Antioxidant capacity of synovial fluid in the temporomandibular joint correlated with radiological morphology of temporomandibular disorders

Available online at www.sciencedirect.com

British Journal of Oral and Maxillofacial Surgery 53 (2015) 114–120

Antioxidant capacity of synovial fluid in the temporomandibular joint correlated with radiological morphology of temporomandibular disorders Kyoko Ishimaru a , Seigo Ohba a , Hitoshi Yoshimura a , Shinpei Matsuda a , Jun-Ichi Ishimaru b , Kazuo Sano a,∗ a b

Division of Dentistry and Oral Surgery, Department of Sensory and Locomotor Medicine, Faculty of Medical Sciences, University of Fukui, Fukui, Japan Department of Oral and Maxillofacial Surgery, Gifu Prefectural General Medical Centre, Gifu, Japan

Accepted 13 October 2014 Available online 7 November 2014

Abstract We investigated the correlation between the antioxidant capacity of synovial fluid and radiological findings of intra-articular structures in patients with disorders of the temporomandibular joint (TMJ). We recruited 21 patients (9 men and 12 women, aged 18–84 years of age) with such disorders, excluding myofascial pain and dysfunction syndrome, or other muscular disorders. The clinical variables recorded included age, sex, interincisal distance, and visual analogue pain scores (VAS). Radiological findings were obtained from diagnostic arthrogram and cone-beam computed tomography (CT). The antioxidant capacity of the synovial fluid was measured by chemiluminescence. Eleven patients were radiologically diagnosed with closed lock, and the remaining 10 with no closed lock. An anchored intra-articular disc was most often seen on cone-beam CT (n = 19) followed by perforated disc (n = 7), osteoarthrosis (n = 7), and anterior disc displacement without reduction (n = 5). Although there were no significant differences between antioxidant capacity and age, sex, VAS, or any findings on cone-beam CT, antioxidant capacity was significantly decreased in the patients with closed lock compared with those who did not have closed lock (p = 0.02). The results suggest an association between the oxidative stress of the synovial fluid and closed-lock in disorders of the TMJ. © 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Keywords: Temporomandibular disorders; Antioxidant capacity; Radiological morphology; Hydroxyl radical; Synovial fluid; Oxidative stress

Introduction Disorders of the temporomandibular joint (TMJ) have been discussed in terms of epidemiology, diagnostic techniques, and treatment. Radiological diagnosis includes arthrograms, double-contrast arthrotomograms, computed tomography (CT), magnetic resonance imaging (MRI), and cone-beam CT. These techniques enable us to visualise the intra-articular structures of the TMJ.

∗

Corresponding author. Tel.: +81 776 61 3111; fax: +81 776 61 8128. E-mail address: [email protected] (K. Sano).

For the classification of disorders, the diagnosis should be based on research diagnostic criteria.1 Additionally, fascial arthromyalgia has been reported as a clinical condition in which the patient complains of chronic pain of unknown origin. It is strongly associated with anxiety and depression.2 Recently considerable attention has been paid to analysing the synovial fluid of the temporomandibular joint (TMJ) to elucidate its aetiology and pathology.3 Several studies have reported that synovial inflammation may be related to the initial stage of a disorder of the TMJ in which inflammatory cytokines, matrix metalloproteases,4,5 disintegrin, and metalloproteinase with thrombospondin motifs are increased, leading to enzymatic degradation of the cartilage matrix, particularly in osteoarthrosis.6

http://dx.doi.org/10.1016/j.bjoms.2014.10.006 0266-4356/© 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

K. Ishimaru et al. / British Journal of Oral and Maxillofacial Surgery 53 (2015) 114–120

Oxidative stress has recently been reported as one of the causes of disorders of the TMJ.7 Human synovial albumin is more oxidised than in serum, and it is more oxidised in patients with disorders of the TMJ than in healthy people with no symptoms.8 Some reports have suggested that reactive oxygen species are generated in the synovial fluid of these patients.7 Generally, reactive oxygen species are represented by superoxide, hydrogen peroxide, and hydroxyl radicals, which are metabolised by superoxide dismutase. However, when their activity is insufficient to metabolise superoxide, toxic hydroxyl radicals will be generated from hydrogen peroxide and may cause cellular damage. Although superoxide is considered to be a major factor that damages tissue,9 we know few reports that have made a quantitative analysis of superoxide in the synovial fluid of patients with disorders of the TMJ. There is little information about the correlation between the detailed morphological and physiological changes in the TMJ and ongoing disease in the synovial fluid. To elucidate the relations between early molecular events and intra-articular disease it is necessary to evaluate the degree of oxidation of synovial fluid in these patients by comparing it with clinical and intra-articular findings. Our aim was to investigate the relations between intraarticular antioxidant capacity and morphological changes by analysing the synovial fluid and radiological images of patients with disorders of the TMJ.

Patients and methods Patients with disorders of the TMJ were selected from among the new patients who presented to the Department of Oral and Maxillofacial Surgery, Gifu Prefectural General Medical Centre. We used research diagnostic criteria for the clinical classification of disorders of the TMJ.1 The diagnosis was made from clinical findings and past history. According to the criteria,1 myofascial pain dysfunction syndrome, facial arthromyalgia, and other muscle disorders associated with anxiety and depression were not included. For further evaluation, age, sex, duration of disease, interincisal distance, and visual analogue scale for pain (VAS), which was assigned a value of 0 for no pain and 100 for the maximum pain ever experienced, were recorded. All procedures were done by the same experienced oral and maxillofacial surgeon. No patient had previously been treated for disorders of the TMJ. For radiological evaluation, all patients had an arthrogram and double-contrast cone-beam CT with radio-opaque fluid or air. Synovial fluid was collected from each TMJ with isotonic saline 1 ml. Collection of synovial fluid Synovial fluid was aspirated under local anaesthesia. After disinfection, a local anaesthetic (2% lidocaine hydrochloride 0.5 ml) was injected into the preauricular subcutaneous

115

tissue. The superior joint space of the TMJ was cannulated, and isotonic saline 1 ml was injected. Then, injection and aspiration were repeated 5 times and the diluted synovial fluid was collected. It was centrifuged at 1600 × g for 3 min, and the supernatant was collected and stored at −70 ◦ C. Conventional arthrogram and double-contrast cone-beam CT Each patient had an arthrogram and double-contrast conebeam CT. After the synovial fluid had been collected, radio-opaque fluid (60% Urografin® ) was injected into the superior joint space through the cannula. The plain arthrogram images were made up of 4 series of articulations of the joint from the intercuspal position, maximal jaw opening, anterior protrusion, and then the intercuspal position. Articular morphology and mobility were examined to find out whether the condyle was anteriorly well-translated, or the disc was displaced, or the condyle was close locked under the mandibular fossa even at maximal mouth opening, or a combination of the three. After the arthrogram, cone-beam CT images were taken using Veraviewepocs3D (Morita Corporation, Osaka, Japan). The radiographic area was extended to 80 mm × 80 mm. The voxel was an isotropic cube as small as 0.125 mm, and the width and pitch of the slice were set between 0.125 and 2 mm. Finally, images of the disc, lamina of the condylar surface, condylar shape, and its position in the mandibular fossa, together with any intra-articular lesions in the superior joint space, were obtained. Evaluation of images Each joint was evaluated by arthrogram and cone-beam CT. The joint was defined as closed lock on the arthrogram when the mandibular condyle did not anteriorly translate across the articular eminence at maximal mouth opening. Similarly, the joint was defined as non-closed lock when the mandibular condyle anteriorly translated across the articular eminence at maximal mouth opening. For the cone-beam CT images, anchored disc in the superior joint space was diagnosed when the joint partially adhered between the temporal surface and the disc, or there was a dense fibrous band, or both. Anterior disc displacement without reduction was diagnosed when the joint was accompanied by an anteriorly displaced disc when the mouth was closed. When there was a morphologically perforated disc, or radio-opaque contrast medium in the inferior joint space, or both, the disc was diagnosed as perforated. When there were any radiological osteoarthrotic bony changes in the mandibular condyle, such as flattening, erosion, sub-cortical cyst, or peripheral bone lapping, osteoarthrosis was diagnosed. Measurement of the antioxidant capacity of synovial fluid in the TMJ The antioxidant capacity of the synovial fluid was measured using specific equipment (Radical Catch® ; Hitachi

116

K. Ishimaru et al. / British Journal of Oral and Maxillofacial Surgery 53 (2015) 114–120

Aloka, Tokyo, Japan) and an AccuFLEX Lumi 400 chemiluminescence reader (Hitachi Aloka, Tokyo, Japan). These systems use the Fenton reaction for assessing the scavenging activity of superoxide, and cobalt (II) chloride was used to produce superoxide because of the stable expression of luminol chemiluminescence with cobalt (II).10 Frozen tubes of samples were collected, prepared at room temperature until defrosted, and centrifuged at 1600 × g for 3 min at 4 ◦ C to remove the large cells and debris. Aliquots of the supernatant of the sample fluid (20 ␮l) were collected using a micropipette. Cobalt solution (50 ␮l) and luminol (50 ␮l) were mixed with the sample solution (20 ␮l). They were incubated for 5 min at 37 ◦ C. Hydrogen peroxide solution (50 ␮l) was then added to the incubated solution for the generation of superoxide. The solution container was placed in the chemiluminescence reader, and light emission was measured at 430 nm for 120 s. The integration of the emission from 80 to 120 s was calculated, and substituted for the light emission volume. Isotonic saline was used as a control. The rate of decrease in the light emission compared with the control was expressed as the antioxidant capacity of the synovial fluid (Fig. 2). Statistical analyses Differences among the antioxidant capacity, sex, and radiological characteristics were analysed using Student’s t test. A simple regression analysis and calculation of the correlation coefficient were used to analyse the relation between the antioxidant capacity and age, duration of disease, interincisal distance, and VAS for pain. All data were analysed using the computer software JMP 10.0.2 (SAS Institute Japan, Tokyo, Japan). Probabilities of less than 0.05 were accepted as significant. The person who measured the antioxidant capacity of synovial fluid in the TMJ was unaware of the clinical and radiographic findings.

Results Twenty-one patients (9 men and 12 women, age range 18–84 years) with disorders of the TMJ were included in the study (Table 1). Clinical symptoms included closed lock (n = 2) and pain (n = 19). Eleven patients were diagnosed with closed lock by arthrogram, and the remaining 10 patients did not have closed lock. Intra-articular anchored disc was most commonly seen in 19 patients by cone-beam CT. Among the patients with anchored discs, 7 were seen to be perforated, and 7 patients had osteoarthrosis. Five patients were diagnosed with anterior disc displacement without reduction, and anchored disc (Fig. 1). In 2 patients, no pathological changes were found. Simple regression analysis failed to show any significant difference between antioxidant capacity and age (p = 0.59) or duration of disease (p = 0.51). There was a slight but not significant link between the antioxidant capacity and VAS

Fig. 1. Cone-beam computed tomographic findings with double-contrast. (*) Anterior disc displacement without reduction was noted in a patient with closed lock by arthrography, accompanied by an anchored disc in the superior joint space.

(p = 0.15), and a moderate correlation between antioxidant capacity and interincisal distance (R2 = 0.22, correlation coefficient = 0.47, p = 0.03). There were no significant differences between sex, clinical symptoms, and antioxidant capacity. However, the arthrogram showed that antioxidant capacity was significantly decreased in the patients with closed lock compared with those who did not have closed lock (p = 0.02). Cone-beam CT showed no significant differences between the antioxidant capacity and anchored disc (p = 0.75), anterior disc displacement without reduction (p = 0.53), perforated disc (p = 0.27), and osteoarthrosis (p = 0.06) (Tables 2 and 3).

Discussion Recently, double-contrast cone-beam CT has been reported to be a reliable and sensitive diagnostic procedure for looking at the TMJ in which an anchored disc is predominant.11 Initially, certain pathological changes in diseases of the TMJ were thought to be the result of simple mechanical load.12,13 The mechanical stress in the intra-articular space of the TMJ

K. Ishimaru et al. / British Journal of Oral and Maxillofacial Surgery 53 (2015) 114–120

117

Table 1 Detailed clinical findings and radiological findings of arthrogram and cone-beam computed tomography (CT). Case No. Age (years) Sex Symptom Duration of disease (weeks)

ID

VAS Clinical diagnosis by RDC

Findings on arthrogram

Findings on cone-beam CT

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

32 30 42 22 50 34 37 28 20 26 27 27 24 32 38 40 46 42 33 53 40

0 22 6 42 20 30 61 30 62 50 26 32 53 0 70 40 46 42 33 53 40

CL CL Non-CL CL Non-CL CL Non-CL CL Non-CL Non-CL CL CL Non-CL CL Non-CL Non-CL CL CL Non-CL Non-CL CL

AD AD AD AD – AD AD AD – AD AD AD AD AD AD AD AD AD AD AD AD

18 73 59 72 61 33 65 53 18 67 57 84 43 40 47 65 72 72 60 75 28

M F F F M M M F F M F F F M F F M F M M F

CL Pain Pain Pain Pain Pain Pain Pain Pain Pain Pain Pain Pain CL Pain Pain Pain Pain Pain Pain Pain

24 3 2 4 2 4 4 8 3 4 2 32 24 24 44 6 24 12 4 3 24

IIb IIb/IIIb IIIa IIb/IIIb IIIa IIb IIIa IIb/IIIb IIIa IIIa IIb/IIIb IIb IIIa IIb/IIIb IIIa IIIa/IIIb IIc/IIIb IIc IIIa IIIa IIc

ADDR ADDR – – – ADDR – – – – – – – – – – ADDR ADDR – – –

– – DP DP – – – DP – – DP DP DP – – – DP – – –

Antioxidant capacity (%) – OA – OA – – – OA – – OA OA – OA OA – – – –

58.7 23.0 62.5 35.0 45.1 57.5 77.0 67.8 62.7 33.6 29.2 47.2 48.9 8.8 68.2 52.1 46.2 38.8 85.4 63.0 22.2

AD, anchored disc; ADDR, anterior disc displacement without reduction; CL, closed lock; DP, disc perforation; ID, interincisal distance (mm), non-CL, non-closed lock; OA, osteoarthrosis; RDC, research diagnostic criteria; VAS, visual analogue pain score.

may produce free radicals through direct tissue damage and hypoxia-reperfusion.14 Nitzan proposed that increased friction of the contiguous parts breaks the lubrication system in the intra-articular space, which may produce free radicals.15 Superoxide radicals in the synovial fluid of the TMJ in such patients were identified using electron spin resonance.9,16 The effect of free radicals in the TMJ is thought to

depolymerise hyaluronan, leading to a direct attack on proteoglycan and degradation of cartilage, which stimulates expression of osteoclasts. Iron-dependent generation of superoxide has a crucial role in the pathogenesis of these disorders.9 The radical is produced by the Fenton reaction17 and reacts catalytically with iron compounds and divalent hydrogen peroxide under acidic conditions. Intra-articular

Fig. 2. Diagram showing the broad principle of measuring the antioxidant capacity of synovial fluid in the temporomandibular joint using the Fenton reaction. The greater the antioxidant capacity, the lower the luminol light emission. SF, synovial fluid.

118

K. Ishimaru et al. / British Journal of Oral and Maxillofacial Surgery 53 (2015) 114–120

Table 2 Antioxidant capacity according to clinical variables. (A) Age (years), duration of disease, interincisal distance, and visual analogue pain score. Variable r2 SCC T(20) p value Age (years) Duration of disease Interincisal distance Visual analogue score

0.001 0.023 0.220 0.106

−0.04 −0.153 0.47 0.33

−0.18 −0.67 2.32 1.50

0.86 0.51 0.03 0.15

(B) Sex Antioxidant capacity (%) Men (n = 9) Mean (SD) 95% CI Women (n = 12) Mean (SD) 95% CI p value

52.8 (22.9) 35.2 to 70.3 47.3 (16.1) 37.0 to 57.5 0.53

SCC, Spearman’s correlation coefficient.

pathological changes are therefore not only the result of simple mechanical load, but also biochemical changes in the TMJ, and early molecular events can be provoked by free radicals.18 Güven et al. reported that the activity of superoxide dismutase may result from insufficient scavenging by free-radicals.18 The remaining antioxidant capacity can be used to evaluate antioxidant component activity and substituted for quantitative analysis of free radicals.10,19,20 Recently, a relation between antioxidant capacity and some diseases has been reported.19,21,22 We focused in this study on the antioxidant capacity in the synovial fluid, which we correlated with the radiological morphology, or morbidity, or both, of the TMJ by using an antioxidant potential measuring system based on the Fenton reaction for assessing superoxide scavenging activity, and there was a significant difference (p = 0.02) in antioxidant capacity between those with closed lock and those who did not have closed lock. Etöz et al. reported that the total antioxidant capacity of the synovial fluid in such patients with limited mouth opening was significantly lower than in those who did not complain of limited mouth opening.23 We produced similar findings and support the conclusion of the

previous study.23 When considering those two sets of results, we suggest that TMJ with closed lock can be more oxidised than those without. The antioxidant capacity was compared among groups with disorders of the TMJ but not healthy controls, because it is not acceptable to collect and analyse synovial fluid from healthy people for ethical reasons. Antioxidant capacity was therefore analysed using patients’ samples correlated with detailed radiological changes in the TMJ. Most patients had anchored discs in the superior joint space, and all patients with closed lock also had anchored discs with reduced antioxidant capacity. These results suggest that intra-articular pathological changes can be initiated from superficial damage to the articular tissue, resulting in articular fibrillation and then an anchored disc in the genesis of early disorders of the TMJ. Certainly, an anchored disc is the most common pathological change in each stage of disorder of the TMJ.24 Adhesion predominantly develops in patients with locked joints and less wide mouth opening, and increased internal derangement results in a worse adhesive grade in the TMJ.24 Mechanically-stressed TMJ may produce free radicals in the intra-articular space that subsequently cause synovial inflammation. We hypothesise that extreme mechanical stress and release evoke a hypoxia-reperfusion mechanism in the intra-articular space. This hypoxia-reperfusion mechanism can account for the generation of free radicals in the TMJ. Highly reactive superoxide can be generated in a reaction catalysed by ferrous ion (Fe2+ ) with the interaction of superoxide and hydrogen peroxide, which is known as the Fenton reaction: Fe2+ + O2 ⇒ Fe3+ + • O2 − •O

2−

+ O2 − + 2H+ → H2 O2 + O2

Fe2+ + H2 O2 ⇒ Fe3+ + OH− + • OH The redox active ion is contained in haemoglobin, which can contribute to a redox reaction leading to tissue

Table 3 Antioxidant capacity (%) compared with radiological characteristics as shown by arthrography and cone-beam computed tomography (CT). Variable Findings on arthrogram Closed lock (n = 11) No closed lock (n = 10) Findings on cone-beam CT Anchored disc (n = 19) No anchored disc (n = 2) Anterior disc displacement without reduction (n = 5) No anterior displacement (n = 16) Perforated disc (n = 7) No perforated disc (n = 14) Osteoarthrosis (n = 7) No osteoarthrosis (n = 14)

Mean (SD)

95% CI

p value

40.4 (18.5) 59.8 (15.1)

26.5 to 52.9 48.6 to 68.8

0.02

49.2 (19.8) 53.9 (12.5) 44.8 (14.7) 51.2 (20.3) 42.9 (19.6) 53.0 (18.5) 38.9 (19.3) 55.0 (17.0)

39.7 to 58.7 −58.0 to 165.8 26.5 to 63.1 40.3 to 62.0 24.9 to 61.1 42.3 to 63.7 21.0 to 56.7 45.2 to 64.9

0.75 0.53 0.27 0.06

K. Ishimaru et al. / British Journal of Oral and Maxillofacial Surgery 53 (2015) 114–120

119

Fig. 3. The molecular mechanism for oxidative stress in the temporomandibular joint. ROS, reactive oxygen species; SOD, superoxide dismutase; TMJ, temporomandibular joint.

damage.14,25 The oxygen species are released by enzymatic reaction and play an important part in the early molecular events of disorders of the TMJ (Fig. 3). Our results showed a significant difference in antioxidant capacity between the groups with and without closed lock. One possible explanation is an intra-articular circulatory disturbance between the synovial fluid and blood plasma. Synovial fluid is the component of plasma that is supplied by movement of the joint. If the condyle is locked, the anti-oxidative contents of plasma, such as superoxide dismutase, barely permeate into the synovial fluid. A significant correlation between antioxidant capacity and interincisal distance suggests that the greater the mouth opening, the healthier is the TMJ. We therefore suggest that the closed lock should preferably be released as early as possible to free it from intra-articular oxidative stress, which leads to healthy locomotive conditions.

Conflict of interest We have no conflict of interest.

Ethics statement/confirmation of patients’ permission The study was approved by the Clinical Ethics Committees, General Medical Centre and Faculty of Medical Sciences at the University. All patients provided written informed consent. References 1. Dworkin SF, LeResche L. Research diagnostic criteria for temporomandibular disorders: review, criteria, examination and specification, critique. J Craniomandib Disorders 1992;6:301–55. 2. Madland G, Feinmann C, Newman S. Factors associated with anxiety and depression in facial arthromyalgia. Pain 2000;84:225–32.

3. Herr MM, Fries KM, Upton LG. J Oral Maxillofac Surg 2011;69:41–7. 4. Mizui T, Ishimaru J-I, Miyamoto K, et al. Matrix metalloproteinase-2 in synovial lavage fluid of patients with disorders of the temporomandibular joint. Br J Oral Maxillofac Surg 2001;39:310–4. 5. Sato J, Segami N, Nishimura M, et al. Expression of interleukin 8 in synovial tissues in patients with internal derangement of the temporomandibular joint and its relationship with clinical variables. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:467–74. 6. Luan Y, Kong L, Howell DR, et al. Inhibition of ADAMTS-7 and ADAMTS-12 degradation of cartilage oligomeric matrix protein by alpha-2-macroglobulin. Osteoarthritis Cartilage 2008;16:1413–20. 7. Cai HX, Luo JM, Long X, et al. Free-radical oxidation and superoxide dismutase activity in synovial fluid of patients with temporomandibular disorders. J Orofac Pain 2006;20:53–8. 8. Tomida M, Ishimari J, Hayashi T, et al. The redox states of serum and synovial fluid of patients with temporomandibular joint disorders. Jpn J Physiol 2003;53:351–5. 9. Lee MC, Kawai Y, Shoji H, et al. Evidence of reactive oxygen species generation in synovial fluid from patients with temporomandibular disease by electron spin resonance spectroscopy. Redox Rep 2004;9:331–6. 10. Parejo I, Petrakis C, Kefalas P. A transition metal enhanced luminol chmiluninescence in the presence of a chelator. J Pharmacol Toxicol Methods 2000;43:183–90. 11. Akiyama K, Takumi M, Baba S, et al. Imaging the temporomandibular joint by double contrast cone-beam computed arthrotomography. J Oral Maxillofacial Surg Med Pathol 2012;24:23–6. 12. Roberts CA, Tallents RH, Katzberg RW, et al. Comparison of internal derangements of TMJ with occlusal findings. Oral Surg Oral Med Oral Pathol 1987;63:645–50. 13. Magnusson T, Egermarki I, Carlsson GE. A prospective investigation over two decades on signs and symptoms of temporomandibular disorders and associated variables. A final summary. Acta Odontol Scand 2005;63:99–109. 14. Milam SB, Zardeneta G, Schmitz JP. Oxidative stress and degenerative temporomandibular joint disease: a proposed hypothesis. J Oral Maxillofac Surg 1998;56:214–23. 15. Nitzan DW. The process of lubrication impairment and its involvement in temporomandibular joint disc displacement: a theoretical concept. J Oral Maxillofac Surg 2001;59:26–45. 16. Tomida M, Ishimaru J-I, Murayama K, et al. Intra-articular oxidative stress correlated with the pathogenesis of disorders of the temporomandibular joint. Br J Oral Maxillofac Surg 2004;42:405–9. 17. Sadrzadeh SM, Graf E, Panter SS, et al. Hemoglobin: a biologic fenton reagent. J Biol Chem 1984;259:14354–6.

120

K. Ishimaru et al. / British Journal of Oral and Maxillofacial Surgery 53 (2015) 114–120

18. Güven O, Tekin US, Durak I, et al. Superoxide dismutase activity in synovial fluids in patients with temporomandibular joint internal derangement. J Oral Maxillofac Surg 2007;65:1940–3. 19. Kawamoto A, Sugano N, Motohashi M, et al. Relationship between salivary antioxidant capacity and phases of the menstrual cycle. J Periodont Res 2012;47:593–8. 20. Yildiz G, Demiryürek AT. Ferrous iron induced luminol chemiluminescence: a method for hydroxyl radical study. J Pharmacol Toxicol Methods 1998;39:179–84. 21. Suantawee T, Tantavisut S, Adisakwattana S, et al. Oxidative stress, vitamin E, and antioxidant capacity in knee osteoarthritis. J Clin Diagn Res 2013;7:1855–9.

22. Okubo H, Syddall HE, Phillips DIW, et al. Dietary total antioxidant capacity is related to glucose tolerance in older people: The Hertfordshire Cohort Study. Nutr Metab Cardiovasc Dis 2014;24:301–8. 23. Etöz OA, Akc¸ay H, Nes¸elio˘glu S, et al. Total antioxidant capacity and total oxidant status of synovial fluids in patients with temporomandibular joint pain and dysfunction. Clin Oral Invest 2012;16: 1557–61. 24. Zhang S, Liu X, Yang C, et al. Intra-articular adhesions of the temporomandibular joint: relation between arthroscopic findings and clinical symptoms. BMC Musculoskelet Disord 2009;10:70. 25. Halliwell B, Gutteride JM. Free radicals in biology and medicine. 4th ed. Oxford: Clarendon Press; 2007.