Abstract
Background:
Pain and anxiety are the most reported negative effect of orthodontic treatment which can influence the quality of life and are responsible for the discontinuation of treatment. These unpleasant experiences raise the stress levels reflected by the increase in the concentration of salivary alpha-amylase and nitric oxide. These can be assessed through various subjective methods which have limited value as it depends on the patient's honesty, assessing whether using objective methods are reliable.
Objectives:
To assess pain and anxiety-induced stress levels using both physiological (Salivary alpha-amylase and Salivary nitric oxide) and psychological testing instruments (Visual analog scale and State Trait Anxiety Inventory Y6) during the initial phases of fixed orthodontic treatment.
Materials and Methods:
A total of 30 patients were included. Pain and anxiety instruments were applied and saliva samples were collected from patients before and after separator placement, before and 48 h after archwire insertion and 1 month after archwire insertion.
Results:
The mean values of alpha-amylase (235.3600), nitric oxide levels (2.060) and pain scores (7.8667) were higher 48 h after archwire insertion (T3) which are statistically significant, P = 0.00. Anxiety scores (57.7000) were high before separator placement (T0). No statistically significant correlation was found among pain and alpha-amylase, anxiety and nitric oxide levels.
Conclusion:
Initial alignment phases of fixed orthodontic treatment affect patients' anxiety and stress levels. The findings could be a result of the psychological stress due to procedures in the initial phases of the treatment.
Keywords: Alpha-amylase, anxiety, orthodontic treatment, nitric oxide, visual analog scale
INTRODUCTION
Pain is a subjective reaction that is influenced by a variety of factors such as age, gender, current emotional state, and previous experiences of an individual. Different dental procedures, especially fixed orthodontic treatment, causes tissue trauma and discomfort in 95% of patients, which is influenced by various stress-related factors and individual anxiety levels.[1] Relevant facts suggest that, in some clinical situations, anxiety and pain may be indistinguishable from an individual's perception and have been shown to be a major barrier to providing adequate dental care, leading to the cessation of treatment.[2]
The autonomic nervous system (ANS) is critical in the maintenance of biological stress in stress-related symptoms. There is a variety of ANS markers that may indicate autonomic activation and specific dependent mechanisms.[3] Pain and anxiety are associated with stressors capable of stimulating the autonomic nervous system (ANS), resulting in increased secretion of salivary stress biomarkers, which may make them markers of pain and anxiety,[4] and in the last few decades, there is a dramatic increase in research involving the assessment of biomarkers of salivary stress because they can be easily and reliably measured from saliva.[5] Therefore, monitoring such biomarkers is critical for preventing disease and stress-related pathology and evaluating behavioural strategies to improve an individual's quality of life. However, the literature on studies investigating the link between anxiety and neuroendocrine changes is very limited. As a result, the purpose of this study was to evaluate and compare the stress-related biomarkers salivary alpha-amylase (SAA), salivary nitric oxide (SNO) levels, pain and anxiety levels in patients during the early alignment phases of fixed orthodontic treatment.
MATERIALS AND METHODS
This was a cross-sectional study done on patients requiring fixed orthodontic treatment at Vishnu Dental College. G Power 3.1 software was used to calculate the research sample size. For one group being measured at five different instances at a level of significance set at 5%, the power of the study was set at 80%; and for an expected effect size of 0.100 (obtained from previous studies), 55 subjects at each time interval are required. To compensate for dropouts if any, 60 subjects were taken at each interval. Before the commencement of the study, institutional ethical clearance was obtained (IECVDC/2019/PG01/OP/IVV/66) on 20/12/2019.
Inclusion criteria include the subjects with:
Age 18 to 24 years requiring the fixed orthodontic treatment without any additional appliances.
Requiring bonding of both maxillary and mandibular arches done on the same day.
Absence of deciduous teeth.
Exclusion criteria include the subjects with:
The history of fixed orthodontic treatment, with medical problems and under medication.
With a habit of smoking and drinking alcohol.
After a detailed explanation of the study, written consent was taken from the subjects.
Samples were collected before (T0) and after separator placement (T1), before (T2) and after arch-wire placement (T3) and after 1 month follow-up (T4).
Subjects who visited the orthodontic department were told to relax for 5 min to reduce orofacial movements. They were then made to sit with their heads tilted slightly forward and eyes open. They were instructed not to eat or drink for 1 to 2 h and were asked to rinse their mouths thoroughly with water before sample collection, allowing saliva to pool on the floor of the mouth and passively dripped through a funnel into a sterile test tube by the spit method. The method was continued till 1 ml of unstimulated saliva was collected.[6]
The salivary samples were collected at five intervals [Box 1].
Box 1.
Demonstrating the study design
| On the First Day of the appointment before starting the treatment, T0 (1st day): A pre-prepared State Trait Anxiety Inventory Y6 (STAI Y6) questionnaire was given to subjects at the waiting room and collected the saliva sample T1 (1st day): After separator placement - (Visual Analogue Scale (VAS), STAI Y6), saliva sampling. T2 (7th day): Before arch wire insertion- (VAS, STAI Y6), saliva sampling. T3 (9th day): 48hrs after arch wire insertion (VAS, STAI Y6), saliva sampling. T4 (Follow-up, 4 weeks): After one month - follow up (VAS, STAI Y6), saliva sampling. |
The samples were immediately placed in a thermocol box with ice packs for transit to the laboratory for examination. They were centrifuged for 15 min at 2,500 rpm to remove exfoliated cells and debris, then incubated at room temperature. Analysis of salivary alpha-amylase and salivary nitric oxide levels was done using a biochemical analyser (Carex EA112) within 3 to 4 h of sample collection.
Assessment of anxiety
To assess the anxiety, State Trait Anxiety Inventory Y6 questionnaire (STAI Y6) was used to note their perceptions. STAI-Y6 is a six-item scale with three positive items (calm, relaxed, and content) and three negative items (tension, upset, and worries) to test the attitudes in individual clinical applications by creating a trusting relationship that their responses will be kept confidential.
STAI-Y6 was given at all five intervals (T0, T1, T2, T3, and T4) and the scoring was given as (1- not at all, 2- somewhat, 3- moderately, and 4- very much).[7,8]
After sum up, all the scores with the number of items, based on the value obtained, are considered as:
20–40 mild anxiety
40–60 moderate anxiety
60–80 severe anxiety
STAI score (range 20–80)
Assessment of pain
Pain perception was measured using the VAS scale, with ‘no pain’ and ‘possible worst pain’. All subjects were asked whether they felt pain at four time intervals (T1, T2, T3, T4) and to make a mark on the line closest to the pain he or she felt.
Statistical analysis
All of the collected data were statistically analysed using the Statistical Package for Social Sciences software (SPSS version 22 IBM). The following statistical methods were applied in the study:
Repeated measures of ANOVA for the mean difference between the intervals
Pearson's correlation coefficient test to correlate between anxiety and salivary stress biomarkers, and between pain levels and Salivary stress biomarkers. The significance level was set at P < 0.05.
RESULTS
A total of 60 patients were included in this study, ages ranging between 18 and 24 years.
Salivary alpha-amylase levels
Table 1 shows the means and standard deviations of salivary alpha-amylase. Salivary alpha-amylase levels were statistically significant between intervals (P = 0.00), with the T3 interval having the highest salivary alpha-amylase.
Table 1.
Means and SDs of salivary alpha-amylase levels (g/dl) at each interval
| Time intervals | Mean | Standard deviation | 95% Confidence interval |
P | |
|---|---|---|---|---|---|
| Lower bound | Upper bound | ||||
| T0 | 198.7700 | 193.50712 | 126.513 | 271.027 | 0.000 |
| T1 | 158.8987 | 84.15572 | 127.474 | 190.323 | 0.000 |
| T2 | 170.5567 | 157.74563 | 111.653 | 229.460 | 0.000 |
| T3 | 235.3600 | 125.22465 | 188.600 | 282.120 | 0.000 |
| T4 | 115.1200 | 67.83675 | 89.789 | 140.451 | 0.000 |
Salivary nitric oxide levels
Table 2 shows the average and standard deviations of salivary nitric oxide. Salivary nitric oxide levels were statistically significant between intervals (P = 0.04), with the T3 interval having the greatest salivary nitric oxide.
Table 2.
Means and SDs of salivary nitric oxide levels (g/dl) at each interval
| Time intervals | Mean | Standard deviation | 95% Confidence interval |
P | |
|---|---|---|---|---|---|
| Lower bound | Upper bound | ||||
| T0 | 1.3283 | 1.21026 | 0.876 | 1.780 | 0.004 |
| T1 | 1.4529 | 0.67327 | 1.202 | 1.704 | 0.004 |
| T2 | 1.2121 | 0.60067 | 0.988 | 1.436 | 0.004 |
| T3 | 2.0650 | 1.05811 | 1.670 | 2.460 | 0.004 |
| T4 | 1.1873 | 0.77300 | 0.899 | 1.476 | 0.004 |
State- trait anxiety values
Table 3 shows the means and standard deviations of anxiety values. Anxiety values were statistically significant between the intervals (P = 0.00). T0 anxiety levels were much greater than at subsequent time intervals, followed by the T3 interval.
Table 3.
Means and SDs of anxiety scores at each interval
| Time intervals | Mean | Standard deviation | 95% Confidence interval |
P | |
|---|---|---|---|---|---|
| Lower bound | Upper bound | ||||
| T0 | 57.7000 | 9.48011 | 54.160 | 61.240 | 0.000 |
| T1 | 44.6333 | 8.19791 | 41.572 | 47.694 | 0.000 |
| T2 | 29.5667 | 5.64271 | 27.460 | 31.674 | 0.000 |
| T3 | 43.4667 | 7.57370 | 40.639 | 46.295 | 0.000 |
| T4 | 28.8000 | 5.37170 | 26.794 | 30.806 | 0.000 |
VAS values
Table 4 shows the means and standard deviations of the VAS scores. There were statistically significant variations across the intervals (P = 0.00). At the T3 interval, the peak for pain intensity was observed.
Table 4.
Means and SDs of pain scores at each interval
| Time intervals | Mean | Standard deviation | 95% Confidence interval |
P | |
|---|---|---|---|---|---|
| Lower bound | Upper bound | ||||
| T1 | 6.8000 | 1.56249 | 6.217 | 7.383 | 0.000 |
| T2 | 4.6000 | 1.61031 | 3.999 | 5.201 | 0.000 |
| T3 | 7.8667 | 1.38298 | 7.350 | 8.383 | 0.000 |
| T4 | 3.7000 | 1.78403 | 3.034 | 4.366 | 0.000 |
Table 5 shows the correlation between anxiety and salivary alpha-amylase and nitric oxide. At the T1 interval, there was a statistically significant association between anxiety and salivary alpha-amylase levels, but no such correlation was identified between anxiety and salivary nitric oxide.
Table 5.
Pearson's correlation coefficient between anxiety, salivary alpha-amylase and salivary nitric oxide
| Salivary alpha-amylase | Salivary nitric oxide | |
|---|---|---|
| T0 | ||
| r | -0.178 | -0.163 |
| p | 0.346 | 0.388 |
| T1 | ||
| r | 0.465** | 0.119 |
| p | 0.010 | 0.530 |
| T2 | ||
| r | 0.138 | -0.206 |
| P | 0.466 | 0.275 |
| T3 | ||
| r | -0.152 | 0.228 |
| p | 0.422 | 0.226 |
| T4 | ||
| r | 0.288 | 0.047 |
| P | 0.123 | 0.807 |
Table 6 shows the correlation between the pain values with the salivary alpha-amylase and salivary nitric oxide. At the T2 interval, there was a statistically significant association between pain and salivary nitric oxide levels, but no such correlation was identified between pain and salivary alpha-amylase.
Table 6.
Pearson's correlation coefficient among pain, salivary alpha-amylase and salivary nitric oxide
| Salivary alpha-amylase | Salivary nitric oxide | |
|---|---|---|
| T1 | ||
| r | -0.334 | 0.077 |
| P | 0.071 | 0.687 |
| T2 | ||
| r | -0.252 | 0.427* |
| P | 0.179 | 0.018 |
| T3 | ||
| P | -0.027 | 0.260 |
| r | 0.886 | 0.166 |
| T4 | ||
| P | 0.066 | 0.090 |
| r | 0.730 | 0.636 |
DISCUSSION
Fear and anxiety are important factors in avoiding dental care. Dental anxiety is associated with state anxiety and pain associated with dental procedures, as well as with a variety of conditions.[9] This includes past unpleasant or traumatic events, personal character traits, lack of understanding, body image perception, and a vulnerable position in the dental chair. Such concerns may imply treatment outcomes.
Orthodontic treatment has a good focus on the functional, aesthetic, psychological and social aspects of the patient. During the early stages of fixed orthodontic treatment, the placement of separators, elastics, brackets and archwires can cause mild sustained forces that cause pain and discomfort to the patient, resulting in increased pressure that can lead to treatment interruption.[10]
Stress responses due to the effect of orthodontic appliances where the experimental tooth movement induces a significant pain and change in emotional behaviours have been studied in animals.[11] However, a correlation between the stress responses and orthodontic treatment in humans is not well documented. The intensity of discomfort influences personal anxiety and stress-related factors.
Stress responses are evoked by anxiety-related situations in the human body which can be observed in the form of increased activity of the Hypothalamopituitary axis (HPA) and sympathetic adrenal medullary system (SAMS).[12] The subjective assessment of stress is influenced by human bias. Therefore, evaluation by objective assessment by using various biomarkers is rapidly increasing. Salivary alpha-amylase is a carbohydrate-digesting enzyme generated by the salivary glands, and it contains a significant number of norepinephrine-stimulated beta-adrenergic receptors. During psychosocial stress, SAMS is activated and increases the plasma norepinephrine and in turn, increases the salivary alpha-amylase.[13]
Psychosocial and physiological stress activates the HPA axis and releases the stress hormones such as glucocorticoids which are mediated by nitric oxide synthases and are responsible for the production of nitric oxide in saliva.[14] These biomarkers have shown a good correlation with subjective analysis of anxiety and pain intensity.[15]
Pain is considered to be one of the most cited negative effects of orthodontic treatment. The VAS is one of the most often used methods for assessing pain intensity, and it has been shown to be a valid and reliable tool that is simple, sensitive and highly reproducible.[16] As orthodontic treatment has an influence on a person's anxiety, according to Sari et al.,[17] individuals who are awaiting orthodontic treatment have higher anxiety levels than those who have already received treatment and are used to it. Assessment of these levels can be done by using various questionnaires such as dental anxiety and STAI, as they are more reliable and valid. State trait anxiety inventory is used to determine the person's anxiety at that moment as well as their general baseline anxiety.[18,19]
In the current study, pain intensity, salivary alpha-amylase and salivary nitric oxide levels all increased significantly 48 h after archwire insertion (T3) [Tables 1, 2, and 4], whereas anxiety levels were higher before starting treatment (T0) [Table 3], which was consistent with the study by Aksoy et al.[20] 2018 in which patients started to feel discomfort and pain after separators, band, and archwire insertion. The difference in values is most likely attributable to procedures creating pain and anxiety in patients. After 1 month follow-up (T4), the values were low when compared to all three intervals as patients might have got adapted to appliances. There is a significant positive correlation between the STAI Y6 and salivary alpha-amylase values at the T1 interval [Table 5]. There was no correlation found between the pain intensity and the concentration of salivary alpha-amylase in our study, which differs from the results obtained from the study conducted by Shirasaki et al. and Arai et al.,[21,22] where there is a significant positive correlation found between pain intensity and salivary alpha-amylase.
As there are no studies regarding the assessment of pain-induced stress salivary nitric oxide levels in patients undergoing fixed orthodontic treatment.[23] This is the first research to our knowledge in which SNO was employed as anxiety and pain-induced stress biomarker in orthodontic patients, and we identified a strong positive correlation between pain and SNO during the T2 interval, but no correlation between anxiety and SNO, which is similar to a study done by Omar Salem Gammoh in 2016.[24]
Saliva and its biomarkers have been analysed in this study which can be used as a model for assessing pain and anxiety-induced stress in general dental practice. SAA and SNO are good indicators of pain, anxiety and stress but in this study, we found no association among salivary biomarkers, pain, and anxiety values. many factors including family, work-life stress, hormonal changes and so on, in addition to any kind of dental treatment, procedure can influence stress that can contribute to variation in these levels.
We found that patients had higher salivary biomarker levels, pain scores at T3 interval (48 h after arch-wire insertion) and anxiety scores at T0 interval due to anticipation of treatment procedure, and all the values were again decreased at T4 interval (after 1 month follow up) as they might have adapted to the appliances.
Limitations
This study has few limitations, we used both psychological and physiological testing instruments to quantify pain and anxiety during the beginning phases of fixed orthodontic treatment with limited sample size. Another limitation of this study is that the data presented are partially informed and depend on the honesty of the participants.
CONCLUSION
The present study assessed pain and anxiety-induced stress during the initial alignment phases of orthodontic treatment. Patients' stress levels were higher after archwire insertion, as evidenced by SAA and SNO activity. Salivary biomarker levels showed no significant association with reported pain and anxiety intensity scores, which were insufficient to objectively assess pain and anxiety in patients undergoing orthodontic treatment. However, the progressive increase in the intensity of values observed during the study period suggests that emotional stress overload is more in these patients in response to initiation of treatment.
Declaration of consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
REFERENCES
- 1.Abdelrahman RS, Al-Nimri KS, Al Maaitah EF. Pain experience during initial alignment with three types of nickel-titanium archwires: A prospective clinical trial. Angle Orthod. 2015;85:1021–6. doi: 10.2319/071614-498.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Patients R. Comparing dental stress in new child patients and returning patients using salivary cortisol, immunoglobulin-A and alpha- amylase. J Clin Pediatr Dent. 2017;41:462–6. doi: 10.17796/1053-4628-41.6.8. [DOI] [PubMed] [Google Scholar]
- 3.Dhiman S, Gaur A, Maheshwari S, Khan S. The relevance of physico-chemical and diagnostic properties of saliva during orthodontic treatment. Med Rev. 2014;1:5. [Google Scholar]
- 4.Batista P, Pereira A, Vaz AB. Salivary biomarkers in psychological stress diagnosis. ARC J Pharm Sci. 2017;3:9–18. [Google Scholar]
- 5.Pappa E, Kousvelari E, Vastardis H. Saliva in the “Omics” era: A promising tool in paediatrics. Oral Dis. 2019;25:16–25. doi: 10.1111/odi.12886. [DOI] [PubMed] [Google Scholar]
- 6.Navazesh M. PDFlib PLOP: PDF linearization, optimization, protection Page inserted by evaluation version methods for collecting saliva. Ann N Y Acad Sci. 1993:72–7. doi: 10.1111/j.1749-6632.1993.tb18343.x. [DOI] [PubMed] [Google Scholar]
- 7.Bekker HL. Scoring _ 6item _ STAI _ shortform _ Bekker2015 Spielberger State-Trait Anxiety Inventory (STAI: Y-6 item) 2015 [Google Scholar]
- 8.Bekker HL, Legare F, Stacey D, O'Connor A, Lemyre L. Is anxiety a suitable measure of decision aid effectiveness: A systematic review? Patient Educ Couns. 2003;50:255–62. doi: 10.1016/s0738-3991(03)00045-4. [DOI] [PubMed] [Google Scholar]
- 9.Appukuttan DP. Strategies to manage patients with dental anxiety and dental phobia: Literature review. Clin Cosmet Investig Dent. 2016;8:35–50. doi: 10.2147/CCIDE.S63626. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Jones ML, Richmond S. Initial tooth movement: Force application and pain-A relationship? Am J Orthod. 1985;88:111–6. doi: 10.1016/0002-9416(85)90234-9. [DOI] [PubMed] [Google Scholar]
- 11.Vaida L, Todor BI, Bertossi D, Corega C. Correlations between stress, anxiety and coping mechanisms in orthodontic patients. Iran J Public Health. 2015;44:147–9. [PMC free article] [PubMed] [Google Scholar]
- 12.Smith SM, Vale WW. The role of the hypothalamic-pituitary-adrenal axis in neuroendocrine responses to stress. Dialogues Clin Neurosci. 2006;8:383–95. doi: 10.31887/DCNS.2006.8.4/ssmith. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Asking B. Sympathetic stimulation of amylase secretion during a parasympathetic background activity in the rat parotid gland. Acta Physiol Scand. 1985;124:535–42. doi: 10.1111/j.1748-1716.1985.tb00045.x. [DOI] [PubMed] [Google Scholar]
- 14.Kordjazy N, Shirzadian A, Mehr SE. Co-occurrence of anxiety and depressive-like behaviors following adolescent social isolation in male mice; possible role of nitrergic system. Physiol Behav. 2015;145:38–44. doi: 10.1016/j.physbeh.2015.03.032. [DOI] [PubMed] [Google Scholar]
- 15.da Silva Campos MJ, Alves CC, Raposo NR, Ferreira AP, Vitral RW. Influence of salivary secretory immunoglobulin A level on the pain experienced by orthodontic patients. Med Sci Monit. 2010;16:CR405–9. [PubMed] [Google Scholar]
- 16.Johnson C. Measuring pain. Visual analog scale versus numeric pain scale: What is the difference? J Chiropr Med. 2005;4:43–4. doi: 10.1016/S0899-3467(07)60112-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Sari Z, Uysal T, Karaman AI, Sargin N, Üre Ö. Does orthodontic treatment affect patients' and parents' anxiety levels? Eur J Orthod. 2005;27:155–9. doi: 10.1093/ejo/cjh072. [DOI] [PubMed] [Google Scholar]
- 18.Beckler K. State-Trait Anxiety Inventory for Adults Sampler Set Manual, Instrument and Scoring Guide. 1983 Consulting Psychological Press Inc Mind Gard Inc; 2010:1–78. [Google Scholar]
- 19.Spielberger CD. State-Trait Anxiety Inventory: A Comprehensive Bibliography. Palo Alto, CA: Consulting Psychologists Press; 1983 [Google Scholar]
- 20.Aksoy A, Gecgelen Cesur M, Harun Dagdeviren B, Alpagan Ozkaynak Y, Karacin G, Gultekin F. Assessment of pain, anxiety, and cortisol levels during the initial aligning phase of fixed orthodontic treatment. Turkish J Orthod. 2019;32:34–40. doi: 10.5152/TurkJOrthod.2019.18043. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Shirasaki S, Fujii H, Takahashi M, Sato T, Ebina M, Noto Y, et al. Correlation between salivary -amylase activity and pain scale in patients with chronic pain. Reg Anesth Pain Med. 2007;32:120–3. doi: 10.1016/j.rapm.2006.11.008. [DOI] [PubMed] [Google Scholar]
- 22.Arai YC, Matsubara T, Shimo K, Osuga T, Ushida T, Suzuki C, et al. Small correlation between salivary a -amylase activity and pain intensity in patients with cancer pain. Acta Anaesthesiol Scand. 2009;53:408. doi: 10.1111/j.1399-6576.2008.01833.x. [DOI] [PubMed] [Google Scholar]
- 23.Sema S, Özcan AL, Ceylan E, Özcan E, Kurt N, Metin E, et al. Evaluation of oxidative stress biomarkers in patients with fixed orthodontic appliances. Dis Markers. 2014;2014:597892. doi: 10.1155/2014/597892. 10.1155/2014/597892. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Gammoh OS, Al-Smadi AM, Ashour AF, Al-Awaida W. Salivary nitric oxide, a biomarker for stress and anxiety? Psychiatry Investig. 2016;13:311–5. doi: 10.4306/pi.2016.13.3.311. [DOI] [PMC free article] [PubMed] [Google Scholar]