ORIGINAL�ARTICLE

ABSTRACT
Objective: To evaluate and compare the chondroprotective effects of Hyaluronic acid and triamcinolone at 
radiographic level in rat model of osteoarthritis.
Study Design: Laboratory based Randomized control trial.
Place and Duration of Study:  This study was conducted in Pharmacology Department, Army Medical College, 
Rawalpindi, from May to July 2019.
Materials and Methods: Osteoarthritis was induced by medial meniscus and anterior cruciate ligament 
resection in right knee joints of twenty-four rats. They were divided in three groups with eight rats in each. 
Group I, II and III were treated with intra articular saline, hyaluronic acid, and triamcinolone once weekly for 
four weeks respectively. After one week, radiographs of corresponding knee joint of anesthetized rats were 
taken.
Results: Collective comparison of radiographs of control, hyaluronic acid and triamcinolone groups exhibited a 
p value of 0.001 While intergroup comparison of group I and II, group I and III and group II and III depicted p 
value of 0.05, 0.01 and 0.01 respectively. 
Conclusion: Intra articular administration of hyaluronic acid and triamcinolone exhibited chondroprotective 
effects at radiological level in a rat model of osteoarthritis. On comparison of treatment groups, it was 
concluded that hyaluronic acid has better chondroprotective effects as compared to triamcinolone.

Key Words: Chondroprotective Effects, Hyaluronic Acid, Osteoarthritis, Rat Model, Triamcinolone.

world. Approximately 10% males and 18% females 
aged over 60 years are suffering from OA all around 
the globe. Basis of OA is degradation and chronic 
inflammation of the connective tissue of the joint, 
including the cartilage. Due to long term damage to 
chondroblasts, chondrocytes and extra cellular 
matrix caused by oxidative stress, as well as 
inflammatory factors and mitochondrial dysfunction 
that cause DNA damage are the leading factors of 
initiation and progression of OA. Currently few 
options are available for treatment and prevention 
of OA and many drugs are in the phase of 

2,3,4
investigation.   treatment of OA in modern era Drug
is still limited to viscosupplement substances, non-
steroidal anti-inflammatory drugs (NSAIDs) and 

5
corticosteroids.  
Hyaluronic acid (HA), a macro molecule of repeating 
unit of D glucuronic acid and D acetyl glucosamine, is 
major constituent of synovial fluid which helps to 
facilitate lubrication and shock absorption in joints. It 
is used in OA for viscosupplementation. It is a 
naturally occurring substance present in synovial 
fluid. It restores the viscoelasticity of the synovial 

Introduction
Osteoarthritis (OA) is the most common joint disease 
of old age, heterogeneous in character, affecting 

1
commonly joints of the hand, knee, hip and spine.  It 
is one of the leading causes of long term pain and 
disabilities. Overall old age population is increasing 
with improvement in health service in modern era. 
So with increase in old age people, Incidence and 
prevalence of OA is progressively increasing. It 
affects around 240 million people all around the 

Radiographic Evaluation and Comparison of Chondroprotective Effects of 
Hyaluronic Acid and Triamcinolone in a Rat Model of Osteoarthritis

1 2 3 4 5
Noaman Ishaq , Muhammad Bilal Shahid , Samia Yasin , Nausheen Ata , Muhammad Waqar Aslam Khan , 

6
Malik Sikandar Mehmood

Correspondence:
Dr. Noaman Ishaq
E-mail: noamanishaq@yahoo.com

1
Department of Pharmacology

Bakhtawar Amin Medical and Dental College, Multan
2
Department of Surgery

Mayo hospital Lahore
3
BHU Chak 60 GB Jaranwala, Primary and secondary health 

department, Punjab
4,6

Department of Pharmacology
Army Medical College, Rawalpindi
5
Department of Pharmacology

CMH Kharian Medical College, Kharian 

Funding Source: NIL; Conflict of Interest: NIL
Received: March 02, 2021; Revised: August 16, 2021
Accepted: August 20, 2021

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165



fluid, may have anti-inflammatory and anti-
nociceptive properties. It also stimulates denovo HA 

6,7
synthesis.  Triamcinolone, an intermediate acting 
synthetic corticosteroid, is quite effective and is one 
of the most common drugs used to relieve symptoms 
in OA. It shows anti-inflammatory activity via 
inhibition of gene expression of prostaglandins and 
other inflammatory substances. So, it not only 
relieves pain but shows functional improvement and 
chondroprotective effects. It also influences 
macrophage activation and eventually decreases 

 8,9
osteophytosis and cartilage degeneration.  
Definite cure of OA is not currently available. HA, 
triamcinolone, and many other drugs are 
investigational and are used to reduce symptoms 
and delay the progression of disease. Aim of this 
s t u d y  i s  t o  e v a l u a t e  a n d  c o m p a r e  t h e  
chondroprotective effects of HA and triamcinolone 
at radiographic level in a rat model of OA.

Material and Methods
It was a Laboratory based randomized control trial 
that was carried out in department of Pharmacology 
and Therapeutics, Army Medical College (AMC), 
Rawalpindi in collaboration with National Institute of 
Health (NIH), Islamabad. Ethical approval certificate 
was endeavored from ethical review committee of 
“Centre for Research in Experimental and Applied 
Medicine (CREAM)”, AMC. Tenure of rat's 
intervention was two months from May 2019 to July 
2019. They were kept and nurtured in animal house 
of NIH during the complete study period. Preliminary 
twenty-four (24) adult male or non-pregnant female 
rats of Sprague Dawley breed, approximately 10 
weeks old and weighing about 500 grams were 
selected through nonprobability convenient 
sampling. They were randomly assigned in three (03) 
groups with eight (08) rats in each group. Group I, II 
and III were labelled as Control, HA and 
triamcinolone groups respectively. Rats were kept in 
standard environment with temperature ranged 
25±5˚C, adequate humidity, and 12 hours day night 
cycle. Free excess to clean drinking water and 
standard rodent diet ad libitum was ensured during 
the whole study period. Surgical procedure was 
performed to induce OA in right knee joint of all rats. 
Before surgery rats were anesthetized with 
intraperitoneal injection of 5% xylazine and 1% 

10
ketamine.  Skin of the joint was shaved in a sterilized 

environment. Then a para patellar incision was made 
on medial side for complete exposure of the joint. 
Anterior cruciate ligament and medial meniscus 
were identified and transected. Aseptic closure of 
wound with surgical stapler was done after the 
completion of the surgery. Animals were allowed to 

11
move freely in the cage for two weeks thereafter.  
Then intra articular drugs were administered in the 
corresponding joint of the rats. Rats of control, HA 
and triamcinolone groups were injected with 0.2 ml 
of Normal saline, 0.2 ml HA, and 70 µl (1.4 mg/ml) 
triamcinolone respectively once weekly for 04 

12,13,14
weeks.  Thereafter Animals were anesthetized 
with intraperitoneal injection of xylazine 10% and 
ketamine 1%, transported to Radiology department 
of a private institute of Rawalpindi where their knee 
joints were radiographed. 500mA digital X ray 
machine China operated at 220 V with a 0.3 sec 
exposure time was used for radiography. Kellgren 
and Lawrence system was considered to grade the 

  
severity of the OA. According to Kellgren and 
Lawrence grading system grade 0 characterizes no 
radiographic features of OA, grade 1 represents 
doubtful joint space narrowing (JSN) and possible 
osteophyte lipping, grade 2 portrays definite 
osteophytes and possible JSN on anteroposterior 
weight-bearing radiograph, grade 3 depicts multiple 
osteophytes, definite JSN, sclerosis, possible bony 
deformity while grade 4 describes large osteophytes, 
marked JSN, severe sclerosis and definite bony 

 15,16
deformity.  After radiographic grading with 
Kellgren and Lawrence system, animals were 
euthanized with toxic dosage of chloroform. 
Obtained data was analyzed using IBM SPSS version 
23.  Groups were analyzed through kruskul wallis test 
followed by Post Hoc Tukey HSD test. The differences 
between two interpretations were considered 
statistically significant if the p value was equal to or 
less than 0.05 (p≤0.05) 

Results
Two radiographs (25%) of group I (control group) 
depicted severe changes of OA and graded as grade 
04, while four (50%) radiographs depicted moderate, 
and two (25%) radiographs depicted minimal 
changes of OA and they were graded as grade 03 and 
grade 02 respectively. Figure 01 is an X-ray of a rat of 
control group that has characteristic features of 
sclerosis and possible joint deformity. Grade of this 

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X-ray is 03. Most of the radiographs of group I feature 
osteophytes, JSN and bone deformity. 
Radiographic changes of group II (HA group) that 
received IA injection of HA described no radiographic 
changes of OA in two radiographs (25%), doubtful 
changes in four (50%) and minimal changes in two 
(25%) radiographs and they were graded as grade 0, 
grade 01, and grade 02 respectively. Figure 02 is an X-
ray of a rat of HA group that depicts the feature of 
minimal changes of OA. Grade of this X-ray is 02. No 
or doubtful changes of OA were the feature of 
radiographs of this group. Radiographic changes of 
group III (triamcinolone group) that received IA 
injection of triamcinolone depicted as moderate 
radiographic changes of OA in four (50%), minimal 
changes in three (37.5%) and doubtful changes in 
one (12.5%) radiograph and were graded as grade 
03, grade 02 and grade 01 respectively. Figure 04 is 
an X-ray of rat of triamcinolone group that depicts 
moderate changes of OA. Grade of this X-ray is 03. 
Most of the radiographs showed features of 
osteophytes and JSN in this group. When Kruskul 
wallis test was applied between all the three groups, 
p value was 0.001 that was statistically significant 
thus claiming the chondroprotective effects of 

hyaluronic acid and triamcinolone at radiographic 
levels. Intergroup comparison of groups via post hoc 
tukey HSD test depicted p values as showed in Table 
01. Comparison of HA group and triamcinolone 
group exhibited p value of 0.01 that confirmed HA 
has superior chondroprotective effects as compared 
to triamcinolone.

Fig 1: Radiograph of a rat of group I
(Control group)

Fig 2: Radiograph of a rat of group II 
(Hyaluronic Acid group) 

Fig 3: Radiograph of a rat of group III 
(Triamcinolone group) 

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Discussion
Viscosupplement substances, nonsteroidal anti-
inflammatory drugs (NSAIDs) and corticosteroids are 
group of drugs that are investigational but frequently 

17 
used in the management of HA, a osteoarthritis.
viscosupplements, is a natural component of 
synovial fluid that helps to facilitate lubrication and 
shock absorption in joints. It is one of the many 
investigational molecules in the treatment of OA. 
Viscosupplementation with intra articular HA has 
positive outcome in pain alleviation and joint 

18
function improvement in OA.  Triamcinolone, a 
synthetic corticosteroid is being used in the 
management of OA for decades. Triamcinolone 
binds to intracellular glucocorticoid receptors and 
down regulates the expression of genes in 
prostaglandins synthesis and leukotrienes release. 
Moreover, it also enhances lipocortin expression 
that modulates anti-inflammatory effects. These 
factors ultimately lessen the inflammation of 

19,20
synovium and other articular structures in OA.
 

This animal study is planned to evaluate and 
compare the chondroprotective effects of HA and 
Triamcinolone. Human admissible dosage of these 
drugs that was proficiently effective in rat model was 
selected by substantial search and literature review.  
OA was induced by medial meniscus and anterior 
cruciate ligament resection in the rats. After that 
they were randomly divided in control group, HA 

group and triamcinolone group with eight rats in 
each group. Control group was intra articularly 
t re a t e d  w i t h  s a l i n e  w a t e r  w h i l e  H A  a n d  
triamcinolone groups were treated with intra 
articularly HA and triamcinolone respectively. Later, 
when intervention protocol was completed, 
chondroprotective effects of these drugs were 
analyzed by radiographs. When radiographic grades 
of treatment group were statistically compared with 
control group, we found a significant p value of 0.001 
that confirmed the chondroprotective effects of HA 
and triamcinolone.  
 Our research work is supported by 2016 research 
work of Zhiwei Zhang who found that HA reduces 
radiographic osteophytosis grading (p<0.05) as 
compared to saline treated rats' model of 

21
osteoarthritis.  Likewise Ai Tong worked to evaluate 
the chondroprotective effects of cocktail of 
mesenchymal cells and HA in chemical induced rat 
model of OA. He assessed rats through Magnetic 
resonance imaging and found significant (p <0.05) 

22
chondroprotective effects of this mixture.  Their 
results also favor our outcome regarding HA. 
Similarly, research work of Yunus Emre proved that 
HA exhibit chondroprotective effects at histological 

23
level (P =0.04) in rat model of OA.
In 2017 Yashashri C. Shetty did research work on 
chemical induced models of rats. His results 
confirmed that triamcinolone reduces the 
histopathological severity of disease (p<0.01) as 

24
compared to disease control group.  Likewise 
research of Jeffrey S. Kroin declares that 
triamcinolone lessens allodynia as compared to mice 
of control group (p value <0.01). Their work at tissue 
level expressed that triamcinolone has potent anti-
inflammatory effects (p value <0.01) via inhibiting 

25
expression of IL- 1β and TNFα.  Furthermore in vitro 
research of E. Frank also confirmed that 
triamcinolone has some chondroprotective effects 
on damaged and swollen cartilage (p value <0.05) via 
i n h i b i t i n g  s u l f a t e  a s s i m i l a t i o n  a n d  
glycosaminoglycan loss. Results of these studies 
s t r e n g t h e n i n g  o u r  o u t c o m e  r e g a r d i n g  

2 6
chondroprotective effects of triamcinolone.  
Consequently chondroprotective effects of HA and 
triamcinolone that is confirmed by current research 
and supported by previous research favor the use of 
these investigational drugs in patients of OA and 

Table I: Intergroup Comparison of Radiographs When 
Post Hoc Tukey Test HSD is Applied

Graph 1:  Kellegren and Lawrence Grading of OA Knee 
Joints of Rats of all Groups

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provides a template for future studies.
When Radiographs of HA group was statistically 
compared with triamcinolone group, it was found 
that HA is more efficacious than triamcinolone. 
These results are according to the research work of 
Yashashri C. Shetty who found that HA has better 
chondroprotective effects than triamcinolone in 

27
chemically induced murine model of OA (p <0.001).  
Likewise, human study by Soad A Elsawy portrayed 
better chondroprotection offered by HA as 

28 
compared to triamcinolone (p=0.01). Similarly, 
meta-analysis by Egemen Ayhan and colleagues also 
verified that HA has superior chondroprotective 

29
efficacy as compared to triamcinolone.

Acknowledgement 
We are highly obliged to Dr. Hussain Ali, Head of 
research animal Facility, National Institute of Health 
(NIH), Islamabad for his supervision regarding 
osteoarthritis induction and intra articular drug 
administration in rats. We also owe special thanks to 
Pharmacology department, Army Medical College, 
Rawalpindi, for providing us opportunity to carry out 
this research work.

Conclusion
Intra-articular administration of 0.2 ml (10mg/ml) 
hyaluronic acid and 70 µl (1.4 mg/ml) triamcinolone 
once weekly for 04 successive weeks reduced 
severity of osteoarthritis in rat model at radiographic 
level. Upon Comparison of hyaluronic acid and 
triamcinolone, it was concluded that former has 
better chondroprotective effects as compared to 
triamcinolone in rat model of osteoarthritis.

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