Chondroprotective effect of N-acetylglucosamine and hyaluronate in early stages of osteoarthritis: an experimental study in rabbits.
Materials and Methods
N-acetylglucosamine was purchased from Sigma (St Louis, Missouri). It was dissolved in saline and sterilized by filtration through 0.22 pm filter (Millipore Corporation, Bedford, Massachusetts). Sodium Hyaluronate (Hyalgan) was purchased from Sanofi-Synthelabo (New York, New York). Hyalgan is a visous solution consisting of a high molecular weight (500.000-730.000 daltons) fraction of purified natural sodium hyaluronate in buffered physiologial sodium chloride, having a pH of 6.8 to 7.5.
Thirty-two New Zealand white rabbits weighing 2.8 to 3.6 kg were used in this study. The animals were housed in seperate metal cages (35x50x35 cm), where they were allowed free access to a solid diet and tap water.
The animals were anesthetized with an intramuscular injection of 10 mg/kg xylazine (Rompun[R], Bayer) and 50 mg/ kg ketamin (Ketalar[R], Pfizer Warner Lambert). After the right knee joint was shaved off and sterilized, the skin was incised longutidinally from superior pole of the patella to the tibial tuberosity. Then a medial parapatellar arthrotomy was made and the anterior cruciate ligament was macroscopically seen and resected (Fig. 1). After the resection, anterior instability was confirmed by an anterior drawer test. Finally, the articular capsule and skin were sutured after the joint cavity was irrigated with physiological saline solution, and the animals were allowed free cage activity postoperatively. After the operation, the rabbits were randomly allocated into four groups each consisting of eight rabbits. The first group was composed of rabbits receiving intraarticular injections of N-acetylglucosamine once a week, starting 2 weeks postoperatively and continuing for a period of 5 weeks. The dose of N-acetylglucosamine was 150 mg/0.3 ml for each injection.
[FIGURE 1 OMITTED]
The second group consisted of rabbits receiving intraarticular injections of hyaluronan (0.3 ml per joint once a week), starting 2 weeks postoperatively and continuing for a period of 5 weeks.
The third group included rabbits receiving an intraarticular injection of hyaluronan (0.3 ml per joint once a week), and simultaneously intramuscular injections of N-acetylglucosamine once a week, starting 2 weeks postoperatively and continuing for a period of 5 weeks. The dose of intramuscular N-acetylglucosamine was 300 mg/kg.
The fourth group served as a control group and consisted of rabbits receiving an intraarticular saline solution of 0.3 ml once a week for a period of 5 weeks.
All injections were performed by the same investigator. The rabbits were sacrificed 8 weeks after the surgery with high dose intravenous thiopentone sodium (Pentothal[R], Abbott) after intramuscular anesthesia with intramuscular xylazine and ketamine.
Gross Morphological Assessment
Gross morphological assessment of the knee joints was performed in a blinded fashion and included tibial plateaus and femoral condyles. The distal femur and proximal tibia were harvested, keeping the 3 to 3.5 cm shaft of the bones. The articular cartilage surface of the each specimen was stained with India ink. The following grading system for articular cartilage assessment was used:
Grade 1: Intact surface (joint surface does not retain India ink);
Grade 2: Minimal fibrillation (joint surface retains India ink as light grey patches);
Grade 3: Overt fibrillation (areas which are velvety in appearance and retain India ink as intense black patches); and
Grade 4: Erosion (loss of cartilage exposing the underlying bone).
Distal femur from the rabbit knee joints were fixed in 10% buffered formalin, decalcified in TBD-2 solution (ThermoShandan, Pittsburg, California), and embedded in paraffin blocks. Sagittal sections of medial femoral condyles were used for the histological analysis. Tissue sections were stained with hematoxylin and eosin for cellular asssessment and with Safranin O for the assessment of glycosaminoglycan content. Histological assessment was done according to Mankin's grading system, which assigns separate scores to structure, cell distribution and density, Safranin O staining, and the integrity of the tidemark. (3) The scores in each of these subcategories were totaled for each sample; zero value represented normal cartilage while a high score represented damage to cartilage.
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Statistical analysis was performed using GraphPad Prisma V3 program. A Chi-square test was used for the comparison of groups. Dunn's multiple comparison test was used for the comparison of subgroups. A P value of less than 0.05 was considered statistically significant.
[FIGURE 4 OMITTED]
No postoperative infection was observed, and no rabbits were lost during the study period. Rabbits were randomized into four groups; intraarticular glucosamine, intraarticular hyaluronate, intraarticular hyaluronate plus intramuscular glucosamine, and intraarticular saline (control group). Gross morphological analysis of the femoral condyles and tibial plateaus after staining with India ink revealed that the joint surface was normal in appearance (grade 1) (Fig. 2A) in all of the glucosamine group. The joint surface was normal in appearance in 37.5% of the hyaluronate group, with minimal fibrillation (grade 2) (Fig. 2B) in the rest of the group. The joint surface showed no abnormal appearance (grade 1) in 62.5% of the hyaluronate plus glucosamine group, with minimal fibrillation (grade 2) in the rest of the group. Overt fibrillation was observed in all joint surfaces (grade 3) (Fig. 2C) of the saline group. A statistically significant difference was found in the comparison of the results of the macroscopic grading between the intraarticular glucosamine, intraarticular hyaluronate, intraarticular hyaluronate and intramuscular glucosamine and the saline (control) group (p < 0.001). The gross morphological analyses are summarized in Figure 3. The overall Mankin score was 5 in the glucosamine group, 5.5 in the hyaluronate group, 5.45 in the hyaluronate plus glucosamine group, and 9.63 in the saline group. Overall there were statistical differences between the glucosamine group, the hyaluronate group, the hyaluronate plus glucosamine group, and the control group with respect to cartilage structure, cellular abnormality, and Safranin O staining. No statistically significant difference with respect to tidemark integrity was observed between groups (Table 1). There were statistically significant differences between the glucosamine and saline groups with respect to cartilage structure (p < 0.01), cellular abnormality (Fig. 4) (p < 0.05), matrix staining (p < 0.05), and gross morphology (p < 0.001). In a comparison of the hyaluronate and saline groups, statistically significant differences were observed with respect to matrix staining (Fig. 5) (p < 0.05) and gross morphology (p < 0.05), and the average scores of the hyaluronate group obtained from the assessment of structure and cells were lower when compared to the saline group.
[FIGURE 5 OMITTED]
[FIGURE 6 OMITTED]
Mankin's grading system showed that the average scores for cartilage structure and cellular abnormality were lower in hyaluronate plus glucosamine group when compared to the saline group. Statistically significant differences were found between the hyaluronate plus glucosamine group and saline group with respect to cartilage structure (p < 0.05) (Fig. 6), cellular abnormality (p < 0.05), and gross morphology (p < 0.01). The hyaluronate plus glucosamine group also showed a beneficial therapeutic trend in matrix staining.
There were no statistically significant differences between the glucosamine and hyaluronate groups, between the glucosamine and hyaluronate plus glucosamine groups, and between the hyaluronate and hyaluronate plus glucosamine groups in the assessment of cartilage structure, cellular abnormality, matrix staining, tidemark integrity, and gross morphology (Table 2).
Osteoarthritis is the most common form of arthritis and has an immense socioeconomic impact in all societies. Drugs for the treatment of osteoarthritis have been classified as symptom or structure modifying drugs. Although no drug has been included in the structure modifying group, compounds that are intended to prevent, retard, stabilize, or reverse the development of the morphological changes of osteoarthritis are being researched. (4,5) Despite the widespread use of intraarticular hyaluronate injection and oral supplementation of N-acetylyglucosamine by patients, only a limited amount of experimental data is available to demonstrate their activities and effects on hyaline cartilage.
Hyaluronic acid is a type of glycosaminoglycan and is a major ingredient of synovial fluid. It has viscous and elastic properties and acts as a shock absorber during fast movements and as a lubricant during slow movements. (6) It reduces the secretion of arachidonic acid from synovial fibroblasts and stimulates the tissue inhibitor of metalloproteinase-1 in articular chondrocytes. (7) It has also been demonstrated to stimulate synovial cells isolated from osteoarthritic joints to synthesize hyaluronate in vitro. (2,7) Takahashi and colleagues (8) investigated the effect of intraarticular injections of hyaluronate on nitric oxide production in synovium and meniscus using an experimental model of osteoarthritis in rabbits. Nitric oxide production in the meniscus and synovium of the hyaluronate group was significantly lower than in the control group. They pointed out that inhibition of nitric oxide production might be a mechanism for the therapeutic effect of hyaluronic acid. (8) Guidolin and associates (9) investigated the effect of hyaluronic acid on human cartilage by morphological analysis of articular cartilage biopsies taken from osteoarthritic knees. They reported reconstitution of the superficial layer and improvement in chondrocyte density, providing further evidence for potential structure modifying activity of hyaluronate. (9) In our experimental model of osteoarthritis, macroscopic evaluation revealed statistically significant cartilage preservation in the hyaluronate group when compared to the saline group. Similar to Guidolin's findings, histopathological evaluation in our study revealed that cartilage surfaces are preserved in the hyaluronate group when compared to the saline group. Overall, macroscopic and microscopic assessment in the current study highlights the chondroprotective activity of intraarticular hyaluronate.
Glucosamine is an amino monosaccharide nutrient and precursor of the disaccharide unit of glycosaminoglycan, which is the building block of the proteoglycan component of the cartilage matrix. It has been demonstrated that N-acetylglucosamine inhibits nitric oxide, cyclooxygenase-2 (COX-2), and IL-6 production in cultured human articular chondrocytes. (10) Oegema and coworkers11 investigated the effect of oral glucosamine on knee joint cartilage after acute damage by a protease. They reported that glucosamine treatment increased glycosaminoglycan content in the cartilage of damaged knees. (11) Also, in an animal model of osteoarthritis, oral administration of glucosamine has been shown to attenuate the changes in the early stages of experimental osteoarthritis. (12)
Anterior cruciate ligament transection was initially developed using dogs, (13) and it has become a well established technique in the rabbits. (14) Unstable joints lead to cartilage degeneration, with the microscopic evidence appearing 2 weeks after anterior cruciate ligament transection. (15,16) In rabbits transection of the anterior cruciate ligament leads to gradual progressive changes in the articular cartilage that are characteristic of human osteoarthritis (16-18); the "... partial-thickness cartilage lesion resulted in early markers of degenerative changes resembling the human situation." (19) Therefore, intraarticular injections in our study were initiated 2 weeks after the anterior cruciate ligament transection, and the rabbits were sacrificed 8 weeks after the surgery. Shikhman and colleagues examined the therapeutic efficacy of N-acetylglucosamine in rabbits with experimentally-induced osteoarthritis. (20) They investigated the efficacy of N-acetylglucosamine in three different groups: intramuscular N-acetylglucosamine (200 mg/kg) three times a week, intraarticular N-acetylglucosamine once a week, and intraarticular N-acetylglucosamine twice a week. The administered dose for each intraarticular injection of N-acetylglucosamine was 80 mg. Intramuscular and intraarticular injections were started one week after the anterior cruciate ligament transection and continued for a period of 7 weeks. They reported that intramuscular administration of N-acetylglucosamine did not have chondroprotective effects, and once a week intraarticular N-acetylglucosamine injections did not demonstrate therapeutic efficacy, while twice a week intraarticular N-acetylglucosamine reduced cartilage degradation. In our study, the dose for each intraarticular N-acetylglucosamine injection was 150 mg once a week for a period of 5 weeks, starting 2 weeks after the operation. Our morphological assessment has similarities with the results of Shikhman's twice a week intraarticular N-acetylglucosamine group. The reason that we found once a week glucosamine administration effective may be the higher dose (150 mg N-acetylglucosamine) that was administered. In our study, morphological evaluation revealed that articular cartilage was preserved in all rabbits in the glucosamine group; no cartilage damage was observed macroscopically. Microscopic evaluation revealed that glycosaminoglycan content and cartilage structure were significantly preserved in the glucosamine group when compared to saline group. Overall, macroscopic and microscopic evaluation in our study demonstrated the chondroprotective activity of intraarticular glucosamine and indicated that glucosamine slows the disease progression in early osteoarthritis.
Our study included a third group in order to investigate the efficacy of simultaneous administration of intramuscular N-acetylglucosamine and intraarticular hyaluronate. Macroscopic and microscopic evaluation revealed that simultaneous administration of intraarticular hyaluronate and intramuscular glucosamine enhanced chondroprotective activity. Although synchronous treatment of intramuscular glucosamine and intraarticular hyaluronate was found to have a chondroprotective effect, no statistically significant difference was found when the hyaluronate plus glucosamine group was compared with the hyaluronate group and the glucosamine group. The reason for this may be the failure of intramuscular administration to achieve a sufficient concentration of glucosamine at the joint.
In conclusion, this study showed that intraarticular glycosamine and intraarticular hyaluronate have a role in slowing the progression of the degenerative process and protecting the cartilage surface during the early stages of osteoarthtritis.
None of the authors have a financial or proprietary interest in the subject matter or materials discussed, including, but not limited to, employment, consultancies, stock ownership, honoraria, and paid expert testimony.
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Feyza Unlu Ozkan, M.D., is in the Department of Physical Medicine and Rehabilitation, Fatih Sultan Mehmet Education and Training Hospital, Istanbul, Turkey. Korhan Ozkan, M.D., is in the Department of Orthopaedics and Traumatology, Goztepe Education and Training Hospital, Istanbul, Turkey. Saime Ramadan, M.D., is in the Department of Pathology, Anatolian Health Center, Istanbul, Turkey. Zeynep Guven, M.D., is a Professor in the Department of Physical Medicine and Rehabilitation, Marmara University, Istanbul, Turkey.
Correspondence: Korhan Ozkan, M.D., Barbaros mah atayurt sit B blok daire 20 Uskudar, Istanbul, Turkey; korhanozkan@ hotmail.com.
Table 1 Mean Scores of the Groups According to the Mankin Scale Glucosamine Hyaluronate Cartilage structure 1.5 [+ or -] 0.53 2 [+ or -] 0.93 Cells 1.25 [+ or -] 0.46 1.38 [+ or -] 0.52 Safranin O staining 1.38 [+ or -] 0.52 1.38 [+ or -] 0.52 Tidemark integrity 0.88 [+ or -] 0.35 0.75 [+ or -] 0.46 Hyal+Glu Saline p Cartilage structure 1.75 [+ or -] 0.8 4 [+ or -] 1.2 0.05 Cells 1.25 [+ or -] 0.5 2.25 [+ or -] 0.46 0.019 Safranin O staining 1.56 [+ or -] 0.53 2.38 [+ or -] 0.52 0.024 Tidemark integrity 0.89 [+ or -] 0.33 1 [+ or -] 0 0.515 Table 2 Comparison Between the Groups Cartilage Macroscopy structure Cells Glucosamine/Hyaluronate P > 0.05 P > 0.05 P > 0.05 Glucosamine/Hyal+Glu P > 0.05 P > 0.05 P > 0.05 Glucosamine/Saline P < 0.001 P < 0.01 P < 0.05 Hyaluronate/Hyal+Glu P > 0.05 P > 0.05 P > 0.05 Hyaluronate/Saline P < 0.05 P > 0.05 P > 0.05 Hyal+Glu/Saline P < 0.01 P < 0.05 P < 0.05 Safranin O Tidemark staining integrity Glucosamine/Hyaluronate P > 0.05 P > 0.05 Glucosamine/Hyal+Glu P > 0.05 P > 0.05 Glucosamine/Saline P < 0.05 P > 0.05 Hyaluronate/Hyal+Glu P > 0.05 P > 0.05 Hyaluronate/Saline P < 0.05 P > 0.05 Hyal+Glu/Saline P > 0.05 P > 0.05 Figure 3 Gross morphological analyses were summarized. Grade 1 Grade 2 Grade 3 Glucosamine 100% Hyaluronate 37,50% 62,50% Hyal+glu 62,50% 37,50% Control 100% Note: Table made from bar graph.
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|Author:||Ozkan, Feyza Unlu; Ozkan, Korhan; Ramadan, Saime; Guven, Zeynep|
|Publication:||Bulletin of the NYU Hospital for Joint Diseases|
|Date:||Oct 1, 2009|
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