Salivary oxidative stress during and after rapid maxillary expansion
How to cite this article: Caglayan Topal S, Ozmeric N, Elgun S, Baloş Tuncer B. Salivary oxidative stress during and after rapid maxillary expansion. APOS Trends Orthod, doi: 10.25259/APOS_137_2021
The objectives of the study were to evaluate oxidative stress biomarkers during a rapid maxillary expansion (RME).
Material and Methods:
Fourteen patients were treated with an acrylic RME device, and after treatment, all were followed for 3 months. Saliva samples were collected before activation (baseline), 1st, 10th days after the first activation, and after retention. Periodontal indexes were recorded at baseline and after retention. Nitric oxide (NO) and malondialdehyde (MDA) levels were evaluated.
NO levels were elevated on the 10th day compared to baseline (P < 0.01), revealing a decrease after retention (P < 0.01). MDA levels were increased on the 10th day and after retention periods compared to baseline (P < 0.01, respectively). Both plaque and gingival indexes increased after retention relative to baseline (P < 0.01, respectively).
Initial stages of orthopedic force increased salivary oxidative stress biomarkers. Long-term results showed decreased levels of NO, but still high MDA levels. The initially increased NO expression decreased after retention, despite the increase in microbial load at this period which might show the effect of mechanical stimuli to be more effective than the microbial load. MDA levels remained at high levels during the post-retention period, arising a possible consideration for the impact of material properties or deficiencies of oral hygiene. Future long-term evaluations for oxidative stress status and orthodontic appliances would be useful.
Rapid maxillary expansion
Orthodontically induced tooth movement is achieved by damage/repair process with inflammation-like reactions, activities of leukocytes, macrophages, and synthesis of pro-inflammatory cytokines in the periodontal structures. Oxidative stress is directly involved in the inflammatory process and is mediated by reactive oxygen species (ROS) and ROS-derived radicals, which have damaging effects on cellular components of the human body. In this respect, nitric oxide (NO) is a free radical related to the defensive response to inflammation by affecting bone remodeling.[3,4] Oxidative stress index has been evaluated previously in patients with periodontitis, by estimating malondialdehyde (MDA) levels to measure the oxidative damage of the lipids occurring from free radicals.
Orthodontic forces comprise optimal force application to provide the desired tooth movements without unwanted reactions to the periodontal structures. However, orthopedic forces, such as rapid maxillary expansion (RME), are more powerful and act on the basal parts of the jaws.
RME induces orthodontic and orthopedic treatment through the expansion of the mid-palatal suture, leading to the correction of posterior crossbites,[6,7] the coordination of the dental arches before orthopedic or functional treatment, and to increase in the arch perimeter. The previous histological data presented that the stage of skeletal maturation at the mid-palatal suture is important for the treatment of maxillary transverse deficiency using an orthopedic device, therefore, the literature search provides studies of the growth and maturation of the intermaxillary sutural system.[10,11] Mechanical loading creates cellular reactions induced by an aseptic inflammation in periodontal tissues. Tooth movement is created by cellular mechanisms. There are few studies in the literature related to oxidative stress levels during fixed orthodontic treatment. We believe that an issue that has not yet been adequately assessed in the literature is the effect of an orthopedic therapeutic procedure such as RME on the role of oxidative stresses. Therefore, patients who needed a transversal maxillary expansion were observed in this study during orthopedic maxillary expansion. RME has been used to stimulate sutural growth and to correct the transverse maxillary deficiency and it has been declared that orthopedic expansion stimulates the synthesis of structural proteins, and triggers DNA synthesis, cell proliferation, and bone deposition. RME involves an active stage in which the orthopedic forces are applied and treatment is followed by a retention period to provide bone remodeling.
The literature search reveals a limited amount of knowledge considering the levels of oxidative stress markers during orthodontic treatment. In a previous study, Atuğ Özcan et al. examined the oxidative damage during fixed orthodontic treatment. However, the effect of orthopedic forces concerning oxidative damage is not clear. We hypothesized that mechanical forces through RME therapy might create oxidative stress and further lead to changes in dental and periodontal tissues. For this aim, to understand the potential impact of mechanical loading on oxidative stress, this study planned to evaluate the levels of salivary oxidative stress biomarkers during active and retention periods of RME. We aimed to measure the levels of biochemical parameters indicating current oxidative stress changes due to RME therapy to elucidate the pathogenesis of orthopedic forces exerted on palatal tissues. The correlations between clinical periodontal parameters and oxidative stress levels were also assessed.
MATERIAL AND METHODS
This study was approved by the Ethics Committee of Gazi University (25901600-5565). All patients were informed regarding their treatments and consent forms were obtained from all patients. The sample size was calculated to detect significant differences at the 0.05 significance level, and the number of patients was required to be a minimum of 12. The immunological properties were evaluated previously, and the study comprised eight female and six male patients, with a mean age of 12.9 ± 0.6 years, who had been seeking orthodontic treatment at the Department of Orthodontics, Faculty of Dentistry, University of Gazi. The inclusion criteria contained that all patients required RME treatment due to the existence of transverse maxillary arch deficiency. Importance was given to the presence of good periodontal health without any evidence of bone loss, no signs of gingival inflammation, and no previous periodontal or orthodontic treatment. All patients were systemically healthy and did not take any anti-inflammatory agents, antibiotics, or immunosuppressants in the past 6 months. Saliva samples were collected before activation (baseline), 1st and 10th days after the first activation, and after a retention period of 3 months. Again, all received only RME treatment during the study with no additional braces or any other orthodontic appliances. All patients were informed to activate the appliance on the same predetermined days.
The study protocol is presented in [Figure 1]. All patients initially received periodontal prophylaxis with scaling and polishing. They were informed about the importance of oral hygiene and instructed to brush their teeth twice a day for a minimum of 3 min. No oral antiseptic solutions or mouthwashes were used, but motivations for maintaining oral hygiene were repeated throughout the study. After 2 weeks from the first visit, the status of the periodontal tissues was evaluated by clinical periodontal assessments including plaque index (PI), gingival index (GI), probing depth (PD), bleeding on probing (BOP), and clinical attachment level (CAL) which were recorded as baseline measurements. All measurements were repeated at the end of retention by the same researcher (SÇT). PD was measured with a periodontal sond (Hu Friedy, Chicago, IL, USA) positioned parallel to the long axis of the tooth with no pressure. BOP was measured as the percentage of sites with bleeding. Recordings were performed around each tooth from the mesiobuccal, buccal, distobuccal, mesiopalatal, palatal, and distopalatal surfaces. Average scores of each parameter were assessed.
At the baseline visit, the RME appliance was placed and the first activation has been performed. The acrylic bonded RME device consisted of a Hyrax screw (Leone orthodontic products, Sesto Fiorentino, Firenze, Italy), two 0.045ʺ stainless steel wires extending to the palatal surfaces of the premolars and molars, and an acrylic covering the occlusal surfaces of posterior teeth extending 2–3 mm away from the gingiva. Care was taken to ensure that the arms of the screw were parallel to the palatal mucosa. The Hyrax screw was activated twice per day with a one-quarter turn in the morning and in the evening until sufficient expansion and overcorrection were obtained. After the active phase, the screw was fixed and the appliance was left in place as a retainer for 3 months. All patients received only RME treatment during the study with no additional braces or any other orthodontic appliances. All patients were informed to activate the appliance on the same predetermined days. The expansion time was 18–21 days, with 8 ± 1.2 mm of expansion.
Collection of saliva samples
Each sampling was held in the morning, between 9.00 am and noon to avoid diurnal variation, and the participants refrained from eating, drinking, and oral hygiene procedures for 2 h before saliva collection. The participants rinsed their mouths thoroughly several times with distilled water and were rested for 3 min. Unstimulated whole saliva (3 ml) was collected concerning the spitting method. Each participant spitted the accumulated saliva into an ice-chilled sterilized test tube every 60 s. Samples were checked for a possible blood leakage into saliva, not to affect the diagnostic use of saliva. Samples were centrifuged for 10 min at 15,000× g at 4°C to remove any particulate matter. The supernatant was frozen at −20°C until analyzed.
NO assay method
The quantitative measurement of NO was performed using a colorimetric non-enzymatic assay. Metallic cadmium was used for quantitative conversion of nitrate to nitrite before quantitation of nitrate using Griess reagent. Following the completion of the reaction, the absorbance of the colored azo dye product of the Griess reaction was measured at 540 nm and the results were expressed as µmol/mg of protein.
MDA assay method
The Salivary MDA level, which provides information on lipid peroxidation, was determined by a method based on reaction with thiobarbituric acid (TBA). MDA, an end product of fatty acid peroxidation, reacts with TBA to form a colored complex, and absorbance was measured at 532 nm. Results were expressed as nmol/mg protein. The analytical imprecision (i.e., the coefficient of variation expressed as a percentage, CV%) of NO and MDA analyses were determined. Within-run and day-to-day NO analyses yielded 4.3% and 5.7%, MDA analyses 4.5% and 6.0%, respectively. The analyses were performed by the same operator. The limit of detections of both analyses has been determined to be 1.0 µM.
The Statistical Package for the Social Sciences SPSS version 20.0 (USA) was used for data analysis. Sample size estimation was carried out to achieve an 80% probability of power to detect a difference of 3.2° of maxillary molar angulation concerning a previous report with a significance level of 0.05. Numerical variables were presented as mean and standard deviation (SD). The normality of data distribution was verified by the Shapiro–Wilk test. The differences between the time intervals within each group were analyzed with Mann–Whitney U-test, for variables that were not normally distributed. When the distribution was normal, paired t-test was used. The correlation between changes in the levels of oxidative stress biomarkers and changes in clinical periodontal parameters was assessed by Spearman’s correlation analysis. P < 0.05 was considered to be statistically significant.
The expansion screw was activated twice a day with a mean activation period of 20 days until the required overcorrection. The mean expansion was 8 mm. After the activation period, the appliances were passively kept for 3 months in the mouth for retention. The descriptive measurements (mean, median, SD, minimum, and maximum values) of clinical periodontal parameters are shown in [Table 1]. Results demonstrated that PI and GI were significantly increased at the end of the retention period relative to baseline (P < 0.01, respectively).
|Baseline (n=14)||Post-retention (n=14)||P-value|
|X (SD)||Median||Min-Max||X (SD)||Median||Min-Max|
|Plaque index||0.70 (0.41)||0.60||0.24–1.33||0.80 (0.39)||0.70||0.35–1.38||0.001|
|Gingival index||0.90 (0.48)||1.10||0.26–1.48||1.00 (0.47)||1.20||0.39–1.53||0.004|
|Probing depth (mm)||2.10 (0.47)||2.00||1.46–2.86||2.10 (0.49)||2.00||1.46–2.90||0.302|
|Bleeding on probing (%)||33.36 (14.12)||28.12||16.20–52.00||33.40 (15.07)||29.18||16.09–53.00||1.108|
The mean and SD of salivary oxidative stress markers are presented in [Table 2]. NO levels declared a significant increase on the 10th day when compared to baseline (P < 0.01). However, there was a significant decrease in the 3rd month relative to the 10th day (P < 0.01). MDA levels demonstrated an increase on the 10th day concerning baseline at a significance level of < 0.01. The highest level was found in the 3rd month concerning baseline (P < 0.01) [Figure 2].
|Baseline||1st day||10th day||3rd month|
Correlation analysis revealed no significant interaction between changes in the clinical periodontal parameters with NO and MDA levels (P > 0.05) [Table 3].
|NO levels||MDA levels|
|Bleeding on probing|
Orthodontic or orthopedic loading creates an aseptic inflammation in periodontal tissues and induces localized cell reactions by the release of mediators, prostaglandins, and/or cytokines to stimulate bone remodeling. These cellular mechanisms may raise questions about whether these forces induce oxidative damage in the supporting tissues. In this respect, the present study aimed to evaluate the effects of RME on salivary oxidative stress levels. We wanted to investigate early responses by detecting results on the 1st and 10th days after initial force loading, and long-term responses during the following retention period of 3 months. As far as we know, there are a limited number of studies relative to oxidative stress levels in patients with fixed orthodontic treatment.[12,21] Our findings revealed changes in salivary oxidative stress levels with orthopedic loading. The clinical contribution that can be obtained from this study demonstrates the importance of the reduction of NO levels after the retention period and that with the removal of the orthopedic force, oxidative stress levels decreased to baseline levels and remained within physiological limits. On the other hand, the high MDA levels obtained throughout the study also highlighted that orthopedic force and materials such as polymethyl methacrylate (PMMA) resins can cause oxidative damage to the lipid components of tissues in the first 3 months of RME treatment. A sampling of saliva provides easy, non-invasive, and easily tolerated evaluations, and also the whole saliva reflects a pooled sample containing variable amounts of substances from all sites, which can be used as a diagnostic tool for periodontal diseases.[22,23] During our study, oral hygiene motivations were periodically given for effective hygiene; however, we found significant increases in PI and GI scores at the end of retention, which might be linked with the long duration of the study design and/or the existence of bonded appliances. Despite the finding that none of the patients had bone or attachment loss at the end of this period, the increased periodontal index scores declared the importance of effective tooth cleaning in the presence of such orthodontic appliances.
The active expansion period of RME includes heavy forces, while the retention period involves a certain duration to achieve adequate bone remodeling. One of our major findings was the significant elevations of NO levels on the 10th day after force loading. A previous study examined the gingival response by examining endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) levels during the distalization of canines in humans, and they found their apparent role during the initial stages of orthodontic treatment, similar to our finding. Again, another study reported that human pulps subjected to orthodontic force were inflamed in the first 15 days with high levels of iNOS and low levels of eNOS, followed by a decrease of iNOS and increase of eNOS levels after 30 days. This result ascertains the differential response of NOS. Inline, the present results declared a significant decrease in the 3rd month relative to the 10th day. Since mechanical loading influences NO production, this finding might be related to the decreased loading and/ or inflammatory response after the retention period.[25,26] Accordingly, Nilforoushan and Manolson found that the high levels of NO isoforms returned to baseline levels during the later stages of orthodontic tooth movement in rats. They concluded that PDL cells respond to early orthodontic tooth movement through the production of NO by different expressions at the tension and pressure sides. The differential response of NOS has also been stated by another study, declaring the importance of iNOS and eNOS in bone remodeling during orthodontic treatment. Han et al. stated an association with the high levels of salivary NO and severe periodontitis and suggested that salivary NO might provide beneficial knowledge for monitoring periodontitis. In our study, plaque and GIs were increased; however, NO levels were decreased at the end of retention. This result showed that a decrease in NO levels occurred despite the increase in microbial load at this period. This interesting finding might point out the early influence of the orthopedic force to be more effective than the microbial load. The previous studies also suggested that human PDL cells produce NO through eNOS which is activated by cyclic tension forces and that the magnitude of the orthodontic force enhances NO production.
As mentioned in the literature, the oxidative damage of the lipids leads to the production of MDA, which is the most often measured evaluation for lipid peroxidation. In this study, the high levels of MDA were found on the 10th day, and the highest level was evident in the 3rd month relative to baseline. In contrast to our findings, Atuğ Özcan et al. could not detect the presence of a significant difference in levels of MDA and NO during fixed orthodontic treatment. They declared that orthodontic forces and the fixed appliances did not expose oxidative damage in the first 6 months of orthodontic tooth movement. Recently, Portelli et al. also stated that fixed orthodontic appliances did not affect oxidative stress during the first 10 weeks of treatment. The differences between our results and previous studies might be due to the differences in force amounts, the appliance designs, and the materials.
Analysis of data suggested that higher salivary MDA levels were found in patients with periodontitis compared to healthy subjects, and a correlation was found between high MDA levels and the clinical periodontal status. In contrast, Portelli et al. did not find any correlation between oxidative stress and dental hygiene level. The high levels of MDA in the present study might be linked with the status of periodontal health, but similar to Portelli et al.’s results, we could not find a correlation between periodontal parameters and oxidative stress markers. In addition, no evidence of periodontitis was found in patients at the end of retention. This arises a possible consideration for the impact of the material properties used for the RME device on the increased MDA levels. However, the possible impact of oral hygiene should also be taken into account when interpreting the results.
The previous studies have examined the relations between oxidative stress and the metal ions released from several types of brackets.[33,34] Buljan et al. mentioned orthodontic brackets to be a source of oxidative stress in vitro, regardless of the materials. An acrylic device was applied in this study. From this viewpoint, further reasons for the high levels of MDA in our study might be related to the cytotoxicity of PMMA dentin resins, which are usually used in such intraoral appliances. These materials have potentially adverse effects on oral epithelial cells and may induce apoptosis. Methyl methacrylate has been reported to destroy cellular components and oxidative stress. The close contact between oral epithelial tissues and the resins may cause an inflammatory reaction that conceives clinical relevance. The changes observed in oxidative stress biomarkers could be also the result of tooth movement and remodeling of nearby alveolar bone and the periodontal tissues since saliva reflects all the changes in biological events that happened in the oral cavity. However, this could not be the case in the present study since periodontal tissues involved in RME therapy were healthy at baseline and through the evaluation period.
Taken together, the imbalance between the production of free radicals and antioxidants in favor of oxidants causes oxidative stress. Therefore, the antioxidant capability of saliva is essential for the maintenance of oral cavity balance. However, we did not evaluate the protective antioxidant enzymes present in saliva in this study. We believe that this requires further long-term evaluations concerning the effects of orthodontic appliances on the periodontal and oral environment. The potential limitations of this study included a small sample size and a lack of evaluation of the antioxidant markers together with the oxidative stress biomarkers. Another limitation of the present study is that the interpretation of the results in the molecular level could not be possible since the histological evaluation of the tissues could not be performed due to ethical issues.
The future studies would provide more beneficial data regarding the oxidative stress status with orthodontic appliances, especially after long periods of observation that may allow for repair processes.
Significant elevations in oxidative stress biomarkers were found at the early stages of RME treatment. NO may serve as an indicator of inflammation and/or bone response to mechanical loading, so the decreased levels after the retention period despite the increase in microbial load might be interpreted as a possible effect of the orthopedic force to be more effective than the microbial load. MDA levels were elevated from the early periods until the end of retention, declaring a possible adverse effect of the acrylic resin properties, but the possibility of inadequate oral hygiene should also be considered.
Declaration of patient consent
Patient’s consent not required as there are no patients in this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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