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Joint Diseases and
Related Surgery
Jt Dis Relat Surg
20 23;34(1):166-175
ORIGINAL ARTICLE
Received: October 25, 2022
Accepted: November 27, 2022
Published online: January 06, 2023
Correspondence: Aras Erdil, DDS, PhD. Uşak Üniversitesi Diş
Hekimliği Fakültesi, Ağız Diş ve Çene Cerrahisi Anabilim Dalı,
6420 0 Uşak, Türkiye.
E-mail: erdil.aras@gmail.com
Doi: 10.52312/jd rs.20 23.901
Osteoarthritis (OA) is a chronic, degenerative joint
disease with multifactorial etiologies affecting the
articular cartilage and bone surfaces. Although
the load-bearing joints such as the knee, hip, and
spine in the body are most affected, it can also be
observed in the joints such as the shoulder and
temporomandibular joints (TMJ).[1] Osteoarthritis
is characterized by pathological changes on the
articular surfaces, including cartilage defibrillation,
erosion of the joint surfaces, chondrocyte
proliferation, subchondral sclerosis, disruption of
Objectives: This study aims to evaluate the efficacy of high-
molecular-weight hyaluronic acid (HMWHA) and lactoferrin
(LF) injections on temporomandibular joint (TMJ) car tilage
and subchondral bone in mono-iodoacetate (MIA)-induced
temporomandibular joint osteoarthritis model in rats.
Materials and methods: In this in vivo study, a total of
24 rats were divided into three groups as follows: saline
group (Group 1), HMWHA group (Group 2), and LF group
(Group 3) including eight rats in each group. The intra-articular
injections were administered once a week for three weeks
after osteoarth ritis was induced. All animals were euthanized
28 days after induction of osteoarthritis, and TMJs were
harvested for histomorphometric, immunohistochemical, and
micro-computed tomography (CT) analysis.
Results: There was no significant difference between the
HMWHA and LF groups in terms of the histomorphometric
and immunohistochemical analysis results (p>0.05). According
to the micro-CT analysis, the LF group had the highest mean
bone volume fraction (74.9±0.5) and trabecular thickness
(0.122±0.002), while the saline group had the lowest mean
valu es (55.0±0.3 a nd 0. 071± 0.0 02, respectively) (p <0.001).
There was no significant difference between the HMWHA and
LF groups according to the micro-CT analysis (p>0.05). Both
groups had better healing effects than the saline group in all
analyses.
Conclusion: Lactoferrin has a healing effect at least as much as
HMWHA in MIA-induced TMJ osteoarthritis. We suggest that
LF may be evaluated in future clinical studies as a promising
agent in the treatment of osteoarthritis.
Keywor ds: High-molecular-weight hyaluronic acid, lactoferr in,
micro- computed tomog raphy, matrix metalloproteinase-3, osteoart hritis,
temporomandibula r joint.
AB STR AC T
Comparison of the efficacy of intra-articular injections
of hyaluronic acid and lactoferrin in mono-iodoacetate-
induced temporomandibular joint osteoarthritis:
A histomorphometric, immunohistochemistry,
and micro-computed tomography analysis
Cansu Gül Koca, DDS, PhD1, Bengisu Yıldırım, DDS, PhD1, Özlem Özmen, MD2,
Muhammed Fatih Çiçek, DDS, PhD1, Mehmet İğneci, DDS1, Özge Kırarslan, DDS1,
Aras Erdil, DDS, PhD1
1Department of Oral and Maxillofacial Surgery, Uşak University Faculty of Dentistry, Uşak, Türkiye
2Department of Pathology, Burdur Mehmet Akif Ersoy University Faculty of Medicine, Burdur, Türkiye
Citation: Gül Koca C, Yıldırım B, Özmen Ö, Çiçek MF, Iğneci
M, Kırarslan Ö, Erdil A. Comparison of the efficacy of intra-
articular injections of hyaluronic acid and lactoferrin in mono-
iodoacetate-induced temporomandibular joint osteoarthritis:
A histomorphometric, immunohistochemistry, and micro-
computed tomography analysis. Jt Dis Relat Surg 2023;34(1):166-
175. doi: 10.52312/jdrs.2023.901
This is an open access article under the terms of the Creative
Commons Attribution-NonCommercial License, which permits use,
distribution and reproduction in any medium, provided the original
work is properly cited and is not used for commercial purposes
(http://creativecommons.org/licenses/by-nc/4.0/).
©2023 All right reserved by the Turkish Joint Diseases Foundation
Intra-articular TMJ OA treatments
167
the cartilage matrix and suppression of the synthesis
of its components.[2]
The data on the efficacy of pharmacological
anti-inflammatory therapies or non-pharmacological
methods such as occlusal splints for long-term
pain management is inconclusive.[3] Intra-articular
injections are relatively less invasive procedures, and
materials such as hyaluronic acid (HA), platelet-rich
plasma, and corticosteroids can be administered.[4]
It has been reported that HA-based scaffolds and
adipose tissue-derived stromal vascular fraction
treatment increased hyaline cartilage formation and
cartilage thickness in osteochondral defects in a
rabbit model.[5]
Lactoferrin (LF) is an 80 kDa non-heme
iron-binding glycoprotein that belongs to the
transferrin family[6] and, recently, its proliferative
and anti-apoptotic properties have been shown.[7, 8]
Chondrocyte apoptosis has been observed in the
pathogenesis of OA. Osteoarthritic cartilage damage
is always associated with increased apoptosis of the
joint chondrocytes, and its suppression reduces the
severity of OA.[9,10]
In the present study, we aimed to compare the
effectiveness of LF and HA in a mono-iodoacetate
(MIA)-induced osteoarthritic rat TMJ with a null
hypothesis that LF and HMWHA could have an
equal healing effect on the subchondral and articular
structure of TMJ.
MATERIALS AND METHODS
This study included a total of 24 male 10-week-old
Sprague-Dawley rats weighing 300 to 320 g with
complete skeletal development. To avoid hormonal
factors and the effects of these factors on bone and
articular cartilage, male rats were preferred instead
of female rats. The rats were randomly divided into
three groups with eight rats in each group. All rats
were housed in wire mesh bottom cages at 22±2°C
with a daily 12-h light/12-h dark cycle. They were
supplied with a regular diet and tap water throughout
the experiment phase ad libitum.
Experimental procedures
General anesthesia was administered by
intraperitoneal injections of 80 mg/kg of ketamine
hydrochloride (Alfamine, Ege-Vet, Izmir, Türkiye)
and 12 mg/kg of xylazine hydrochloride (Alfazyne,
Ege-Vet, Izmir, Türkiye) into each experimental
animal. The trichotomies were performed on the skin
of the left TMJ regions and disinfected with a 10%
povidone-iodine solution after general anesthesia.
In all animals, a 50-µL quantity of 3 mg/mL sodium
MIA (Sigma-Aldrich, Saint Louis, MO, USA) was
injected into the left joints. After waiting four
weeks for the formation of OA, the high-molecular-
weight HA (HMWHA) (Hylan GF-20, Synvisc,
Wyeth, Genzyme, Biosurgery Ridgefield, NJ, USA)
and LF (Sigma-Aldrich Bovine Lactoferrin L-9507,
Saint Louis, MO, USA) injections were administered
once a week for three weeks. The groups were as
follows:
Group 1 (sham injection): n=8, 50 µL saline
administration
Group 2: n=8, 50 µL of 3 mg/mL Hylan GF-20
administration
Group 3: n=8, 50 µL of 100 µg/mL LF admini stration.
The intra-articular injections were administrated
with a 29-gauge needle attached to a 1-mL plastic
insulin syringe. The TMJ was palpated 5 mm
anterior to the external auditory canal and
5 to 10 mm posterior to the lateral canthus of the left
eye, while the mandible was manipulated.[11] When
the mandibular condyle was identified, the needle
was inserted from a posterosuperior direction with
an angle of 30 to 40 degrees to the sagittal plane
under the zygomatic arch. The needle was inserted
2 to 3 mm, until it came into contact with the
posterolateral aspect of the mandibular condyle.[12]
The content of the syringe was administered after
negative aspiration. All injections were performed
by a single surgeon using the same technique
for both inducing OA and administering the
experimental agents.
All animals were euthanized by anesthesia
overdose 28 days after induction of OA in the TMJs.
The TMJ capsule, articular disc, the mandibular
condyle, and the retrodiscal tissues were
harvested for micro-computed tomography (CT),
histomorphometric, and immunohistochemical
analyses.
Micro-CT analysis
All specimens were placed in the holders of
the micro-CT (Scanco Medical µCT50, Switzerland)
and scanned at 70 kVp energy, 114 µA intensity,
300 ms integration time, and 20 µm voxel size.
A volume of interest (VOI: 0.4¥0.4¥0.6 mm) was
selected in each sagittal position of the condyles.
These values were determined for ideal visualization
of the condylar surface and cancellous structure.
All specimens were scanned with an 8 to 10-mm
diameter, and the micro-CT Evaluation Program
version 6.5 (Scanco Medical, Wangen-Brüttisellen,
Switzerland) was utilized for analysis.
Jt Dis Relat Surg168
The morphological changes in the bone tissue
were analyzed with a trabecular bone volume to total
volume fraction (BV/TV). The parameters used to
detect the alterations in the trabecular structure were
as follows:
• The trabecular number (Tb.Nb), calculated
as the average number of trabeculae per unit
length;
• The trabecular thickness (Tb.Th), calculated as
the mean thickness of trabeculae; and
• The trabecular separation (Tb.Sp), calculated
as the mean distance between trabeculae.
Histomorphometric methods
Harvested joint samples were fixed in a 10%
neutral formalin solution for histopathological
and immunohistochemical examinations. After
two days of fixation, samples were decalcified in
ethylenediaminetetraacetic acid (EDTA) (0.1 M)
solution for two weeks. Then, tissue samples were
washed in running water for 8 h and routinely
processed using automatic tissue processing
equipment (Leica ASP300S, Leica Microsystem,
Nussloch, Germany). The samples were embedded in
paraffin wax, and all blocks were sectioned at three
different levels by a fully automatic rotary microtome
(Leica 2155, Leica Microsystem, Nussloch, Germany).
Then, the sections were stained with hematoxylin and
eosin (H&E) stain for histopathological examinations.
Chondrocyte histology, articular cartilage tissue,
and lesions of all sections at the osteochondral and
subchondral bone tissue were examined. Articular
cartilage was evaluated for thickness (regular, thick,
thin), chondrocyte histology (normal, hypocellular,
clustered), and subchondral bone architecture
(regular, increase in trabecular bone). In addition,
osteochondral junctions were evaluated according to
their microscopic appearance as usual, invaginated,
and weak junctions.[13] Histopathological evaluations
were performed in a blinded manner by an
experienced pathologist who was unaware of which
groups the specimens pertained to.
Immunohistochemical examination
The streptavidin-biotin complex peroxidase
method was used for immunohistochemical
examination to coat sections taken from the TMJs
on poly-L-lysine slides. Matrix metallopeptidase-3
(MMP-3) antibody (anti-MMP-3, ab52915; Abcam,
Cambridge, UK) was used for this procedure at a
1/100 dilution. An UltraVision Detection System Anti-
Polyvalent HRP kit (TP-060-HL) (Thermo Shandon
Limited, Cheshire, UK) was used as the secondary
kit, and 3,3´-diaminobenzidine (DAB) was used
as the chromogen (Abcam, Cambridge, UK).
Immunohistochemical analysis was performed
according to the manufacturer's recommendations.
Harris hematoxylin was used as a counterstain, and
the slides were examined under a light microscope
(Olympus CX41, Olympus Corp., Tokyo, Japan).
Antibody dilution solution was used i nstead of primary
antibodies for negative controls. Microphotography
and morphometric analysis were performed
using the Database Manual Cell Sens Life Science
Imaging Software System (Olympus Corp., Tokyo,
Japan). Immunohistochemical staining was scored
semi-quantitatively between 0 and 3: 0= negative,
1= mild expression, 2= moderate expression, and
3= high expression. Statistical analysis was performed
on the scores, and differences between groups were
evaluated.
Statistical analysis
The study power analysis and sample size
calculation were performed using the G*Power
version 3.1 software (Heinrich-Heine-Universität
Düsseldorf, Düsseldorf, Germany).[14] The sample size
was estimated using data from a previous study.[15]
With an alpha value of 0.05 and statistical power of
80%, a minimum of 24 animals were required.
Statistical analysis was performed using the IBM
SPSS version 21.0 software (IBM Corp., Armonk,
NY, USA). Descriptive data were expressed in
mean ± standard deviation (SD) or number and
frequency, where applicable. Considering the
micro-CT and histomorphometric results, one-way
analysis of variance (ANOVA) was used for
normally distributed data, and Kruskal-Wallis and
Mann-Whitney U tests were used for non-normally
distributed data. The Duncan test evaluated
differences between the groups obtained in the
immunohistochemical analysis. A p value of <0.05
was considered statistically significant.
RESULTS
The condylar surface and trabecular structure
analysis
Statistically significant differences were found among
the groups for BV/TV (p<0.001). The LF group showed
the highest mean BV/TV value (74.9±0.5), while the
saline group showed the lowest (55.0±0.3) (p<0.001).
There were also statistically significant differences
among the groups for Tb.Th (p=0.001). The LF group
had the highest mean Tb.Th (0.122±0.002), while the
saline group had the lowest mean value (0.071±0.002).
We observed statistically significant differences
Intra-articular TMJ OA treatments
169
among the groups for the mean Tb.Nb (p<0.001).
The Tb.Nb for the saline group was significantly
lower than that for the other two groups (p<0.001),
and no significant difference was found between
the HMWHA and LF groups (p=0.918). Likewise,
statistically significant differences were observed
among the groups for Tb.Sp (p<0.001). The Tb.Sp for
the saline group was significantly lower than that of
the other two groups (p<0.001), and no significant
difference was found between the HMWHA and LF
groups (p=0.78) (Table I). The osteolytic degenerations
in bone tissue are illustrated in Figure 1 and Figure 2.
Inflammatory articular cartilage alterations
Histopathological evaluation revealed
inflammatory cell infiltration around the TMJs. The
most severe inflammatory reactions were observed
in the saline group, and HMWHA and LF treatments
relatively caused reduced inflammatory reactions
(Figure 3a-c). The articular cartilage tissues were
markedly thinned in the saline group compared to
the HMWHA and LF groups. The HMWHA and LF
groups were similar to the regular appearance, and
the hypertrophic joint thickness was observed more
TAB LE I
Comparison of micro-CT parameters among groups
95% CI
Micro-CT parameters Study groups nMean±SD Lower bound Upper bound p
BV/TV (%)
Saline 755±0.3a54.7 55.3 < 0.0 01¶
Lactoferrin 77 7.1±0.7 b76.4 77.8
HMWHA 774. 9±0. 5c74.5 75.3
Trabecular thickness
Saline 70.071±0.002a0.069 0.073 0.001‡
Lactoferrin 70.122±0.002b0.120 0.125
HMWHA 70.118±0.001c0.117 0.118
Trabecular number
Saline 744.2 ±1.1a43.2 45.2 Sal vs. LF < 0.0 01†
Lactoferrin 753. 4±1. 3b,c 52.2 54.6 Sal vs. HA <0.001†
HMWHA 752.7±1.5 c51.2 54.1 LF vs. HA =0.918†
Trabecular separation
Saline 70.092±0.002a0.090 0.094 Sal vs. LF <0.0 01†
Lactoferrin 70.054±0.001b,c 0.052 0.055 Sal vs. HA <0.001†
HMWHA 70.056±0.001c0.055 0.057 LF vs. HA=0.78†
CT: Computed tomography; CI: Confidence interval; SD: Standard deviation; BV: Bone volume; TV: Total volume; HMWHA: High-molecular-weight hyaluronic acid;
¶ One-way ANOVA, post-hoc Bonferroni; ‡ Kruskal-Wallis; Different superscript letters represent significant differences among groups (p<0.05).
FIGURE 1. Representative micro-CT images from study groups. (a) HMWHA group, (b) Saline group, (c) LF group.
CT: Computed tomography; HMWHA: High-molecular-weight hyaluronic acid; LF: Lactoferrin.
(a) (b) (c)
Jt Dis Relat Surg170
often than the reduced joint thickness. While the
highest hypocellularity was seen in the saline group,
hypocellularity was slightly higher in the HMWHA
group than in the LF group, but the numbers of
clusters were equal. The rats in all groups exhibited
invagination, and a weak junction was primarily
seen in the saline group and the least in the LF
group. The saline group noted relatively increased
trabecular bone tissue, while the other groups
exhibited a more normal appearance. The HMWHA
and LF groups had better healing than the saline
group (Figure 3a-c). The numerical distribution of
histopathological findings of TMJs and statistical
analysis are shown in Table II.
MMP-3 levels among groups
The immunoexpression of MMP-3 was increased
in the saline group compared to the other groups.
A marked decrease was observed in the LF group,
while a mild increase was observed in the HMWHA
group (Figure 4). Statistical analysis results of the
immunohistochemical scores are shown in Table III.
FIGURE 2. Micro-CT images showing the changes of the bone structure in each group. (a) (HMWHA group), (b) (Saline group) and
(c) (LF group) images were obtained after image recording, and the natural bone structure is shown in blue and osteolytic bone
changes are shown in red.
CT: Computed tomography; HMWHA: High-molecular-weight hyaluronic acid; LF: Lactoferrin.
(a) (b) (c)
FIGURE 3. Microscopical appearance of the joint cartilage between the group, (a) decreased cartilage thickness (double headed
arrow), decreases in articular (thin arrow) and proliferative (medium arrow) zones, lack of hypertrophic and transition zones in
saline group; (b) relatively thickened joint cartilage in HMWHA group with slight increase in articular (thin arrow), proliferative
(medium arrow) and transition (thick arrow) zones and without hypertrophic zone, (c) increase cartilage thickness and marked
articular (thin arrow), proliferative (medium arrow), transition (thick arrow) and hypertrophic (arrow head) zones in LF group (H&E,
Scale bars= 50 µm).
HMWHA: High-molecular-weight hyaluronic acid; LF: Lactoferrin.
(a) (b) (c)
Intra-articular TMJ OA treatments
171
DISCUSSION
Regarding the current results, the intra-articular
injections of HMWHA and LF in the MIA-induced
TMJ OA model revealed statistically improved
outcomes compared to the control group in
histopathological, immunohistochemical, and
micro-CT analyses. The null hypothesis of the study
was accepted, as there was no significant difference
between the outcomes of intra-articular HMWHA
and LF injections, except for hypocellularity and
MMP3 levels. Therefore, the LF injections were
at least as effective as HMWHA injections on
MIA-induced TMJ OA.
The effect of exogenous HMWHA on TMJ OA has
been previously evaluated and the r esearchers argued
that degenerative changes in the articular cartilage
in the HMWHA group were significantly less than
those in the control group.[13] Tolba et al.[16] and Lemos
et al.[17] reported that HMWHA had a protective
effect on cartilage by regulating endogenous
HA, glycosaminoglycans, collagen synthesis, and
preventing proteolytic enzymes, the inflammatory
response, cartilage friction by providing lubrication.
In the current study, articular cartilage was observed
to be regular in four joints, and degenerative changes
were observed in four joints (one thinning, three
thickenings) in the HMWHA group. However, in the
control group, in all samples, degenerative changes
were observed (five thinning, three thickenings).
The difference between these two groups was
considered to be significant. The results of this
study are consistent with the literature and indicate
that HMWHA protects cartilage tissue. Another
factor affecting articular cartilage degeneration is
chondrocyte apoptosis.[13] Several studies have shown
that one of the repair functions of HMWHA in TMJ
OA is its chondroprotective effect.[16,18] Chondrocyte
appearance is an early change in TMJ OA, which
is vital, as hypocellularity is associated with
chondrocyte apoptosis, and clustering is associated
with the repair response.[13,19] In the current study,
normal chondrocyte appearance in four joints,
hypocellularity in two joints, and clustering in two
joints were observed in the HMWHA group.
In contrast, chondrocyte appearance in one joint,
hypocellularity in four joints, and clustering in
TAB LE I I
The distributions of articular cartilage changes in all groups
Study groups
Articular cartilage changes Saline
(n)
HMWHA
(n)
LF
(n)
Cartilage
Normal 044
Thinned 511
Thickened 333
Osteochondral Junction
Normal 345
Invagination 222
Weak Junction 321
Subchondral bone Normal 266
Increase in trabecular bone 622
Chondrocytes
Normal 145
Hypocellularity 421
Cluster 322
HMWHA: High-molecular-weight hyaluronic acid; LF: Lactoferrin.
TABLE III
Comparison of MMP-3 levels among groups
Study groups nMean±SD p
Saline 82.3±0.5a
HMWHA 81.3±0.9 b<0. 001†
LF 81.4±0.5b
MMP-3: Matrix metallopeptidase-3; SD: Standard deviation; LF: Lactoferrin;
HMWHA: High-molecular-weight hyaluronic acid; † denotes the p value
obtained by one-way ANOVA test. The Duncan test was performed to detect
the inter-group differences. The mean ± standard deviation values carrying
the different indices were significantly different.
Jt Dis Relat Surg172
three joints were observed in the control group.
As previously reported, the difference between the
groups was not considered significant.[13] However,
the authors argued that although there was no
significant difference between the groups, the
number of joints that appeared normal and clustered
in the HMWHA group was higher than in the control
group, inducing the repair response of HMWHA
and could be effective in reducing the frequency
of chondrocyte apoptosis. Another degenerative
change observed during the early period of TMJ
OA may occur in subchondral bone tissue. It has
been reported that the decrease in prostaglandin
(PG) levels and some alterations in the cartilage
tissue increase the destructive factor of the force
on the subchondral bone and affect the incidence
of pathological changes.[13, 20] In the literature, it has
been reported that exogenous HMWHA positively
affects subchondral bone tissue by concentrating in
load-bearing areas during TMJ function, reducing
the destructive effect of the incoming load with
its lubricative characteristic, and increasing PG
synthesis.[13] Also, in the current study, six normal
joints and trabec ular bone increases in two joints were
observed in the HMWHA group. Two normal and
trabecular bone increases in six joints were observed
in the control group, and the difference between the
groups was found to be significant. Furthermore,
changes in the subchondral bone and articular
cartilage structure may affect the osteochondral
junction.[13] As a result of structural changes, the
destructive effect of the force on the joint increases,
which may cause some changes in the osteochondral
junction.[13] In the current study, four joints were
observed as regular, and two joints as invaginate
and weak in the HMWHA group. Three joints were
observed as regular, two as invaginate, and three as
weak in the saline group. The difference between the
groups was not considered significant, as previously
indicated.[13] In addition, although the difference
was not significant, the authors considered that the
higher number of joints with normal osteochondral
junctions in the HMWHA group might be due to the
protective effect of HMWHA on articular cartilage
and subchondral bone tissue.
There are studies, albeit limited, indicating the
effects of LF on joints with OA. The anti-inflam matory
effects were indicated with a significant decrease
in interleukin (IL)-1 and IL-6 levels in the joints,
cartilage, and synovial fluid with OA in the LF
group.[ 21] Likewise, Xue et al.[22] induced OA in
rat knees and reported that LF caused a marked
decrease in IL-1 levels, which was achieved by the
anti-inflammatory effect of LF. In this study, the
typical structure of the articular cartilage in four
joints, thickening in three joints and thinning in one
joint in the LF group, and degenerative changes were
observed in all the joints in the control group. In the
literature, the protective effect of LF on the articular
cartilage is significant.
The present study differs from other studies
regarding the amount and frequency of LF
administered and the joint for which OA was induced.
Lactoferrin is a potent regulator of chondrocyte
metabolism and eliminates some mediators that
cause chondrocyte apoptosis.[21,22] In this study,
five normal joints, hypocellularity in one joint, and
clustering in two joints were observed in the LF
group. The typical structure was observed in one
joint, hypocellularity in four joints, and clustering
in three in the control group. Although there was
no significant difference between the groups, we
believe that the low hypocellularity in the LF group
FIGURE 4. MMP-3 expressions in TMJ between the groups. (a) Marked increased in expression in saline group (arrows),
(b) decreased expression (arrows) in HMWHA group, (c) slight expression in LF group (arrow), Streptavidin-biotin peroxidase
method, Scale bars= 20 µm.
MMP-3: Matrix metallopeptidase-3; TMJ: Temporomandibular joints; HMWHA: High-molecular-weight hyaluronic acid; LF: Lactoferrin.
(a) (b) (c)
Intra-articular TMJ OA treatments
173
may be due to its protective effect on chondrocytes
following the literature. Wang et al.[11] reported that
osteoclastic activity triggered by vascular endothelial
growth factor in TMJ OA might be among the factors
causing degenerative changes in subchondral bone
tissue. In this study, subchondral bone was regular
in six joints, while trabeculation was increased in
two joints. In the control group, subchondral bone
was regular in two joints, while trabeculation was
increased in six joints, and the difference between
the groups was significant. We believe that the lower
amount of degenerative changes observed in the
articular cartilage and the possibility of suppression of
osteoclastic activity with the anabolic effect may have
led to fewer degenerative changes in the subchondral
bone. In the LF group, the osteochondral junction was
regular in five joints, invaginated in two joints, and
weak in one joint, while it was regular in three joints,
invagination in two joints, and weak in three joints in
the control group. Although there was no significant
difference between the groups, articular cartilage
and subchondral bone tissue in the LF group were
significantly healthier regarding the number of joints
with normal osteochondral junctions.
The histomorphometric results of this study
revealed non-significant differences between the
LF and HMWHA groups in articular cartilage,
chondrocyte appearance, and the subchondral bone
or osteochondral junction. Also, both agents had a
positive effect on OA. Since there is no similar study
available in the literature, we speculate that these
results were achieved with the specified effects on
the joint for both agents. In addition, HA has the
unique characteristic of providing lubrication on the
TMJ and being the primary component of synovial
fluid. Achieving successful results in joints with
OA validates these characteristics of exogenous HA.
A previous study reported that LF stimulated HA
synthesis in dermal fibroblasts.[23] In this study, the
possibility of exogenously administered LF affecting
HA synthesis might be a factor leading to similar
results for the two agents. Another factor may be
that LF is a component of synovial fluid, such as HA,
and its levels become elevated through exogenous
administration. In the present study, LF deficiency
was an essential factor in joints with rheumatoid
arthritis, and treatments with exogenous LF were
successful.
One of the primary changes seen in TMJ OA is a
significant increase in MMP expression. In particular,
MMP-3 is primarily produced by synovial cell lines
and activates many MMP family members, playing
a role in many degenerative functions, including
articular cartilage destruction.[16] Tolba et al.[16]
reported that HMWHA significantly decreased
MMP-3 levels in the joint where TMJ OA was induced.
A significant difference was observed in this study
between the HMWHA group (1.25±0.88) and the
control group (2.25±0.53). Also, Yan et al.[21] showed
that LF significantly suppressed the expression of
the cartilage-degrading enzymes MMP-1, MMP-3,
and MMP-13 and exerted anti-inflammatory and
anti-catabolic effects. In this study, the LF group
(1.37±0.51) significantly suppressed MMP-3 expression,
and this finding is consistent with the effect of LF on
MMP-3, as reported in the literature.
Kim et al.[24] and Shi et al.[25] demonstrated that
the decrease in BV/TV value directly affected the
bone quality. In addition, the BV/TV value, not
alone, but its conformity with other parameters,
provided a complimentary analysis of the effect of
OA on the TMJ microstructure (Tb.Th, Tb.Sp, Tb.Nb).
Significant decreases in BV/TV, Tb.Th, Tb.Nb values
and a significant increase in Tb.Sp values indicate
bone loss and structural degeneration.
Additionally, a decline in Tb.Th values, while
BV/TV and Tb.Nb values increased in the TMJ with
OA indicate an endochondral maturation pathway,
but not an increase in osteoblastic activity.[26] In the
current study, except for Tb.Sp, all parameters were
significantly higher in the LF and HMWHA groups
than the saline group, indicating a more protective
effect on the joint in terms of bone quality and loss.
Also, the LF group had significantly higher BV/TV
and Tb.Th values than the HMWHA group indicating
the possible superior effect of LF on the repair
response in the TMJ.
To the best of our knowledge, the current
study is the first to compare the efficacy of
intra-articular injection of LF with HMWHA in an
MIA-induced TMJ-OA. The consistency between
histomorphometric, immunohistochemical, and
micro-CT findings of the study increases the
authenticity of the healing effect observed in
response to LF and HMWHA on TMJ OA. LF, which
has been reported to have no toxic effects in studies
conducted thus far and is already a part of synovial
fluid and is a promising agent in treating TMJ OA.
Nevertheless, this study has certain limitations.
First, the long-term effects of LF and HMWHA on
TMJ OA were unable to be evaluated, as the study
is not longitudinal in nature. Another limitation is
that LF was used as a single dose with a uniform
administration frequency and, therefore, no
information concerning the ideal dose or frequency
Jt Dis Relat Surg174
of administration could be obtained. Also, there are
some methodological limitations such as induction
of OA. In the current study, TMJ OA was chemically
induced. In the MIA-induced OA model, diffuse cell
death and rapid destructive changes were observed,
unlike spontaneous or posttraumatic OA.[28] As a
result, although clinical features can be mimicked in
rapidly progressive pathological changes such as in
the current model, severe immediate histopathological
changes observed in human OA were absent.[29]
The absence of TMJ OA-related histopathological
changes, such as disc perforation, subchondral bone
exposure, and vertical splitting in the cartilage,[30] can
be considered a disadvantage in this model. It should
be kept in mind that this feature of the model may
impact the effects of the treatment methods applied.
In addition, the effects that may vary according
to dose-dependent MIA administrations were not
investigated in the current study. Furthermore, the
animals used in the present study were 10-week-old
male rats. However, whether age-related efficacy
changes in MIA-induced models is an issue that needs
further investigation.
In conclusion, our study results indicated that
the administration of 50 µg of LF at 100 µg/mL a nd
150 µg of HMWHA once a week for three weeks
in a MIA-induced rat TMJ OA model resulted
in significant therapeutic effects, without any
differences between the study groups. Based on
these findings, we believe that LF may be as
effective as HMWHA in the treatment of TMJ OA by
determining the appropriate doses and frequency
of administration.
Ethics Committee Approval: The study protocol was
approved by the Afyon Kocatepe University, Animal
Research and Ethics Committee (date: 03.06.2020, no:
AKUHADYEK-221-20). The study was conducted in accordance
with the principles of the Declaration of Helsinki.
Data Shar ing Statement: The data that support the findi ngs
of this study are available from the corresponding author upon
reasonable request.
Author Contr ibutions: Provided input into the concept
and design of the study: C.G.K., B.Y.; Provided the materials,
analyzed the data, carried out literature review: C.G.K.,
B.Y., Ö.Ö., Ö.K., A.E.; Supervision of the study: C.G.K., Ö.Ö.;
Collected and assembled the data: M.F.Ç., M.İ.; Wrote the
article: C.G.K., B.Y., A.E.; All authors have critically revised
the article, read and approved the final version at the time of
submission.
Conflict of Interest: The authors declared no conflicts of
interest with respect to the authorship and/or publication of
this article.
Funding: The authors received no financial support for the
research and/or authorship of this article.
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