Abstract: Orthodontic treatment may cause dental root resorption. The risk of dental root resorption increases in orthodontic treated-traumatised teeth. An approach to monitor root resorption is by monitoring inflammatory markers. Therefore, we observed the profiles of inflammatory markers namely Creatine Kinase (CK), nitric oxide (NO), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) to determine their importance in orthodontic treatment especially in traumatised and nontraumatised incisors. A total of 13 subjects (7 female, 6 male) participated in this study. Samples were collected from gingival crevicular fluid at baseline (M0), 2 weeks after 0.014 NiTi archwire (M1) and 2 weeks after 0.018 NiTi archwire (M2). All enzyme activities were measured spectrophotometrically at 340 nm. Griess assay was used to measure nitric oxide level. The NO concentration and activities of CK, LDH and AST were not significantly different between traumatised and nontraumatised incisors groups during observation period (p>0.05). Compared to M1, NO concentration decreased significantly in M2. Other comparisons of all four markers over period of archwire changes in both traumatised and nontraumatised incisors groups showed no significant differences. Inflammatory markers showed different activities or level as responses to previous archwire usage and archwire changes. All markers are not sensitive enough to be categorized as inflammatory markers during orthodontic treatment with two archwire changes.
INTRODUCTION
Orthodontic treatment may cause dental root resorption known as Orthodontically Induced Inflammatory Root Resorption (OIIRR) (Kokich, 2008). The risk of root resorption increases in the traumatised teeth (Malmgren et al., 1982; Brin et al., 1991). However, root resorption risk is also influenced by genetic predipostition, force and root morphology (Hartsfield et al., 2004).
Early detection is essential for identifying teeth with risk to root resorption (Levander and Malmgren, 1988). Radiographic techniques, the choice to monitor root resorption has disadvantages. Therefore, dentine sialoprotein (DSP) was proposed as a marker to monitor dental root resorption (Kereshanan et al., 2008). However, DSP does not specifically represent the dentine matrix released from dentin since DSP was also found in control (Balducci et al., 2007). The need for identification of other markers of dental root resorption should be based on the pathogenesis of dental root resorption.
Local and sterile inflammation was reported to occur in orthodontic treatment and may result in dental root resorption (Hartsfield et al., 2004). In addition, severe root resorption incidence during orthodontic treatment and progress of the pulp tissue inflammation was related with substance P, a neuropeptide involved in inflammation (Yamaguchi et al., 2008). Therefore, another approach to monitor root resorption is by monitoring inflammatory markers. Here, we observed the profiles of several inflammatory markers to see their importance in orthodontic treatment especially in traumatised and nontraumatised incisors. In this present study, we reported our preliminary investigation on several potential inflammatory markers namely Creatine Kinase (CK), Nitric Oxide (NO), Lactate Dehydrogenase (LDH) and aspartate aminotransferase (AST).
MATERIALS AND METHODS
Study design and patients selection: This is a prospective longitudinal study. All patients were selected from patients seeking treatment in Department of Orthodontics, Faculty of Dentistry, Universiti Kebangsaan Malaysia in Kuala Lumpur. The selection was based on the inclusion criteria as stated in Table 1. Traumatised and nontraumatised maxillary incisors were determined from the patient’s history, clinical examination and periapical radiographs (Fig. 1). All patients were prohibited from taking any anti-inflammatory drugs throughout the study period. Optimal oral health was achieved in all subjects prior to the study. Informed consents were obtained from all participants or guardians (for patients under 18 years of age). Ethical approval was obtained from the Research Ethical Committee of Universiti Kebangsaan Malaysia No. UKM1.5.3.5/244/ERGS/1/2012/SKK11/UKM/02/05).
GCF sample collection: GCF sample was collected before archwire application (M0), 2 weeks after 0.014” NiTi archwire application (M1) and 2 weeks after 0.018” NiTi archwire application (M2). Before collection of GCF samples, any plaque was removed and sampling sites were gently washed with water, isolated using cotton rolls to minimize saliva contamination and dried with an air syringe. The isolation of the teeth was obtained using a self-retaining retractor, suction and cotton rolls. An pre-cut absorbent paper strip (periopaper™ 593520, oraflow, New York) was inserted into gingival sulcus at a depth of 1 mm for 3 min. Each site was sampled 3 times at a minute interval. The periopapers were cut, placed in a microtube and added with 110 μL deionized water and centrifuged at 1500 g, 4°C, 10 min using Heraeus Fresco 21 centrifuge (Thermo Scientific) to extract the samples.
Creatine kinase assay: A total of 10 μL diluted GCF samples were reacted with 100 μL reconstituted reagent of the creatine kinase assay kit (KA1665, Abnova) and were incubated in 37°C for 20 min. The absorbance of creatine produced in this reaction was measured at 340 nm at 20 and 40 min using Varioskan® Flash spectrophotometer (Thermo Fischer Scientific) after reconstituted reagent addition to diluted GCF samples. Absorbance of deionized water and calibrator were measured at 40 min.
| Fig. 1: | A periapical radiograph of a traumatised upper left permanent central incisor with an enamel-dentine fracture (as indicated by the arrow) |
| Table 1: | Inclusion and exclusion criteria for patient selection |
Griess assay: The nitric oxide level was assayed using standard Griess assay. A 50 μL of diluted GCF sample was mixed with 50 μL 1% sulfanilamide (S-9251, Sigma) in 5% phosphoric acid and 50 μL 0.77 mM N-(1-Naphthyl) ethylenediamine dihydrochloride solution (NED) (NB0650, Biobasic, Canada). The absorbance of blank, standard solutions and diluted samples were read at 540 nm using Varioskan® Flash spectrophotometer (Thermo Fischer Scientific). The approximate concentration of nitric oxide in samples was determined by the equation of the linear line of standard curve generated from known serial dilutions of concentrations of 100 mM sodium nitrite (S-2252, Sigma-Aldrich).
Lactate dehydrogenase assay: A 10 μL of diluted GCF samples were incubated for 5 min in a substrate of 16.2 mmol L-1 pyruvate (Sigma, EC No. 204-024-4), 0.2 mmol L-1 reduced nicotinamide adenine dinucleotide (NADH) (N-8129, Sigma), 54.0 mmol L-1 phosphate buffer (pH 7.5±0.1 at 30°C), in a total volume of 180 μL. In the presence of LDH, pyruvate is reduced to L-lactate with the simultaneous oxidation of NADH. The rate of decrease in absorbance at 340 nm using Varioskan® Flash spectrophotometer (Thermo Fischer Scientific), representing the NADH consumed, is directly proportionate to the LDH activity in the sample. A value of 6.22 was considered as the NADH millimolar absorptivity.
Aspartate aminotransferase assay: A total of 200 μL solution was added into well containing 20 μL of diluted GCF samples to result in final concentration of 100 mM L-aspartic acid (Acros-Organics, 105045000), 12 mM 2-oxoglutarate (Merck, 1.05194.01000), 0.2 mM b-NADH (N-8129, Sigma) and 0.96 U mL-1 malate dehydrogenase (442610, Calbiochem®) and 100 mM Tris (2895B90, Amresco) (pH 7.8). In the presence of AST, L-aspartate and 2-oxoglutarate are first converted to glutamate and oxaloacetate which is then converted by malate dehydrogenase to produce malate and NAD+. The conversion of the NADH to NAD+ product, measured at 340nm using Varioskan® Flash spectrophotometer (Thermo Fischer Scientific) is proportional to the level of AST activity in the sample. The activity of AST in sample will be directly determined from the changes in absorbance at 340 nm.
Statistical analysis: Statistical data were analysed using SPSS ver. 20. The data distributions were examined using Shapiro-Wilk Test. The comparisons between the traumatised incisors and nontraumatised incisors groups in the same period of archwire application were examined using Mann-Whitney U Test. The comparisons between the same (traumatised or nontraumatised incisors) groups in the different periods of archwire application were analysed using Wilcoxon test.
RESULTS
A total of 13 subjects (7 female and 6 male) participated in this study. Average age at M0 was 17.8 years (min 13.69 and max 23.16). Mean time from M1 to M2 is 93±40.5 day.
| Fig. 2: | Mean creatine kinase activity from 13 subjects before archwire application (M0), 2 weeks after 0.014” NiTi archwire application (M1) and 2 weeks after 0.018” NiTi archwire application (M2). Trauma: Traumatised incisors groups, Nontrauma: Nontraumatised incisors group |
| Fig. 3: | Mean NO concentration from 13 subjects before archwire application (M0), 2 weeks after 0.014” NiTi archwire application (M1) and 2 weeks after 0.018” NiTi archwire application (M2), Trauma: Traumatised incisors groups, Nontrauma: Nontraumatised incisors group, *: Significant difference |
CK activity in traumatised incisors groups decreased over period of archwire changes while in the nontraumatised incisors groups, CK activity increased (Fig. 2). When comparing between the traumatised incisors and nontraumatised incisors group, CK activities in each period of archwire change did not show any significant difference (p>0.05). Non significant differences were also observed in groups of traumatised incisors when compared with CK activity of every archwire change. Non significant differences were also observed when comparing groups of nontraumatised incisors.
NO concentration increased slightly in the traumatised incisors group at M1 (p>0.05) and decreased significantly (p<0.05) at M2 (Fig. 3). In the nontraumatised incisors group, NO concentration increased slightly to the highest level in M1 and subsequently decreased significantly to below the level of M0 and M1.
| Fig. 4: | Mean LDH activity from 13 subjects before archwire application (M0), 2 weeks after 0.014” NiTi archwire application (M1) and 2 weeks after 0.018” NiTi archwire application (M2), Trauma: Traumatised incisors groups, Nontrauma: Nontraumatised incisors group |
| Fig. 5: | Mean AST activity from 13 subjects before archwire application (M0), 2 weeks after 0.014” NiTi archwire application (M1) and 2 weeks after 0.018” NiTi archwire application (M2), Trauma: Traumatised incisors groups, Nontrauma: Nontraumatised incisors group |
There is no significant differences (p>0.05) in NO concentrations on the comparisons of traumatised and nontraumatised incisors groups in each period of archwire change.
LDH activity of traumatised and nontraumatised incisors groups increased in M1 and M2 (Fig. 4). However, comparisons among traumatised and nontraumatised incisors groups, traumatised incisors groups over period of archwire changes, or nontraumatised incisors groups over period of archwire changes resulted in no significant differences (p>0.05).
Traumatised incisors groups showed highest AST activity in M1 and decreased AST activity in M2 (Fig. 5). Non traumatised incisors showed slightly increased AST activity over period of archwire change. However, there were no significant differences in comparisons between traumatised and nontraumatised incisors groups and between changes in archwire in both groups.
DISCUSSION
Dental trauma may induce inflammation and root resorption. Inflammatory mediators act synergistically to enhance root resorption. Study on first and second premolars of subjects showed that the incidence of external apical root resorption was higher in the asthma group than healthy group (McNab et al., 1999). However, several studies reported that incisors with clinical signs or patient reports of trauma had essentially the same prevalence of moderate to severe OIIRR as those without trauma (Levander et al., 1994; Brin et al., 2003; Mandall et al., 2006). These raised the importance of the investigation on the corelation of inflammation process and root resorption. CK, NO, LDH and AST are among the well known inflammatory markers. However, our study showed all four markers did not show any significant differences when compared between traumatised and nontraumatised incisors.
Creatine kinase is an enzyme used for detection of acute infarction. In myocardial infarction cases, creatine kinase increased after onset (Watanabe et al., 2009). The apperarance of CK is to be indirect marker of muscle damage (Baird et al., 2012). Meanwhile in the periodontal tissues, gingival fibroblasts exhibited CK-MM (muscle form) as a predominant isoenzyme and CK-BB (brain form) as a minor fraction (Huang et al., 1990). Therefore, we suggest that CK activity in GCF samples mostly come from gingival fibroblasts damage due to archwire force.
In this present study, traumatised incisors group showed decreased CK activity while non traumatised incisors showed increased CK activity over period of archwire change. There might be relatively low amount of cells residing in the surrounding tissues of traumatised incisors. Therefore, archwire change has little effects to the traumatised incisor surrounding tissues and cells. However, there might be relatively large amount of healthy tissue residing in surrounding tissues of the nontraumatised incisors groups so that once the archwire was applied, the tissues necrotized and released more CK to GCF.
NO, formerly known as endothelial derived relaxing factor, acts as a vasodilator agent (Allen et al., 2009) and is intermediate substance produced by Nitric Oxide Synthase (NOS). NO regulates the synthesis of several mediators of inflammatory reaction and functions of inflammatory cells (Moilanen and Vapaatalo, 1995). Inflammatory reaction stimulates NO production in osteoclast and osteoblast (Herrera et al., 2011). NO could also attenuate vascular integrity in human chronic inflammation (Hama et al., 2008).
There are three types of NOS namely eNOS, iNOS and nNOS. iNOS inhibition prevents alveolar bone loss in a rat model of ligature-induced periodontitis, thus confirming that iNOS-derived NO plays a crucial role in the pathogenesis of periodontitis, probably by stimulating osteoclasts differentiation and activity (Herrera et al., 2011). However, the use of an iNOS selective inhibitor can accelerate the healing process in periapical lesions (Farhad et al., 2011). This raised question for us about the concentration of NO responsible for different actions.
Changes in blood vessel number and density correlated with the direction of tooth movement. The vascular changes were dependent on the site of evaluation and the size of the blood vessel. The periodontal vascular changes in distribution and density can be: increased after application of orthodontic force, transient decrease subsequent to removal of force, transient increase during reactivated distal drift and normalization (Murrell et al., 1996). In response to vascular change (Murrell et al., 1996) and local hypoxia (Veberiene et al., 2009; Chae et al., 2011) due to orthodontic treatment, periodontal tissues react by increasing NO level (M1). In M2, NO level decreased could be due to the necrosis in periodontal tissues. The increase level of CK activity might explain the low level of NO in nontraumatised incisors. In addition, so far there is no report about NO concentration in traumatised and non traumatised incisors group during orthodontic treatment with two archwire changes.
Orthodontic force causes compression and tension of the periodontal ligament tissues and hypothetically change tissue respiration and local hypoxia (Veberiene et al., 2009). In hypoxic condition, LDH activity increased by more than 2-fold. In this present study, LDH actvity of nontraumatised incisors groups increased slightly in M1 and M2. Therefore, we suggest that NO release, as a vasodiator agent, cannot support the surrounding tissues from local hypoxia and subsequently change the metabolic rate as represented by CK activities. AST level in serum have been used for many years as an aid in the diagnosis of infarction and necrosis (Koss et al., 2009). AST is intracellular enzyme and will be released during cells damage or death. AST activity levels in GCF reflect the biological activity which occurs in the periodontium (Perinetti et al., 2003). AST activity levels in the test tooth group were significantly greater than the one in the control tooth on day 14 (Perinetti et al., 2003). In this present study, AST activity in both groups increased in M1 and decreased in M2 representing the cell necrosis.
The dimension of archwire resulted in different reaction patterns in surrounding tissues. The 0.014” NiTi archwire resulted in M1 pattern. However, the M2 showed periodontal tissue reactions after application of the 0.014” NiTi archwire and effects of 2 weeks 0.018” NiTi archwire usage.
CONCLUSION
Inflammatory markers showed different activities or level as responses to previous archwire usage and archwire changes. All markers are not sensitive enough to be categorized as inflammatory markers during orthodontic treatment with two archwire changes.
ACKNOWLEDGMENTS
We would like to thank Department of Higher Education, Ministry of Education, Malaysia for the Grant FRGS/1/2011/SG/UKM/02/13 and ERGS /1/2012/SKK11/UKM/02/5 and Ministry of Science, Technology and Innovation, Malaysia for the Grant 02-01-02-SF1052. We would also like to thank Universiti Kebangsaan Malaysia for the Grant UKM-DLP-2012-001, UKM-DLP-2012-025 and UKM-DPP-2013-024.