Abstract
The aim of this study was to determine the efficacy of Hylan G-F 20 on experimentally induced osteoarthritic changes in rabbit temporomandibular joint (TMJ). A 3 mg/ml concentration of sodium mono iodoacetate (MIA) had been injected into both joints of 24 rabbits to create osteoartrosis. The study group was injected with Hylan G-F 20 in one joint and saline in the contralateral joint as a control (once a week for 3 weeks). Histological changes in articular cartilage, osteochondral junction, chondrocyte appearance and subchondral bone were determined at 4, 6, and 8 weeks. Regarding cartilage, there was a statistically significant difference between the two groups at 4 weeks. Degenerative bony changes to subchondral bone were significantly higher in the controls. No statistical difference was found in the study group at 6 weeks. A positive correlation was found between osteochondral junction and subchondral bone in the study group at 8 weeks. The changes in chondrocyte appearance were significantly decreased in the study group at all follow-up times. Intra-articular injection of Hylan G-F 20 decreased cartilage changes in early stage TMJ osteoartrosis and clustering of chondrocytes showed the chondroprotective effects of Hylan G-F 20 caused by hypertrophic responses.
Osteoarthrosis (OA) is a disease characterised by deterioration and abrasion of articular cartilage and disc surfaces, and thickening and remodelling of the underlying bone. The term ‘osteoartrosis’, a synonym for OA in the medical orthopaedic literature, has recently come to be identified in the dental temporomandibular joint (TMJ) literature with a low-inflammatory arthritic condition that results in similar degenerative changes to OA. The concept of TMJ osteoartrosis introduced by S tegenga et al. emphasizes the role of progressive cartilage degradation in the formation of OA in the TMJ . They have indicated that when external loading forces on the TMJ exceed the adaptive capacity of the chondrocytes to produce matrix, degradation exceeds repair and leads to cartilage breakdown, synovial inflammation, impaired biomechanics and disc movement . The cartilage degeneration can be a result of continuous stimuli caused by functional overloading of the joint and the response of intra-articular tissue to mechanical stress, hypoxia–reperfusion, neurological inflammation, free radicals and direct mechanical damage, and can be pathological at the molecular level . Osteoarthritic changes in the joints are characterised by fibrillation and erosion in the cartilage, chondrocyte proliferation and osteophyte formation at the joint margins and sclerosis of subchondral bone .
Hyaluronic acid (HA), a major natural component of synovial fluid and articular cartilage, plays a major role in joint lubrication, protection of cartilage, nutrition of avascular parts of cartilage and disc and it also has a structural role in cartilage formation . It is mostly concentrated around the articular surfaces and provides lubrication. Recent reports have pointed out the importance of joint lubrication for correct joint function, also hypothesizing that abnormalities of the joint lubrication system may play a role in the onset of TMJ OA . In the presence of TMJ inflammation, the local decrease in molecular weight and concentration of HA in synovial fluid changes the metabolism and biomechanical properties of cartilage. The functions of viscosity and the molecular barrier are weakened, which makes it difficult to protect proteogylcan in the cartilage matrix and to restrain the inflammation reaction . It is thought that exogenously applied HA can be effective in providing joint lubrication, stimulating the synthesis of endogenous high molecular weight HA (HMWHA) and maintaining the normal rheological properties of the synovial fluid . Exogenous HA controls the synthesis and apoptosis of endogenous HA, proteoglycan (PG), glycosaminoglycan (GAG) and collagen by affecting chondrocyte metabolism and plays a role in stimulating a repair response in the articular cartilage by inhibiting the proteolytic enzymes and thus preventing an inflammatory reaction .
The intra-articular injection of low molecular weight HA in the treatment of OA has been proved to have a positive effect on the TMJ and the other joints and is widely used in clinical practice . There are numerous studies evaluating the effects of the different molecular weights of HA on OA, but there is no consensus on the concept of molecular weight. The successful results of HMWHA injection in other joints in animals and humans is thought to be related to HMWHA’s rheological properties, its longer retention period in comparison to low molecular weight HA, the resistance of free radical degradation due to its cross-link with HA, and the smaller rate of injection . The biological efficacy of HMWHA has been well documented in other joints, but the beneficial effects in TMJ cartilage have not been clarified. To the best of the authors’ knowledge, there is no animal study investigating the histological and radiological changes in TMJ cartilage after intra-articular HMWHA injection. The aim of this study was to determine the efficacy of HMWHA (Hylan G-F 20) on experimentally induced osteoarthritic changes in rabbit TMJ.
Materials and methods
All experimental procedures were approved by Istanbul University Institutional Animal Care and Use Committee (2007/25). 24 New Zealand white rabbits (male, on average 18 weeks old and 3.5 kg) were used in this study. All rabbits were housed with a 12–12 h light–dark cycle (artificial lighting), free access to water and food and with an ambient temperature of 21 °C and 50% humidity. The study began after a 2 week adjustment period for the animals.
All rabbits were anaesthetized by intramuscular injection with a mixture of 5% ketamine (20 mg/kg) and 2% xyladine (0.1 mg/kg) during all experimental procedures. The area was shaved and disinfected with povidone iodine. Each intra-articular injection was performed using an arthrocentesis technique. Briefly, the location of the joint was identified by palpating its lateral aspects with the mouth opened and closed several times, and a syringe with 29 G needle was inserted into the lower joint cavity.
Early osteoarthritic changes in rabbit TMJ, defined by histological analysis, were found at 4 weeks and with a 3 mg/ml concentration of sodium mono iodoacetate (MIA) . 4 weeks after bilateral intra-articular MIA injection, 0.5 ml of 4 mg Hylan G-F 20 (sodium hyaluronate, Synvisc, Wyeth, Genzyme, Biosurgery Ridgefield, NJ, USA) was injected into a single joint of each rabbit (the study group consisted of 24 joints). 0.5 ml of 0.9% saline solution was injected into the contralateral joint to serve as a control. All injections on both joints were performed once a week for 3 weeks. The molecular weight of Hylan G-F 20 was 6 × 10 6 Da and the concentration of HA was 8 mg/ml. The product is available in 2 ml vials. Each vial was stored at 4 °C until use to increase viscosity.
Histological evaluation
The 24 rabbits were randomly divided into 3 groups and killed with an overdose of pentobarbital at the fourth, sixth and eighth weeks after the last intra-articular injection of Hylan G-F 20 or saline. All joints were removed and fixed in buffered formalin immediately for histological examination. The specimens were prepared for undecalcified histology as described in a previous study . The histological changes to the articular cartilage, osteochondral junction, chondrocyte appearance and subchondral bone were determined in both groups at all follow-ups. The changes to the articular cartilage (normal, thinning, thickening), the appearance of the chondrocytes (normal, hypocellularity or clusters), and the osteochondral junction (normal, invagination, weak junction) and changes to the trabecular structure of the subchondral bone (normal, trabecular bone increase) in each joint of both groups were recorded at all follow-up periods.
Statistical evaluation
SPSS/PC (17.0 version, SPSS Inc, Chicago, IL, USA) was used for statistical analysis. A Spearman rank correlation test was used for determining if significant correlations amongst the data existed. Comparisons were made using the Wilcoxon signed rank test and paired t tests. The χ 2 test was used to determine the association between the computed tomography (CT) and histological findings. Comparison was considered significant at P < 0.05.
Results
The histological cartilage changes for both groups at all follow-ups are shown in Table 1 . Histological findings at 4, 6, and 8 weeks after Hylan G-F 20 injection were found in cartilage (50%, 25%, 25%), the osteochondral junction (62.5%, 50%, 37.5%), the appearance of chondrocytes (87.5%, 37.5%, 12.5%), and subchondral bony changes (62.5%, 50%, 50%), respectively.
| Articular cartilage changes ( N = 24) | Controls ( N = 24) | Study groups ( N = 24) | ||||
|---|---|---|---|---|---|---|
| 4 week | 6 week | 8 week | 4 week | 6 week | 8 week | |
| Cartilage | ||||||
| Normal | 2 (25%) | 5 (62.5%) | 6 (75%) | 4 (50%) | 6 (75%) | 6 (75%) |
| Thinning | 3 (375%) | 1 (12.5%) | 1 (12.5%) | 4 (50%) | 1 (12.5%) | 0 (0%) |
| Thickening | 3 (37.5%) | 2 (25%) | 1 (12.5%) | 0 (0%) | 1 (12.5%) | 2 (25%) |
| Osteochondral junction | ||||||
| Normal | 3 (37.5%) | 4 (50%) | 4 (50%) | 3 (37.5%) | 4 (50%) | 5 (62.5%) |
| Invagination | 3 (37.5%) | 2 (25%) | 4 (50%) | 1 (12.5%) | 3 (37.5%) | 2 (25%) |
| Weak junction | 2 (25%) | 2 (25%) | 0 (0%) | 4 (50%) | 1 (12.5%) | 1 (12.5%) |
| Subchondral bone | ||||||
| Normal | 1 (12.5%) | 4 (50%) | 3 (37.5%) | 3 (37.5%) | 4 (50%) | 4 (50%) |
| Trabecular bone increase | 7 (87.5%) | 4 (50%) | 5 (62.5%) | 5 (62.5%) | 4 (50%) | 4 (50%) |
| Chondrocyte | ||||||
| Normal | 2 (25%) | 3 (37.5%) | 6 (75%) | 1 (12.5%) | 5 (62.5%) | 7 (87.5%) |
| Hypocellularity | 2 (25%) | 3 (37.5%) | 0 (0%) | 3 (37.5%) | 1 (12.5%) | 0 (0%) |
| Cluster | 4 (50%) | 2 (25%) | 2 (25%) | 4 (50%) | 2 (25%) | 1 (12.5%) |
The histological cartilage images for both groups at 4 weeks are shown in Fig. 1 . At 4 weeks, a statistically significant difference was found between the two groups for cartilage changes (P = 0.034, r = 0.745). Degenerative cartilage changes in the study group at 4 weeks were lower than those for the controls. Whilst no statistically significant difference was found between the changes in cartilage, osteochondral junction and subchondral bone (P > 0.05), the degenerative bony changes in subchondral bone were significantly higher in the controls compared with the study group (P = 0.034). In the study group, there was a positive correlation between cartilage and the osteochondral junction (P = 0.000) and subchondral bone (P = 0.024).
At 6 weeks, there was no statistically significant difference between the two groups for cartilage, osteochondral junction and subchondral bony changes. A positive correlation was found between cartilage and osteochondral junction (P = 0.044, r = 0.720), and subchondral bony changes (P = 0.017, r = 0.802) in controls, whilst no statistically significant difference was found in the study group (P > 0.05). A negative correlation was found between the changes to the osteochondral junction (P = 0.002) in both groups and subchondral bone (P = 0.000) in the controls. The histological cartilage images for both groups at 6 weeks are shown in Fig. 2 .
At 8 weeks, there was no statistically significant difference between the two groups in cartilage, osteochondral junction and subchondral bony changes (P > 0.05). The cartilage changes to the control joints were significantly decreased at 8 weeks (P = 0.006, r = 0.864). In the study groups, a positive correlation was found between osteochondral junction and subchondral bony changes (P = 0.030, r = 0.756). A negative correlation was found between the subchondral bone in controls and cartilage in the study joints (P = 0.034). A significant decrease of cartilage changes was found in controls between 4 and 6 weeks; a significant increase of cartilage changes was found in controls at 4 weeks compared to the study joints at 8 weeks (P = 0.017, r = 0.800). Significant decrease of cartilage changes was found in controls between 4 and 8 weeks (P = 0.024, r = 0.775). There was no statistically significant difference between the groups at 8 weeks (P > 0.05). The histological cartilage images of both groups at 8 weeks are shown in Fig. 3 .
The distribution of chondrocyte appearance in both groups at each follow-up is shown in Table 1 . The changes to chondrocyte appearance at the three follow-ups were 6 (75%), 5 (63%) and 2 (25%) in the controls, and 7 (88%), 3 (38%) and 1 (13%) in the study group, respectively. Hypocellularity and cluster in the chondrocytes at all follow-ups were observed in 5 (21%), and 8 (33%) of the controls and in 4 (17%), and 7 (29%) of the study group, respectively. A negative correlation was found for the change in chondrocyte appearance between the controls at 6 weeks and the study group at 4 weeks (P = 0.009, r = −0.842). The changes in chondrocyte appearance were significantly decreased in the study group at all follow-ups (P = 0.034).
The distribution of chondrocyte appearance according to the cartilage changes in both groups at all follow-ups is shown in Table 2 . At 4 weeks, chondrocyte clusters were observed on 2 of the 3 control joints that showed cartilage thickening. Whilst on the study joints, which showed the cartilage thinning, hypocellularity and clusters were equally observed. At 6 weeks, in joints with cartilage thickening, hypocellularity and clusters were observed in the controls, whereas the chondrocyte appearance of one study joint was normal. At 8 weeks, normal cartilage and chondrocyte appearance in both groups were found in 5 and 6 joints, respectively. In all follow-ups, no statistically significant difference was found between the changes in cartilage and chondrocyte appearance in both groups (P > 0.05).
| Cartilage changes | Chondrocyte appearance | ||
|---|---|---|---|
| Normal | Hypocellularity | Cluster | |
| 4th week | |||
| Control | |||
| Normal | 1 | – | 1 |
| Thinning | 1 | 1 | 1 |
| Thickening | – | 1 | 2 |
| Study group | |||
| Normal | 1 | 1 | 2 |
| Thinning | – | 2 | 2 |
| Thickening | – | – | – |
| 6th week | |||
| Control | |||
| Normal | 3 | 2 | – |
| Thinning | – | – | 1 |
| Thickening | – | 1 | 1 |
| Study group | |||
| Normal | 4 | 1 | 1 |
| Thinning | – | – | 1 |
| Thickening | 1 | – | – |
| 8th week | |||
| Control | |||
| Normal | 5 | – | 1 |
| Thinning | – | – | 1 |
| Thickening | 1 | – | – |
| Study group | |||
| Normal | 6 | – | – |
| Thinning | – | – | – |
| Thickening | 1 | – | 1 |
The distribution of the osteochondral junction changes and chondrocyte appearance in both groups at all follow-ups is shown in Table 3 . At 4 weeks, the distribution of invaginations in the osteochondral junction according to chondrocyte appearance was observed in 3 control joints with clusters and hypocellularity and one joint with a weak junction showed hypocellularity. Invagination was observed in one study joint, which showed cluster and a weak junction, whilst 4 study joints showed clusters and hypocellularity. At 6 weeks, hypocellularity was observed in 1 of the 2 control joints that showed invaginations in the osteochondral junction. Chondrocyte clusters were observed in 2 control joints with a weak junction. In the study joints, clusters and hypocellularity were observed in 2 of 3 joints with an invagination. A weak junction was observed in one joint with a normal chondrocyte appearance. At 8 weeks, chondrocyte clusters were observed in 1 of the 4 control joints with an invagination. In the study joints, chondrocyte clusters were observed in 1 of the 2 joints with an invagination. A weak junction was observed in one joint with a normal chondrocyte appearance. In all follow-ups, no statistically significant difference was found between osteochondral junction changes and chondrocytes appearance in both groups (P > 0.05).
| Osteochondral junction | Chondrocyte appearance | ||
|---|---|---|---|
| Normal | Hypocellularity | Cluster | |
| 4th week | |||
| Control | |||
| Normal | 1 | – | 2 |
| Invagination | – | 1 | 2 |
| Weak junction | 1 | 1 | – |
| Study group | |||
| Normal | 1 | 1 | 1 |
| Invagination | – | – | 1 |
| Weak junction | – | 2 | 2 |
| 6th week | |||
| Control | |||
| Normal | 2 | 2 | – |
| Invagination | 1 | 1 | – |
| Weak junction | – | – | 2 |
| Study group | |||
| Normal | 3 | – | 1 |
| Invagination | 1 | 1 | 1 |
| Weak junction | 1 | – | – |
| 8th week | |||
| Control | |||
| Normal | 3 | – | 1 |
| Invagination | 3 | – | 1 |
| Weak junction | – | – | – |
| Study group | |||
| Normal | 5 | – | – |
| Invagination | 1 | – | 1 |
| Weak junction | 1 | – | – |
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