, whereas in the 24 weeks after MIA
injection, the hyperalgesia could well correspond to the subsequent
pronounced destruction of condylar cartilage and subchondral
bone erosion. When the synovitis was alleviated and cartilage
damage was repaired by fibrous tissue and the subchondral bone
underwent a sclerotic change, the nociceptive responses
correspondingly returned to baseline. This was consistent with known
clinical features. For example, patients often experience severe
pain during the active destructive phase of TMJOA with synovitis
 and feel alleviation over time [47,48]. However, the
hyperalgesia in our TMJOA model recovered to the control level
within 6 weeks, whereas last-long hyperalgesia was observed in the
MIA-induced knee joint OA model . Although the reasons for
this difference are unknown, it might be related to the difference in
the degree of cartilage damage induced by MIA in different joints,
since the same dose of MIA induces more severe cartilage loss in
the knee joint than in the TMJ . It might also be related to the
properties of the different types of cartilage, i.e., the TMJ is
covered with fibrocartilage and the knee joint with hyaline
cartilage. Since the hyperalgesia of the TMJ correspondingly
reflected the degree of lesions induced by MIA, our results also
suggested that MIA-induced TMJOA can be used for evaluating
osteoarthritic pain in the TMJ.
MIA induced TMJOA through chondrocyte apoptosis and the
disturbance of cartilage and subchondral bone metabolism. MIA
could sensitively induce chondrocyte apoptosis as early as 1 day
after MIA injection and condylar apoptosis reached a peak on day
3, leading to hypocellular changes in the cartilage and disc.
Chondrocyte apoptosis in the early stages could be an important
initiator of cartilage degeneration. Genes of the death receptor
family, such as Fas and FasL, have been reported to be related to
chondrocyte apoptosis [50,51,52]. Gene expression of the death
receptor family and IHC staining of caspase3 further showed that
the apoptotic process appeared to be caspase-dependent. This is
consistent with previous studies of OA in the knee [18,22] and
discectomy-induced TMJOA . In addition, cartilage
degeneration also results from the imbalance between anabolism and
catabolism due to increased matrix degrading proteases and
decreased synthesis of matrix . Although the genes expression
was evaluated from the condylar head containing both cartilage
and subchondral bone, the results showed that the catabolic genes
MMP3, MMP13, and ADAMTS5 were elevated in the condylar
head, whereas the anabolic genes aggrecan and collagen I and II
were decreased in the condylar head. The observed changes in
gene expression were similar to previous reports of experimental
OA or clinical OA [55,56]. Therefore, MIA-induced imbalances
in gene expression with regard to cartilage metabolism and
subchondral bone could also be an important factor contributing
to condylar deterioration.
The present model of TMJOA has advantages and
disadvantages. Lack of severe histopathologic changes associated with
TMJOA, such as vertical splitting in the cartilage, exposure of
subchondral bone, and disc perforation, could be one of the
disadvantages for MIA-induced TMJOA model. In contrast,
advantages include the signs of reconstruction, including
hypertrophic reactions in the cartilage surrounding lesions, fibrous
restoration as represented by a-SMA  overexpression in the
proliferative cells, and hypertrophy of the chondrocyte layer at 4
weeks post-MIA injection. In addition, sclerosis of subchondral
bone and osteophyte formation were observed in the later stages,
which mimics the typical clinical features . Lesion repair
following MIA injection was also reported in a rabbit model
[13,23]. TMJOA is a self-limiting disease and reconstruction plays
Figure 6. Changes in gene and protein expression in condyle following MIA injection were evaluated by real-time PCR and IHC,
respectively. A. Two weeks after MIA injection, anabolism-associated aggrecan and collagen I and II were downregulated compared with the
control group. Catabolism-associated MMP3, MMP13, and ADAMTS5 were upregulated and TIMP2, but not TIMP1, was correspondingly
downregulated. B. Two weeks after MIA (0.5 mg) injection, apoptosis-associated genes of the death receptor family, such as, TNFa, Fas, FasL,
caspase8, caspase3, and BAX, but not caspase2 and caspase9, were significantly elevated in the MIA injection group; PCNA and a-SMA, representing
proliferation and fibrous restoration, respectively, were upregulated (mean 6 SEM; n = 6; **P,0.01; *P,0.05). C. There were very few chondrocytes
left in the lesion labeled as L 2 weeks after MIA (0.5 mg) injection. MMP3 was mainly expressed in the hypertrophic layer in the control cartilage (a).
Diffuse staining of MMP3 was observed in the chondrocytes adjacent to the lesion (L) at 2 weeks (b). Caspase3 was rarely expressed in the control
cartilage (c). Enhanced staining of caspase3 was observed in the proliferative and hypertrophic layers adjacent to the lesion (L) at 2 weeks (d).
Expression of a-SMA was mainly in the hypertrophic chondrocytes in the control group (e). Stronger staining of a-SMA was observed adjacent to the
lesion (L) at 4 weeks (f). (Bar = 40 mm).
an important role . However, reconstruction is rarely seen in
TMJOA models induced by methods other than MIA. Although
several animal models of TMJOA have been established, our
multi-level data suggest that the present rat model accurately
mimicked most of the clinical features of TMJOA.
In conclusion, the present study demonstrated a reliable and
simple rat model of TMJOA induced by intra-articular injection of
MIA into the upper compartment. The histopathologic,
radiographic, behavioral, and molecular changes of this model will help
us to understand the progression of TMJOA and to facilitate
future TMJOA-associated researches.
Conceived and designed the experiments: XDW XXK YHG YHZ.
Performed the experiments: XDW XXK MMZ DQH ZM RYB YL JNZ.
Analyzed the data: XDW XXK YHG YHZ. Contributed reagents/
materials/analysis tools: XDW XXK YHG YHZ. Wrote the paper: XDW
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