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DOI: 10.5628/aehd.v4i2.155 Original Research Manuscript The effect of moderate exercise with and without glucosamine
supplementation on rat's knee osteoarthritis

Mohammadi MF1, Mohammadi ZF2, Mirkarimpour H3
1Department of Sport Traumatology and Corrective Exercises, School of Physical Education and Sports Sciences, University of Kharazmi, Tehran, Iran 2Department of Exercise Physiology, School of Physical Education and Sports Sciences, University of Mazandaran, Babolsar, Iran 3Department of Sport Traumatology and Corrective Exercises, School of Physical Education and Sports Sciences, University of Tehran, Tehran, Iran Objectives: As the most common form of arthritis, osteoarthritis is considered to be one out of
the ten major causes of disability in the world. The objective of the present study was to
investigate the effect of treadmill exercise alone, and in combination with glucosamine
supplementation on male rats' knee osteoarthritis induced by intra-articular injection of
monosodium iodoacetate. Methods: Twenty-five male Wistar rats (weight of 173±1 g, 8 weeks
old) were randomly divided into 5 experimental groups (n=5): intact control, monosodium
iodoacetate (MIA) only, exercise, glucosamine, and exercise plus glucosamine. The
osteoarthritis model was induced by intra-articular injection of MIA. Subjects then followed a
moderate intensity exercise program and glucosamine supplementation (250 ml/kg/day) for 28
days. Rats were killed after 28 days and histological assessment performed on their knee joints.
One-way ANOVA (P<0.05) and post-hoc Tukey test were used for the statistical analysis.
Results: Histological assessment including 1) Depth Ratio of Lesions (F=71.7, P=.001), 2) Total
Degeneration Width (F=18.1, P=.001), and 3) Significant Degeneration Width (F=12.3, P=.001)
demonstrated a beneficial influence of the moderate exercise combined with glucosamine
supplementation group, but this influence was not superior to the moderate exercise alone
group. Conclusions: This study shows that a moderate exercise program and glucosamine
supplementation, either alone or in combination, exert a beneficial influence on rats' knee
osteoarthritis. But it appears that moderate exercise alone is more effective.
Arch Exerc Health Dis 4 (3): 313-319, 2014
Key Words: Moderate Exercise; Glucosamine; Osteoarthritis; Rat


margins and center of the joint (4). Progressive degeneration of articular cartilage is accompanied by malformation of subchondral bone, which may lead to musculoskeletal disorder and it is considered as a osteophyte formation around the joint and ultimately major cause of pain and disability in the United States alteration of the joint's range of motion (2). and Australia (1). It is reported that 45 to 75 percent of Although its nature and symptoms are well-defined people above 55 years have OA symptoms (2). The and its related risk factors have been studied precisely, number of patients diagnosed with OA was estimated there is no known cure for OA (1,5). Treatment to be 6 million from 1995 to 2005 and it is predicted modalities that are used for OA include that this number will be doubled by 2020 (3). pharmacological, non-pharmacological, and surgery. Osteoarthritis is mainly characterized by articular Today, using nutraceuticals such as glucosamine is cartilage degeneration and callous formation in very common and thus various studies have been
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Corresponding author:
2012 CIAFEL. This is an Open Access article distributed under Mohammad Fallah Mohammadi: Department of Sport Traumatology the terms of the Creative Commons Attribution License and Corrective Exercises, School of Physical Education and Sport (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en). You Sciences, Kharazmi University, Tehran, Iran • Tel: 098 911 211 are free: to share, to copy, distribute and transmit the work, 6938 • Email: mohammad1671@yahoo.com provided the original author and source are credited. http://ciafel.fade.up.pt/ojs/index.php/AEHD/index Exercise and Glucosamine on Rat's Knee Osteoarthritis conducted on it (6,7,8). However, there is still no dark cycle (light-on period, 6:00 AM-6:00 PM) in a general consensus about the effectiveness of controlled temperature of 22 ± 2 °C and 50 ± 5% glucosamine. For instance, one of the studies (2010) humidity on sawdust bedding. They were fed a examined the effects of glucosamine sulphate on rat's standard diet in pellet forms and had access to tap knee OA. Researchers suggested that oral water ad libitum. The animals were randomly divided administration of glucosamine sulphate (250 ml/kg) into five groups (n = 5): 1) Intact control, 2) MIA only for 10 weeks could significantly reduce cartilage (OA), 3) training, 4) glucosamine, and 5) training plus degeneration compared to the control group (9). But a meta-analysis by Wandel (2010) that investigated the The animals in the training group were habituated on a effects of glucosamine, chondroitin, and their motor-driven treadmill at a speed of 10m min-1 for 10 combination on patients with hip and knee OA showed min/day for 1 week to reduce their stress regarding the different results. After examining 10 studies new environment (13). OA was induced by intra- performed on 3,803 patients, researchers concluded articular injection of monosodium iodoacetate (MIA); that separate or combined usage of these two its injection into joints inhibits glyceraldehydes-3- supplements could not improve joint pain and has no phosphate dehydrogenase activity in chondrocytes, effect on joint space narrowing in comparison with leading to disruption of glycolysis and eventual cell placebo (6). But the results of several meta-analyses death. For this purpose, the animals were anesthetized indicated that the effects of glucosamine range from with ketamine (90 mg/kg, i.p.) and xylazine (20 short-term effect on pain to structural efficacy (8). mg/kg, i.p.); MIA (Sigma-Aldrich, Germany) was On the other hand, exercise is a proven non- injected with a U-100 insulin needle containing 1 mg pharmacologic treatment for knee OA (8). Some of iodoacetate diluted in 50 µL saline solution into studies have determined intense exercises to have an animals' right knee, while 50 µL saline solution was adverse role (10,11,), although most of them agree that injected (19) into their left knee. exercise with moderate intensity is ideal (13,14). The training program started 24 hours after OA According to recent guidelines of Osteoarthritis induction. After the adaptation period, a program of Research Society International (OARSI) in 2010, moderate physical training was performed once a day optimal treatments of knee and hip OA should include for 4 weeks with a speed of 18 m min-1 for 30 non-pharmacologic min/day (14). To assess its therapeutic effects, modalities (15). However, reviewing past studies, glucosamine (Serva, USA) supplementation was there are few investigations on the combination of the administered orally by gavage in doses of 250 two modalities. It should be noted that these studies mg/kg/day for 28 days (9). On day 28, animals were evaluated the effects of interventions using killed by cervical dislocation under anesthesia. Whole questionnaires or assessing some physical fitness knee joints were dissected, fixed in 10% formaldehyde measures. Hence the effects of treatment protocols on solution in 50 cc vials, and sent to a pathology the joint's cartilage remain nearly unknown, because laboratory. The samples decalcified with 5% formic the researchers were unable to assess biochemical acid, dehydrated through a descending series of properties of the tissue in vivo (16). For this reason, ethanol with the use of an automated tissue-processing there is a need to perform histopathological apparatus. After embedding in paraffin, serial sections assessments on animal models of OA that resemble the with a thickness of 7 µm were prepared for condition in human models. According to our studies, histological examination. Frontal and sagittal intra-articular injection of monosodium iodoacetate sectionings were prepared from tibiofemoral joints. (MIA) in animal models (such as rats), results in The sections were stained with hematoxylin-eosin to pathological changes closely resembling those seen in observe cellularity. human OA (17,18). The severity of OA lesions was graded on a scale So the aim of the present study was to examine the adopted from OARSI histopathology instructions. effects of moderate-intensity exercise in combination Three histological measures used in this study with glucosamine supplementation on rats' knee OA. according to OARSI recommendation included 1) depth ratio of lesions (DR), 2) total cartilage MATERIAL AND METHODS
degeneration width (TDW), and 3) significant cartilage degeneration width (SDW) (12). DR is a Twenty-five male Wistar rats (173 ± 1 g, 8 weeks old) measurement of the depth of cartilage degeneration were obtained from Pasteur Institute (Amol, Northern (e.g., including areas of chondrocyte and proteoglycan Iran). The maintenance and care of the experimental loss, which may have good retention of collagenous rats were in accordance with the guidelines of the matrix and no fibrillation) that is taken at the midpoint Helsinki convention. Subjects were kept in standard in each of the three zones across the tibial surface. condition in individual plastic cages in a 12:12 light- TDW is the total width of the area of articular cartilage Arch Exerc Health Dis 4 (3): 313-319, 2014 Exercise and Glucosamine on Rat's Knee Osteoarthritis affected by any type of degenerative change (matrix Total Degeneration Width (TDW)
fibrillation/loss, proteoglycan loss with or without chondrocyte death). And SDW is a measurement of Regarding results related to TDW, there were the width of the tibial cartilage in which 50% or significant differences between training plus greater of the thickness (from surface to tidemark) is glucosamine group and training group (P=0.001), seriously compromised. Histomorphological scores in whereas there was no significant difference between training plus glucosamine and MIA group (P=0.551). measurements for statistical analysis (11). The difference between training plus glucosamine and The data was analyzed using the SPSS statistical glucosamine groups was not significant (P=0.187). software version 16. The One-way ANOVA (P<0.05) Also significant differences were observed between and post-hoc Tukey test were used for the statistical the glucosamine and MIA groups (P=0.008), but the difference between the glucosamine group and the training group was not significant (P=0.065). Moreover, significant differences were seen between training and MIA groups (P=0.001). These findings The findings of histological analysis on each are presented in Figure 2. experimental group include 1) Depth Ratio of Lesions (F=71.7, P=0.001), 2) Total Degeneration Width
(F=18.1, P=0.001), and 3) Significant Degeneration
Width (F=12.3, P=0.001). Moreover, theses results are
separately presented for each group.
Depth Ratio of Lesions (DR)
For this measure, there was significant difference
between training plus glucosamine group and training
and MIA groups (P=0.001). But the difference
between training plus glucosamine and glucosamine
groups was not significant (P<0.05). Also, there was a
significant difference between glucosamine, MIA, and
training groups (P=0.001). Finally, the difference
between training and MIA groups was significant
(P=0.001). These findings are presented in Figure 1.

Figure 2.
The mean ± SD of histological scores related to
Total Degeneration Width measured in micrometers. Higher
scores show greater severity of lesions. * Significant
difference with Training and MIA groups. ** Significant
difference with MIA group.

Significant Degeneration Width (SDW)
Also for this measure, there were no significant
differences between training plus glucosamine group
and training group (P=0.054), but there was a
significant
glucosamine and MIA group (P=0.048). The difference between training plus glucosamine and glucosamine groups was not significant (P<0.05). Significant differences were observed between ! glucosamine and MIA groups (P=0.048), but there Figure 1. The mean ± SD of histological scores related to
were no significant differences between glucosamine Depth Ratio of Lesions measured in micrometers. Higher and training groups (P=0.077). Also, differences scores show greater severity of lesions. * Significant between training and MIA groups were significant difference with Training and MIA groups. ** Significant (P=0.001). These findings are presented in Figure 3. difference with MIA group. Arch Exerc Health Dis 4 (3): 313-319, 2014 Exercise and Glucosamine on Rat's Knee Osteoarthritis DISCUSSION
The present study aimed at investigating the
therapeutic effects of moderate exercise alone and
combined with glucosamine supplementation on rat's
knee OA. In order to induce osteoarthritic symptoms
in rats' knee joints, the authors used intra-articular
injection of MIA. This model is validated by other
studies (17,18), but many limitations exist in utilizing
MIA models to correlate human OA due to inherited
variation, such as the biomechanical differences
between two-legged and four-legged species (13). The
results showed that treadmill exercise with moderate
intensity alone had remarkable effects and could
surprisingly almost treat histological symptoms of
rat's knee OA including Depth Ratio of Lesions, Total
Degeneration Width, and Significant Degeneration Figure 3. The mean ± SD of histological scores related to
Width. However, combination of moderate exercise Significant Degeneration Width measured in micrometers. with glucosamine supplementation did not result in Higher scores show greater severity of lesions. * Significant additive effects. Furthermore, supplementation of difference with Training and MIA groups. ** Significant glucosamine alone had significant effects but it was difference with MIA group. not superior to that of moderate exercise. Several studies investigating the efficacy of dietary supplementation on joint diseases such as OA have led Furthermore, photomicrographs of histomorphological to contradictory results. In fact, international medical changes of joint cartilage stained by Hematoxylin- And Figure 5 provides methods of assessing Eosin for subjects in experimental groups are provided histopathological measures used in this study. Figure 4. Photomicrographs of histomorphological changes of knee joint sections of subjects in each experimental group.
Cellularity and surface integrity were evaluated by Hematoxylin-Eosin staining (the original magnification is ×10). A: MIA
only; OA lesions can be seen with tibial cartilage surface clefts and decrease in cellularity. Note the pink-red sites of lesions.
B: Intact control; there are no observable changes in cartilage surface. C: Training; chondrocytes can be observed in many
isogenic groups, which are the indicators of cell division stimulation. D: Glucosamine; the degenerated area is seen with
chondrocytes' degradation and eosinophilic matrix. E: Training+Glucosamine; cartilage thickness is decreased, surface cells
are compressed and cartilage matrix is eosinophilic. Arch Exerc Health Dis 4 (3): 313-319, 2014 Exercise and Glucosamine on Rat's Knee Osteoarthritis Figure 5. Method of histopathological assessment. F: Photomicrograph (the original magnification is ×3.2) showing method
of assessing Depth Ratio of Lesions; DD=Degeneration Depth, CD=Cartilage Depth, and DD/CD=Degeneration Depth
Ratio; score 1 stands for the most severe lesion. G: Photomicrograph (the original magnification is ×3.2) showing method of
assessing Total Degeneration Width and Significant Degeneration Width; TDW= Total Degeneration Width and SDW=
Significant Degeneration Width.
societies have not yet reached any agreement overexpression. Another study by Cifuentes and regarding usage of these supplements. Naito and colleagues (2010) was conducted to observe efficacy colleagues (2010) examined the effects of glucosamine of a moderate exercise program on rats' knee OA (13). supplementation on an experimental model of rat's Their results indicated that physical training knee OA (20). They administered 1000 mg contributes to the preservation of joint cartilage in rats glucosamine/kg/day orally (dissolved in tap water). with OA and increases the defense mechanism against oxidative stress. Researchers suggested that this effect chondroprotective potential against OA and can inhibit is presumably because of an increase in activities of degradation of type II collagen and enhance its antioxidant enzymes such as Superoxide Dismutase synthesis in articular cartilage. On the other hand, a (SOD) and Myeloperoxidase (MPO). meta-analysis conducted by Wandel and colleagues Cartilage, in fact, is an avascular tissue, and (2010) examined the effects of glucosamine, chondrocyte metabolism depends on diffusion and chondroitin, and their combination in patients with hip convection of synovial fluid for nutrition. Cyclic and knee OA (6). The authors found that separate or loading induced by physiological and overuse combined usage of these two supplements could not activities produces deformations, pressure gradients, improve joint pain and has no effect on joint space and fluid flows within the tissue. Mechanical stress narrowing in comparison with placebo. has a direct effect on chondrocyte metabolism and can, Moreover, numerous studies have been conducted on under certain conditions, induce anti-apoptotic factors the effectiveness of exercise in management of OA in (14). Additionally, exercise promotes important both human and animal models, and most of them changes in anti-oxidant enzyme activities, reducing agree that moderate intensity exercise has positive oxidative damage and increasing tissue resistance effects (13,14,21,22). However, findings of other against free radicals (24). Some studies have shown studies suggest that intense exercise has an adverse increases in SOD, Glutathione peroxidase, and role (10,11,12,23). In a dose-response study by Galois catalase activities after aerobic exercise training in and colleagues (2004), the effects of an exercise young rats (25,26). program with 3 intensities (low, moderate, and In the present study – according to guidelines of intense) were investigated on progression of ACL OARSI presented in 2010 (15) - and also because in transected rat's knee OA (14). Researchers indicated most of the clinical settings the combination of that low and moderate exercise had positive effects on pharmacologic and non-pharmacologic treatments is severity of chondral lesions, but an intense effort used for improving OA symptoms in patients, the converses this chondroprotective effect. They authors believed that the combination of moderate suggested that positive effects of low and moderate exercise and glucosamine supplementation may have exercise were possibly due to a reduced level of synergistic effects. But our findings indicate that moderate exercise alone exerts beneficial influence on potentials of heat shock protein 70 (Hsp70) histological measures of rat's knee OA. Arch Exerc Health Dis 4 (3): 313-319, 2014 Exercise and Glucosamine on Rat's Knee Osteoarthritis As can be observed in the figures, in the training based approach to the management of knee osteoarthritis: group, chondrocytes can be seen in many isogenic Report of a Task Force of the Standing Committee for International Clinical Studies Including Therapeutic Trials groups, which is the indicator of cell division (ESCISIT). Ann Rheum Dis. 2003; 62: 1145–1155. stimulation (Fig. 4;C). Also, as presented in Figures 1, Bennell KL, Hinman Rana S. A review of the clinical evidence 2, and 3, training protocol used in the present study for exercise in osteoarthritis of the hip and knee. Journal of was beneficial in treating OA symptoms. From these Science and Medicine in Sport. 2011; 14: 4–9. Wandel Simon, Jüni P, Tendal B, Nüesch E, Villiger PM, figures it can be seen that the pathological score of the Welton NJ, et al. Effects of glucosamine, chondroitin, or MIA group is nearly 130 micrometers for histological placebo in patients with osteoarthritis of hip or knee: Network measure of Depth Ratio of Lesions (Fig. 1), 700 meta-analysis. BMJ. 2010; 341: c4675. micrometers for Total Degeneration Width (Fig. 2), Homandberg GA, Guo D, Ray LM, Ding L. Mixtures of glucosamine and chondroitin sulfate reverse fibronectin and 550 micrometers for Significant Degeneration fragment mediated damage to cartilage more effectively than Width (Fig. 3). Furthermore, these scores for the either agent alone. Osteoarthritis and Cartilage. 2006; 14: 793- training group in all 3 measures were zero and equal with healthy controls. Messier SP, Mihalko S. Glucosamine/chondroitin combined with exercise for the treatment of knee osteoarthritis: A The beneficial effects of this exercise program may be preliminary study. Osteoarthritis and Cartilage. 2007; 15: explained by its ability to suppress the signal transduction pathways of proinflammatory/catabolic Wen ZH, Tang CC, Chang YC, Huang SY, Hsieh SP, Lee CH, mediators, while stimulating anabolic pathways. et al. Glucosamine sulfate reduces experimental osteoarthritis and nociception in rats: Association with changes of mitogen- Mechanical strain of low magnitude inhibits activated protein kinase in chondrocytes. Osteoarthritis and Cartilage. 2010; 18: 1192-1202. TNF‐α‐induced 10. Blagojevic M, Jinks C, Jeffery A, Jordan KP. Risk factors for proinflammatory mediators involved in cartilage onset of osteoarthritis of the knee in older adults: A systematic review and meta-analysis. Osteoarthritis and Cartilage. 2010; degradation. This also results in the upregulation of proteoglycan and collagen synthesis that is drastically 11. Kawasaki T, Kurosawa H, Ikeda H, Kim SG, Osawa A, inhibited in inflamed joints (27). Takazawa Y, et al. 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Femtosecond-assisted intrastromal corneal cross-linking forearly and moderate keratoconus M. Balidis,1,2 V.E. Konidaris,2 G. Ioannidis,1,3 A.J. Kanellopoulos4,5 months. Our study demonstrates the safety and efficacy ofthe proposed method. Purpose: To evaluate the effect of Femtosecond-assisted Key words: keratoconus, femtosecond, cross-linking.