Oxidative Stress Levels in Orthodontic Patients and Efficacy of Antioxidant Supplements in Combating Its Effects- A Randomized Clinical Study

Introduction: Orthodontic appliances are considered to be biocompatible although adverse effects attributed to release of nickel ion which are free radicals in oral cavity. These free radical produce damages both in cellular and extracellular components phospholipid membrane, proteins, mitochondrial and nuclear DNA leading to Oxidative stress which is normally counter balanced by the action antioxidant mechanisms. However in higher concentrations of free radicals resulting in cellular death and apoptosis Orthodontic appliances such as brackets , wire, resins has considered as potential allergen leading to release of free radicals. The study was conducted to determine and compare the role of oxidative stress and role of antioxidants in saliva of patients undergoing fixed orthodontic appliances therapy at different time intervals. Materials & Method: A double-blinded, parallel, randomized clinical study was designed consisting of 40 healthy participants, aged 15-30yrs. The samples were divided into two groups Group A without antioxidants supplements and Group B with antioxidants supplements. Salivary MDA levels and gingival health index was recorded at different time intervals from each group. Salivary Lipid peroxidation (Malondialdehyde) level was estimated using Thiobarbituric acid (TBA) method and gingival status was investigated using Silness & Loe gingival index. Intragroup and intergroup comparison was statistically analyzed using student’s paired T test. Result: Increased salivary MDA levels and mild to moderate amount of gingivitis is seen in both groups. This is more pronounced after 24 hours of appliance placement. Improvement in salivary MDA levels and gingival health status is observed during the course of treatment, In Group B the salivary MDA levels and gingival health index score reached below their pretreatment values. However even after a span of 3 months the levels remained higher to their base values in Group A. Conclusion: Improvement in salivary MDA levels and gingival health status is observed following antioxidant therapy during course of treatment, indicating combating nature of antioxidant supplements in orthodontic patients.


INTRODUCTION
Free radicals are highly reactive and have very short span of life. Considering this, the damage produced by a free radical ROS damages all components of the cell, including proteins, lipids, and DNA. 6 However, the most important cellular targets of oxidative stress are the phospholipid membranes. 7 When it reacts with lipids the by-product of the reaction is lipid peroxidation. 8 It is the process in which free radicals "steal" electrons from the lipids present in cell membranes, resulting in cell damage. 9 Lipid peroxides attack on polyunsaturated fatty acids one of the by-product of this reaction is MDA. 10 Saliva, an important physiologic fluid, containing a highly complex mixture of substances, is rapidly gaining popularity as a diagnostic tool. 11 It carries important relevant information in the form of biomarkers and act as a mirror of oral and systemic health. 12 Saliva can be used as a medium to analyse the oxidative stress levels in terms of MDA. 13 Antioxidants neutralize free radicals. They may be classified as enzymatic and non enzymatic. They act as scavengers, helping to prevent cell and tissue damage. 14 The most well research non enzymatic antioxidant includes lipid soluble vitamin E and water soluble vitamin C and vitamin A. 15 Recently, it has been claimed that the imbalances in the levels of free radicals and antioxidants in saliva may play an important role in the onset of periodontal diseases. 15 However literature lack studies whether dietary supplements of antioxidants can combat with the oxidative stress induced by orthodontic appliances and improve the gingival health status of orthodontic patients. Hence this study was undertaken to study the effect of dietary supplements on salivary MDA level and gingival health status of orthodontic patients.

Study Design
The study was designed to be double blinded and randomized consisting of forty healthy patients aged 18-30yrs who were supposed to undergo fixed orthodontic treatment. The patients were randomly selected from the outpatient department of orthodontics using simple random sampling techniques. The CONSORT statement was used as a guide for this study. 16

Ethical Approval and Informed Consent
Ethical approval of this prospective clinical study was obtained from the local ethics committee of our institute and University vide letter no MUHS /PG/E-2/1190/14. Written consent was retrieved before commencement of the study.

Sample Size Calculation
The sample size was determined from a previous study. 17 Our standard difference=0.6 and power= 80; 20 was the number of volunteers needed per group.  The coding of the saliva samples was not disclosed to the laboratory personnel to minimize experimental bias.

Outcomes
The saliva was centrifuged at 3000 rpm for 15 min in

Error of the Method
All the measurements were done by single examiner. The intra examiner reliability for data readings was assessed using Kappa statistics which was found to be 94%.

RESULT
Intragroup and intergroup comparison of salivary MDA levels and gingival health index was statistically analysed using standard paired t test.

Comparison of Mean Level
In Group A, MDA levels were found to be (0.

DISCUSSION
Over the past few years, strong evidence has emerged to implicate oxidative stress in pathogenesis of dental disease. At low concentration, they stimulate the growth of fibroblasts and epithelial cells in culture, but at higher concentrations it may result in tissue injury. 19 An Orthodontic appliance remain in mouth for 2 years or more where saliva is the connecting media, may produce electro galvanic currents that produce a discharge of ions. 20 The harmful effects of free radicals are counterbalanced by an antioxidant mechanism. 21    balance in favour of free radicals deteriorates the oxidative balance and produces oxidative tissue damage associated with oxidative stress. 21,22 Saliva reflects general health status of the human organism and is easy to collect, it can be used as a non-invasive diagnostic tool. 23 This study was undertaken to determine whether oxidative stress are induced by orthodontic appliance, role of antioxidants on oxidative stress and also its effect on gingival health.
In present study MDA levels were significantly increased after 24hrs in comparison of pre-treatment values in both groups. There was constant decline in the MDA levels after seven days to three months but yet not reaching to the base values. This can be attributed to the fact that metal ions release from appliances are higher in first 24hrs. The results were same with findings reported by Nilforoushan et al 24 and Maja khuta et al. 25 The study was also in accordance to D'Attitillo et al 26 and Oltleanu et al 27 who stated that radical ions were higher in early stages of treatment.
In antioxidant group the results were also seen to exceed from base line to seven days and were statistically significant, however after three months, MDA levels were lower than the baseline values and were significant. The antioxidants prescribed to the patients combated with the free radicals formed due to metal ions. These findings were in correlation of Rai et al 28  The MDA level among the two groups showed insignificant difference. However after 3 months there was significant difference in MDA levels among the two groups. (P=0.000). These findings indicate that oxidative stress are built up at 3 months was combated by antioxidants.
The second part of study was to determine the effects of oxidative stress on gingival health and role of antioxidants during the treatment. Oral hygiene was maintained yet gingival inflammation was seen in both the groups. As explained earlier releases of metal ions were higher at initial stages of appliance placement. This increase in oxidative stress levels in the initial period may be the reason of gingival tissues inflammation as suggested by Grimsdottir et al30 and Buljan et al. 31 The results of our study were in accordance to the findings of Ajith Pillai 32  The results of our study are seen in collaboration to the results of study conducted by Benjamin. 17 They reported reduction in BOP, GI in the patients treated with local applicant of antioxidant -essential oil gel. The highly significant difference between the two groups can be explained by the fact that antioxidant supplements prescribed had its effect on combating with oxidative stresses generated by the orthodontic appliance.
The results of our study are in contrast with the results of Elcin Esenlick 34 studied role of (vitamin E) on lipid peroxidation levels in GCF of 50 orthodontic patients may be due to the difference in medication and protocol followed. In our study antioxidants supplements prescribed for 3 months included vitamin A, vitamin C and vitamin E, whereas in Esenlick study only vitamin E was prescribed for a 1 month. Micronutrients such as beta-carotene and vitamins A, C and E can be depleted during inflammation. 6 As mitochondria, release ROS in the cell. In a study in Schectman 35 et al suggested that vitamin C enters the mitochondria and protects against oxidative injury. These vitamins maintens structural and functional integrity of epithelial tissues and physiological or metabolic parameters of periodontal health. 35 Gingival health status is observed during the course of treatment, but do not reach its pre-treatment values. This may be due to oxidative stress induced by the leached metal ions from the appliance. Prescription of antioxidant has shown to deal with this problem in a time span of 3 months. However in this study, no changes in the archwire or ligatures were done throughout the study.

CONCLUSION
Our study concludes that even after thorough oral hygiene maintenance in orthodontic patients, increased salivary MDA levels and mild to moderate amount of gingivitis is seen in both groups. Improvement in salivary MDA levels and gingival health status is observed during the course of treatment, however even after a span of 3 months the levels remained higher to their base values due leaching of ions.
where as in Antioxidant group the salivary MDA levels after