Abstract
Background
Biomarkers can aid in determining the success of complete denture. Fewer studies evaluated the biomarkers in edentulous patients. This can aid in planning of better qualitative care for edentulous patients. The study compared the quantities of nitric oxide (NO), salivary alpha amylase (SAA) and thiobarbituric acid reactive substances (TBARS) before and after denture insertion in complete denture patients.
Methods
The study involved 25 edentulous patients. The saliva samples were obtained from patients prior, 1 h, and 1 week after denture insertion. Standardized procedure was followed in sample management, transportation and storing. The investigations varied with the biomarkers. SAA was estimated by enzymatic assay. TBARS by lipid peroxidation and NO was assessed by Griess reaction. The obtained data were tabled and statistically analyzed.
Results
The results displayed variation in the level of markers between before and after denture insertion. The mean levels of SAA decreased from 8.26 ± 1.32 nm/ml to 3.44 ± 1.25 nm/ml in following denture insertion. The mean of TBARS and NO increased after denture insertion to 14.33 ± 4.72 nm/ml and NO 22.92 ± 5.79 ng/ml. The results were statistically significant with p < 0.05.
Conclusion
The study concluded that denture insertion decreased the levels of psychological stress marker (SAA) and increased the Oxidative stress marker (TBARS, NO).
Keywords: Stress, Biomarker, Prosthodontics, Edentulous, Salivary diagnostic
Introduction
The loss of natural teeth affects the ability to masticate, speech, and may lead to the complex rehabilitative care. The complete dentures restore functional and esthetic deficiencies.1 The edentulism has a great impact on tissues and psychology of the patient. Studies have recognized that psychological stress influences the success of complete denture treatment due to fear and anxiety of complete denture.2, 3, 4 Additionally, the complete denture prosthesis generates forces on supporting tissues causing inflammatory response around the alveolar ridge. This reaction may initiate free radical production leading to development of oxidative stress and can be damaging to supporting tissues. These changes were not evaluated in earlier literature in edentulous patients were observed in this study. These changes can be quantified better with biomarkers. Among the various biomarkers the assessment of salivary biomarkers was better appreciated in intraoral situations. Salivary biomarkers assist as a diagnostic aid is easier, more appreciative, and less invasive than other procedures. The potential salivary stress biomarker that supports the objective are salivary alpha amylase (SAA) and thiobarbituric acid reactive substances (TBARS), nitric oxide (NO) for oxidative stress.5, 6, 7, 8
Studies assessing the influence of biomarkers in the field of prosthodontics remain unclear. The evaluation of these biomarkers can aid in reducing the degeneration caused due to complete denture and aids in determining the psychological influence of the complete denture treatment. The aim of the study was to determine the levels of psychologic stress and oxidative markers in saliva of complete denture patients. This was done by evaluating and comparing the levels of SAA, TBARS, NO before and after complete denture insertion.
Materials and methods
The study was approved by institutional ethical committee and informed consent was obtained from all the subjects. Twenty five edentulous subjects reporting to the Department of Prosthodontics, SRM Dental College, Ramapuram, Chennai were included in the study. The recruited patients were selected with definitive inclusion and exclusion criteria. The intervention group had the edentulous individuals with first time denture wearers, edentulous for not more than 2 months, both genders, free from psychological stress, class 1 skeletal relationship, no oral lesions, non-smokers, no temporomandibular joint (TMJ) disorders, non-alcoholic and no pan chewing habits were considered. The patients with history on systemic diseases, auto immune diseases are excluded from the study. The study has 11 male and 14 female patients with the mean age 53 ± 2 years. The conventional clinical and laboratory procedures were followed in fabricating the heat cure acrylic denture. The levels of psychological stress in individuals included in the study were assessed using State Trait Anxiety Inventory (STAI) and Cohen perceived stress scale questionnaire before the procedure. Patients Denture Assessment (PDA) tool was used to evaluate the patient denture satisfaction before the postoperative data collection.
A sample of 5 ml of unstimulated saliva was collected by passive drooling method from the participants. The salivary samples were collected at three different time intervals preoperative, 1 h after denture insertion and one week after complete denture. The volunteers were requested to avoid drinking or eating anything 30 min before the sample collection. All samples were collected after mouth rinsing with water and at 9.00 am before breakfast. The saliva samples were transferred, centrifuged and stored at 40c for no longer than 2 h before freezing. Collected samples were frozen at −80 °C for four months till the requisite sample size was obtained. The analysis of SAA was based on biochemical enzymatic assay and the resultant colored end product was measured in spectrophotometer (Cary 300 UV; Agilent Technology, California).9,10 The oxidative stress was measured as an index of lipid peroxidation by measuring the concentration of TBARS and NO levels by Griess method. The resultant colored end product was measured in spectrophotometer.11, 12, 13 The optical density obtained was used for calculating the levels of all three stress biomarkers.
Results
The values are tabled and statistically analyzed (Table 1, Table 2, Table 3, Table 4, Table 5, Table 6). The mean SAA was 8.26 ± 1.32 nm/ml before denture insertion and 5.94 ± 1.35 nm/ml, 3.44 ± 1.25 nm/ml at 1 h and 1-week after denture insertion (Table 1). The mean values of α – amylase in second and third interval (Table 1, Table 2) were statistically significant (P < 0.05).
Table 1.
SAA descriptive statistics.
| N | Mean | Std. deviation | |
|---|---|---|---|
| SAA - Before wearing denture | 25 | 8.26∗ | 1.32 |
| SAA - 1 h after wearing denture | 25 | 5.94∗ | 1.35 |
| SAA - 1 week after wearing denture | 25 | 3.44∗ | 1.25 |
Mean value ∗nm/ml.
Table 2.
ANOVA analysis of SAA.
| Source | Type III sum of squares | df | Mean square | F | Sig. | Partial eta squared |
|---|---|---|---|---|---|---|
| Lower-bound | 290.299 | 1.000 | 290.299 | 141.253 | .000 | .855 |
Table 3.
TBARS descriptive Statistics.
| N | Mean | Std. deviation | |
|---|---|---|---|
| TBARS - Before wearing denture | 25 | .20∗ | .046 |
| TBARS - 1 h after wearing denture | 25 | 3.27∗ | 1.64 |
| TBARS - 1 week after wearing denture | 25 | 14.33∗ | 4.72 |
Mean value ∗nm/ml.
Table 4.
ANOVA analysis of TBARS.
| TBARS tests of within-subjects effects | ||||||
|---|---|---|---|---|---|---|
| Source | Type III sum of squares | df | Mean square | F | Sig. | Partial eta squared |
| Lower-bound | 2762.712 | 1.000 | 2762.712 | 156.707 | .000 | .867 |
Table 5.
Nitric oxide descriptive statistics.
| N | Mean | Std. deviation | |
| Nitric Oxide - Before wearing denture | 25 | .58∗ | .29 |
| Nitric Oxide - 1 h after wearing denture | 25 | 6.56∗ | 1.61 |
| Nitric Oxide - 1 week after wearing denture | 25 | 22.92∗ | 5.79 |
Mean value ∗nm/ml.
Table 6.
ANOVA analysis of nitric oxide.
| Source | Type III sum of squares | df | Mean square | F | Sig. | Partial eta squared |
|---|---|---|---|---|---|---|
| Lower-bound | 6689.804 | 1.000 | 6689.804 | 269.866 | .000 | .918 |
The mean TBARS (nm/ml) after denture insertion was 3.27 ± 1.64 nm/ml, 14.33 ± 4.72 nm/ml (Table 3). The NO was .58 ± .29 nm/ml, 6.56 ± 1.61 nm/ml, 22.92 ± 5.79 nm/ml increased after denture insertion (Table 5). The ANOVA analysis (Table 4, Table 6) revealed that results were statistically significant with P < 0.05.
Discussion
The psychological and the oxidative stress are generated with the complete denture rehabilitation. These stress markers with cautious interpretation can aid in determining the success of complete denture treatment. The psychological stress can be evaluated through stress biomarkers which are objectively measured from the biologic, pathogenic processes, or pharmacologic responses to therapeutic intervention. Various biomarkers used in quantification of psychotic stress include cortisol levels, immunoglobulin's, chromogranin-A.14,15 The increase in response of salivary α-amylase to psychological and physical stress through interactions with the autonomic nervous system is a valuable tool in determining the psychological stress in edentulous patients.16,17 Associated with the psychological stress, the oxidative stresses are generated with complete denture treatment procedure. This oxidative stress production occurs due to the biomechanical and traumatic forces generated by complete denture prosthesis. It can be associated with effects like inflammation, trauma, or denture irritation. These forces along with psychological stress may initiate inflammatory responses to underlying tissues. The stimulation of inflammatory process may increase free radical production that might lead to oxidative stress and tissue degeneration underlying complete denture prosthesis. Olteanu et al6 used NO and TBARS as markers for oxidative stress. These markers had significance because of a reduced sensibility, non-invasive, simple and easy method of collection.18, 19, 20, 21, 22 This study showed marked variations in the SAA, TBARS and NO before and after denture insertion.
The psychologic stress in subjects before including them in the study were analyzed with stress assessment questionnaire. The questionnaire aided in determination of the stress levels in elderly individuals. Either counseling was provided to control the stress or the patients were excluded from the study with the qualitative evaluation. This reduced the variability of influences due to other psychological factors not related to denture treatment.
The estimation of α amylase, TBARS and NO revealed that it was normal values in control group. In the intervention group the SAA level was increased and the TBARS as well as NO levels were in lower limits in predenture insertion phase. Analysis of biomarkers 1 h after denture insertion showed decreased levels of α-amylase and increased levels of TBARS and NO in saliva. The levels of biomarkers at one week after denture insertion showed increase in TBARS and NO whereas SAA showed minimal levels in saliva. The mean values of SAA in test group at first interval (before denture insertion) was higher and the values were gradually decreased when reached towards second interval (1 h after denture insertion) and third interval (1 week after denture insertion).
Utmost care was taken in reducing the postinsertion complaints of denture and no discomfort was reported by the patients. The results from biochemical enzymatic assay showed high levels of SAA in saliva in the intervention group before inserting complete denture. Biochemical enzymatic assay in literatures revealed that the levels of stress markers in saliva do not remain static, and concentrations may change in response to a number of influences. Several factors may be of importance depending on the marker of interest and the nature of the study.22 The increase in SAA levels in predenture insertion phase can be related to the patient's anxiety towards complete dentures. Anxiety is also a type of psychological stress that can alter the biomarker levels which decreases over the period of time.
The changes in TBARS and NO could be physiological responses of supporting structures for the dentures. The increase in the oxidative markers can be classified either as an initial response of tissues to complete denture. It can either be a cell damaging reaction or it can be adaptive response on long term. A long-term evaluation of these markers is required for effective interpretation. The evaluation of oxidative biomarkers can reduce the tissue degeneration by supplying antioxidants and the psychological stress can be reduced by appropriate counseling to the edentulous patients before denture insertion. Further research should be directed towards evaluating the long-term response of these markers and the other salivary biomarkers. The evaluation can improve the qualitative care that can benefit the edentulous patients. The use of various denture base materials, procedures, techniques and postoperative care provided should be identified for increase or decrease in biomarkers. The dentures can be modified in accordance to the oxidative and stress levels produced in the tissues and minimize the damage to the underlying tissues. An integrated research at tissue level is required to evaluate how to reduce the stress and tissue damage in postoperative phase of prosthesis.
Further studies to find the influence of various salivary stress markers affecting treatments in the field of Prosthodontics has to be explored and analyzed.
Conclusion
The following conclusions were obtained from this study:
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•
The preoperative mean marker levels for SAA, TBARS, NO were SAA 4.857 ± 10.407 IU/nm/ml, .127 ± .296 IU/nm/ml and 0.147 ± .993 IU/nm/ml.
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The postoperative mean marker levels of SAA, TBARS, NO at 6 months were 1.008 ± 5.566 IU/nm/ml, 6.073 ± 23.256 IU/nm/ml and 12.587 ± 34.266 IU/nm/ml.
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•
The levels of SAA (10.407 IU/nm/ml) decreased after denture insertion. TBARS and NO levels in saliva were increased after denture insertion and the levels increased on denture use.
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•
The decrease in psychological stress markers (SAA) and increase in the oxidative stress markers (TBARS, NO) was observed with the denture insertion.
Disclosure of conflicting interest
The scientific paper was presented before 11th Asian Academy of Prosthodontics conference and the abstract was published in conference proceedings.
References
- 1.Langer A., Michman J., Seifert I. Factors influencing satisfaction with complete dentures in geriatric patients. J Prosthet Dent. 1961;11:1019–1031. [Google Scholar]
- 2.Berg E., Johnsen T.B., Ingebretsen R. Psychological variables and patient acceptance of complete dentures. Acta Odontol Scand. 1986;44:17–22. doi: 10.3109/00016358609041293. [DOI] [PubMed] [Google Scholar]
- 3.Giddon D.B., Hittelman E. Psychologic aspects of prosthodontic treatment for geriatric patients. J Prosthet Dent. 1980;43:1177–1183. doi: 10.1016/0022-3913(80)90204-8. [DOI] [PubMed] [Google Scholar]
- 4.Friedman N., Landesman H.M., Wexler M. The influences of fear, anxiety, and depression on the patient's adaptive responses to complete dentures. Part I. J Prosthet Dent. 1987;58:687–689. doi: 10.1016/0022-3913(87)90419-7. [DOI] [PubMed] [Google Scholar]
- 5.Naumova E.A., Sandulescu T., Bochnig C., et al. Dynamic changes in saliva after acute mental stress. Sci Rep. 2014;4:4884. doi: 10.1038/srep04884. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Olteanu C., Muresan A., Daicoviciu D., Tarmure V., Olteanu I., Keularts I.M. Variations of some saliva markers of the oxidative stress in patients with orthodontic appliances. Physiology. 2009;19:26. [Google Scholar]
- 7.Sculley D.V., Langley-Evans S.C. Periodontal disease is associated with lower antioxidant capacity in whole saliva and evidence of increased protein oxidation. Clin Sci (Lond) 2003;105:167–172. doi: 10.1042/CS20030031. [DOI] [PubMed] [Google Scholar]
- 8.Battino M., Bullon P., Wilson M., Newman H. Oxidative injury and inflammatory periodontal diseases: the challenge of anti-oxidants to free radicals and reactive oxygen species. Crit Rev Oral Biol Med. 1999;10:458–476. doi: 10.1177/10454411990100040301. [DOI] [PubMed] [Google Scholar]
- 9.Winn-Deen E.S., David H., Sigler G., Chavez R. Development of a direct assay for alpha-amylase. Clin Chem. 1988;34:2005–2008. [PubMed] [Google Scholar]
- 10.Arhakis Aristidis, Vassilis Karagiannis, Kalfas Sotirios. Sampling small volumes of saliva for determination of the stress hormone α-amylase: a comparative methodological study. J Behav Brain Sci. 2011;1:194–198. [Google Scholar]
- 11.Celec P., Hodosy J., Celecová V., et al. Salivary thiobarbituric acid reacting substances and malondialdehyde--their relationship to reported smoking and to parodontal status described by the papillary bleeding index. Dis Markers. 2005;21:133–137. doi: 10.1155/2005/693437. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Sun J., Zhang X., Broderick M., Fein H. Measurement of nitric oxide production in biological systems by using griess reaction assay. Sensors. 2003;3:276–284. [Google Scholar]
- 13.Giustarini D., Rossi R., Milzani A., Dalle-Donne I. Nitrite and nitrate measurement by Griess reagent in human plasma: evaluation of interferences and standardization. Methods Enzymol. 2008;440:361–380. doi: 10.1016/S0076-6879(07)00823-3. [DOI] [PubMed] [Google Scholar]
- 14.Tóthová L., Kamodyová N., Červenka T., Celec P. Salivary markers of oxidative stress in oral diseases. Front Cell Infect Microbiol. 2015;20:73. doi: 10.3389/fcimb.2015.00073. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Wang J., Schipper H.M., Velly A.M., Mohit S., Gornitsky M. Salivary biomarkers of oxidative stress: a critical review. Free Radic Biol Med. 2015;85:95–104. doi: 10.1016/j.freeradbiomed.2015.04.005. [DOI] [PubMed] [Google Scholar]
- 16.Nater U.M., Rohleder N. Salivary alpha-amylase as a non-invasive biomarker for the sympathetic nervous system: current state of research. Psychoneuroendocrinology. 2009;34:486–496. doi: 10.1016/j.psyneuen.2009.01.014. [DOI] [PubMed] [Google Scholar]
- 17.Koracevic D., Koracevic G., Djordjevic V., Andrejevic S., Cosic V. Method for the measurement of antioxidant activity in human fluids. J Clin Pathol. 2001;54:356–361. doi: 10.1136/jcp.54.5.356. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Gillard B.K., Marksman H.C., Feig S.A. Direct spectrophotometric determination of alpha-amylase activity in salive, with p-nitrophenyl alpha-maltoside as substrate. Clin Chem. 1977;23:2279–2282. [PubMed] [Google Scholar]
- 19.el-Saadani M., Esterbauer H., el-Sayed M., Goher M., Nassar A.Y., Jürgens G. A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent. J Lipid Res. 1989;30:627–630. [PubMed] [Google Scholar]
- 20.Saral Y., Coskun B.K., Ozturk P., Karatas F., Ayar A. Assessment of salivary and serum antioxidant vitamins and lipid peroxidation in patients with recurrent aphthous ulceration. Tohoku J Exp Med. 2005;206:305–312. doi: 10.1620/tjem.206.305. [DOI] [PubMed] [Google Scholar]
- 21.Panjwani S., Bagewadi A., Keluskar V., Malik R., Rai S., Misra D. Estimation and comparison of levels of salivary nitric oxide in patients with oral lichen planus and controls. Int J Prev Med. 2013;4:710–714. [PMC free article] [PubMed] [Google Scholar]
- 22.Granger D.A., Kivlighan K.T., el-Sheikh M., Gordis E.B., Stroud L.R. Salivary alpha-amylase in biobehavioral research: recent developments and applications. Ann N Y Acad Sci. 2007;1098:122–144. doi: 10.1196/annals.1384.008. [DOI] [PubMed] [Google Scholar]