- Email: [email protected]
Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars
YIJOM-3532; No of Pages 7
Int. J. Oral Maxillofac. Surg. 2016; xxx: xxx–xxx http://dx.doi.org/10.1016/j.ijom.2016.10.005, available online at http://www.sciencedirect.com
Clinical Paper Oral Surgery
Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars
A. Gutie´rrez-Corrales, E. Campano-Cuevas, G. Castillo-Dalı´, ´ . Serrera-Figallo, M.-A D. Torres-Lagares, J.-L. Gutie´rrez-Pe´rez Department of Stomatology, School of Dentistry, University of Seville, Seville, Spain
A. Gutie´rrez-Corrales, E. Campano-Cuevas, G. Castillo-Dalı´, M.-A´. SerreraFigallo, D. Torres-Lagares, J.-L. Gutie´rrez-Pe´rez: Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars. Int. J. Oral Maxillofac. Surg. 2016; xxx: xxx–xxx. # 2016 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Abstract. Many authors have studied various different parameters in relation to postoperative anxiety after the extraction of third molars. However, the effect that the acute inflammatory process occurring post extraction could have on these parameters has not been studied. Certain salivary biomarkers, although not specifically inflammatory, may be affected by the acute inflammatory process occurring following the extraction of a retained lower third molar. Three biomarkers were assessed in this study: total protein, immunoglobulin A (IgA), and alphaamylase. A total of 15 patients were recruited. Four samples of saliva were taken from each patient: before extraction, immediately after extraction, at 2 h after extraction, and at 7 days after extraction. The concentrations of the proteins in the saliva were measured. The average values of each marker were compared across the different stages of the study. Statistical analysis revealed that of the three salivary biomarkers, only alpha-amylase was associated with an inflammatory response to the surgery (P < 0.05). These results suggest the possibility that salivary alphaamylase levels may be affected by the acute inflammation occurring post extraction; therefore, this would not be an appropriate marker to use in the study of other situations, unless this interference is controlled for.
The extraction of impacted lower third molars is one of the most common surgical techniques performed in the oral cavity. This procedure may result in postoperative pain, inflammation, and/or trismus. The inflammatory process is usually characterized by soft tissue swelling and subsequent facial deformity, and a degree of trismus is 0901-5027/000001+07
also sometimes observed. The postoperative recovery process generally takes around 7 days, with inflammation being the primary side effect during the healing period.1,2 Multiple factors can influence patient discomfort, including the complexity and duration of the surgery, the surgeon’s
Key words: oral swelling; third molar surgery; alpha-amylase; immunoglobulin A; total proteins. Accepted for publication 13 October 2016
technique, iatrogenic complications, etc. Minimizing these factors increases satisfaction with the treatment, improves the patient’s quality of life, and reduces the fear of surgical interventions.3 Postoperative symptom management has improved considerably over the past few years due to a better understanding of not only the
# 2016 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Gutie´rrez-Corrales A, et al. Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.10.005
YIJOM-3532; No of Pages 7
2
Gutie´rrez-Corrales et al.
pathophysiological causes of pain and inflammation, but also the mechanisms of action and pharmacodynamics of the analgesics and anti-inflammatories used to treat them. Therefore, greater emphasis is now placed on the importance of preventing both pain and inflammation. Pain and inflammation are typically brief and peak in intensity during the early postoperative period, within the first 24 h post extraction; swelling generally appears within 48–72 h after the surgery.4 Human saliva is a biological fluid with enormous diagnostic potential. As it can be collected non-invasively, it presents a viable alternative to blood, serum, or plasma.5–10 Human saliva contains a variety of proteins,11 hormones,12 antibodies,13 drugs,14 and cytokines15 that enter the saliva through the blood, so most compounds found in the blood are also present in saliva. Many of these arrays of proteins are useful for the detection and treatment of oral and systemic diseases.5 As the composition of saliva can be influenced by systemic changes, specific biomarkers could help identify certain disease conditions.11 Recent studies have demonstrated how saliva can aid in the diagnosis of cardiovascular disease, systemic and local inflammation, hepatic damage, autoimmune disease, and insulin resistance.11,16–19 Current clinical diagnostic methods are unable to detect the onset of periodontal inflammation or to identify those patients at greatest risk of periodontal disease progression.20 Biomarkers from oral fluids have been used to evaluate the response to therapies such as periodontal surgery combined with matrix metalloproteinase inhibition.21 The analysis of recent data supports the use of salivary biomarkers associated with matrix destruction, inflammation, the host response, and bone turnover in the diagnosis of periodontal disease progression.22 Salivary alpha-amylase has been proposed as an important biomarker of stress in terms of autonomic dysregulation, as it increases in response to both physical and psychological stress via interactions with the autonomic nervous system.23 On the other hand, one of the major antibodies present in saliva is immunoglobulin A (IgA), which is generally synthesized by plasma cells in the salivary glands and then exported by an epithelial receptormediated mechanism.24 Secretory IgA levels in the saliva have also been proposed as a potentially useful immunological marker of stress.25 Finally, human saliva also contains clinically relevant proteins, many of which come from the
blood and may also prove useful in clinical applications.26,27 After surgery, there is a systemic reaction that encompasses endocrine, immunological, and haematological changes.28 Certain studies have investigated the value of IgA, alpha-amylase, and the total protein in saliva as indicators of dental anxiety during the extraction of retained lower third molars.23,25,29 However, the effect that the acute inflammatory process occurring post extraction could have on the values of these biomarkers has not been studied. In view of this possible relationship, the results of these previous studies should be interpreted with caution, as this possible interference was not controlled for . The present study was designed to evaluate the levels of salivary alphaamylase, IgA, and total protein in the saliva as biomarkers for inflammation after lower third molar surgery. Patients and methods
A total of 20 patients requiring impacted third molar extractions were initially considered for inclusion. The final study sample comprised 15 subjects between 20 and 37 years of age. The other individuals did not complete the study due to a lack of compliance with the protocol. The surgical extractions took place in the oral and maxillofacial clinic of a hospital in Seville, Spain. Third-year residents of a master’s degree programme in oral surgery performed the procedures. Each patient underwent an exhaustive radiographic study (a panoramic radiograph was used to classify the lower third molars based on Winter’s classification: mesioangular, distoangular, vertical, and horizontal impactions) and diagnosis to ensure that the surgery was as simple as possible. In addition, the purpose of the clinical study was explained to each patient to make them aware of the importance of their collaboration. Complete case histories were collected: age, sex, and smoking status (smoker or non-smoker), among others. Written informed consent forms were signed by all patients who agreed to participate in the study. The inclusion criteria for the study were the following: patient with impacted lower third molars with a moderate degree of difficulty (5–7 Koerner classification); no relevant systemic pathology (ASA I as per the American Society of Anesthesiologists classification). A complete periodontal examination was also included; it was imperative that the patient did not have periodontitis or any oral infection prior to the surgery. The study was
approved by the institutional ethics committee. Variables and data measurement
One researcher was responsible for collecting data on the following variables: age, sex, smoking status, depth of impaction using the Pell and Gregory classification, position using Winter’s classification, and whether the patient was taking any medication. One tooth extraction was performed per session, with an interval of 15 days between interventions to allow the tissues to heal and recover from the first extraction. The first specimen of saliva was collected before the third molar was removed. The patient was asked to refrain from smoking and vigorous exercise for 2 h prior to the collection of saliva. Saliva was allowed to flow in the floor of mouth and 1 ml was then collected using an auto-aspiration pipette tip. After this, the patient received local anaesthetic (articaine 4% and epinephrine 1:100,000). Due to the position and localization of the third molar, all cases required an osteotomy, which was performed using a 20,000-rpm hand piece under irrigation . Some cases required tooth sectioning . At the end of the intervention a 3–0 suture was used to facilitate soft tissue healing and avoid infections in the area. The patient demographic data and the Koerner index for the extracted teeth are shown in Table 1. The time taken to perform the extraction ranged from 7 to 25 min (if it was longer than 30 min, the patient was excluded from the study). A second saliva sample was taken immediately after extraction, trying to avoid any blood. The patient then remained in the waiting room for 2 h without eating or drinking anything, after which a third saliva sample was taken. The final saliva sample was taken 7 days later, before the stitches were removed. Ibuprofen (600 mg every 8 h for 7 days) and amoxicillin/clavulanate (875 mg/ 125 mg every 8 h for 5 days) were Table 1. Clinical and demographic patient data. Sex Male 8 patients Female 7 patients Age range 20–37 years Smoker Yes 7 patients No 8 patients Koerner index 5 6 teeth 6 15 teeth 7 9 teeth
Please cite this article in press as: Gutie´rrez-Corrales A, et al. Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.10.005
YIJOM-3532; No of Pages 7
Salivary biomarkers and postoperative swelling prescribed as anti-inflammatory agents during the postsurgical period. Each patient received an explanation of hygiene techniques and recommendations for the postoperative period, and received a booklet in which to record pain, inflammation, complications, and fulfilment of the prescription for the 7 days of follow-up. Sample collection and analysis
As mentioned above, unstimulated whole saliva was collected at four different timepoints: before surgery, immediately after surgery, 2 h later, and 1 week later. Samples were collected in sterile containers and kept at 80 8C in a vertical deep freezer (ULT-1786-V Model, Value Series; REVCO, Marietta, Ohio, United States of America) until testing. The biochemical analyses were performed by the Biology Service of the University of Seville Centre for Research, Technology and Innovation (CITIUS). Total protein was evaluated as per the Bradford method,30 using the Coomassie Plus (Bradford) Assay Kit (Thermo Fisher Scientific, Marietta, Ohio, United States of America). The working standard 1-ml ampoule of albumin served as the most concentrated protein standard (2000 mg/ml) and was used to prepare serial dilution standards (1500, 1000, 500, 250, 25, 15, and 5 mg/ml) with distilled water as the diluent. A test tube with only 1 ml of distilled water served as the dilution blank. Ten microlitres of sample and standard were added to the corresponding well. Three hundred microlitres of Coomassie Plus Reagent was added to each well and incubated for 10 min at room temperature. After incubation, absorbance was measured at 595 nm using a Multi-detection Microplate Reader (Synergy HT Model; BioTek Instruments, Winooski, Vermont, United States of America) and the standard graph was plotted. This graph was then used to estimate the amount of protein present in a given sample. Alpha-amylase levels were estimated using the kinetic method with an amylase assay kit (Abcam, Cambridge, United Kingdom). Fifty microlitres of each saliva sample and standard dilutions were added to a 96-well plate. Then, 100 ml of Reaction Mix was added to each well and
mixed thoroughly. The optical density (OD) was measured immediately (T0) on the Synergy HT microplate reader at 405 nm. After incubation at 25 8C, the output for various times were measured (T1) at the same wavelength. Salivary IgA was measured using a competitive enzyme-linked immunosorbent assay method with the Human IgA Platinum ELISA kit (eBioscience, San Diego, California, United States of America). A 96-well plate coated with anti-IgA was used, and two-fold serial dilutions were prepared for the standard curve. With 20 ml of each sample in duplicate, the standards and blank were added to the corresponding wells. Next, 50 ml of diluted horseradish peroxidase (HRP)-conjugated antibody were added to every well and the plate incubated at room temperature for 1 h on a microplate shaker set at 400 rpm. After washing, 100 ml of 3,30 ,5,50 -tetramethylbenzidine (TMB) substrate solution was pipetted into each well and the plate incubated at room temperature for 30 min. The enzyme reaction was stopped by quickly pipetting 100 ml of Stop Solution into each well. The absorbance of each well was read immediately on the Synergy HT spectrophotometer at a wavelength of 450 nm. Average absorbance values were calculated for each set of duplicate standards and samples. The mean absorbance of each standard concentration was plotted to create a standard curve, which was then used to determine the human IgA concentration of each sample.
Statistical analysis
Data were entered into SPSS version 13.0 software (SPSS Inc., Chicago, IL, USA) and the Kolmogorov–Smirnov test was used to check the normality of the data. With the aim of detecting differences in averages of 10 nmol/ml (alpha-amylase), 400 ng/ml (IgA), and 100 mg/ml (total protein), with a variance of double this difference, an alpha error of 5%, and a beta error of 20%, it was determined that a sample size of 12 was required; a larger sample was recruited to allow for possible losses . Subsequently, a two-by-two comparison of the four values found for each
3
biomarker was performed using the Student t-test for paired samples. Results
A total of 15 patients fulfilled the study criteria; five other patients were ruled out, as they did not meet the inclusion criteria. A total of 30 mandibular molar extractions were performed. Eight of the patients were male (53%) and seven were female (47%); they ranged in age from 20 to 37 years. Only patients with a diagnosis of impacted lower third molars were selected for the study (100%). Seven of the patients were identified as smokers and eight as nonsmokers. The patients completed the proposed treatment . No complications were identified during the postoperative period . The data relating to the healing process and postoperative inflammation were fully compatible with clinical norms . The variables recorded were the salivary concentrations of alpha-amylase (nmol/ml), IgA (ng/ml), and total protein (mg/ml) throughout the four different stages of the study (Table 2). On statistical analysis, the only salivary biomarker that was found to be correlated with the inflammatory response to surgery was alpha-amylase; this was significant at 2 h and at 1-week after extraction (P < 0.05). Thus, the concentration of alpha-amylase after third molar removal was relevant (P < 0.05) (Fig. 1). No statistically significant results were obtained for IgA and total protein. There was no correlation between post-surgical inflammation and the concentration of these two salivary biomarkers. A peak in the values of IgA could be observed at 2 h after the surgical procedure, and values of total proteins increases during post-surgery period. No data linked them directly to postoperative inflammation (Fig. 2). Discussion
The surgical removal of third molars is considered one of the most common surgeries performed in the oral cavity. Impacted lower third molar extraction is usually accompanied by postoperative swelling, pain, restricted ability to open
Table 2. Concentrations of biomarkers at the different study time-points. Alpha-amylase (nmol/ml) IgA (ng/ml) Total proteins mg/ml *
Before surgery
Post-surgery
After 2 h
After 7 days
100.55 3.1* 4662.43 1492.6 689.15 424.8
105.46 9.0 3686.18 2015.7 926.63 367.4
114.15 17.1*,** 4348.58 2383.2 1104.55 587.9
102.13 3.0** 4562.23 1495.1 1050.98 356.5
P < 0.01. P < 0.005.
**
Please cite this article in press as: Gutie´rrez-Corrales A, et al. Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.10.005
YIJOM-3532; No of Pages 7
4
Gutie´rrez-Corrales et al.
Fig. 1. Concentration of alpha-amylase (nmol/ml) throughout the perioperative period (before surgery to 7 days after surgery).
the mouth, and, in rare cases, the possibility of complications such as alveolar osteitis, infection, nerve injury, abscesses, or mandible fracture. Regarding the surgical difficulty of extracting impacted third molars, Akadiri and Obiechina concluded in their literature review that the scientific community has reached a consensus on the assessment of difficulty, which has been classified into different variables.31 The most relevant radiographic factors in the assessment and prediction of surgical difficulty are the depth of impaction, molar angulation, and root morphology. Of course, other variables also influence the degree of surgical difficulty, with the current literature reporting surgeon experience and the type of intervention (duration of surgery) as having the greatest impact. In terms of demographic variables, patient age is the principal variable to consider, and although sex, weight, and ethnicity have also been mentioned in the scientific literature as factors influencing
the immediate reaction to third molar surgery, an objective assessment is lacking. Carvalho and do Egito Vasconcelos reported a prospective cohort study of 285 patients undergoing impacted lower third molar extraction, with a total of 473 surgeries performed.32 They concluded that only some variables of surgical difficulty must be taken into account when predicting complications. Root number and morphology, tooth position, periodontal space, and the relationship to the second molar were identified as the variables influencing surgical difficulty; meanwhile sex, body mass index, associated pathologies, and the relationship to the mandibular canal were not relevant predictors. The majority of patients undergoing third molar surgery in the present study were between 20 and 37 years old. Based on data concerning the age of patients undergoing third molar extractions, it is clear that the procedure is most frequently performed on patients between the ages of 18 and 30 years. This is because younger
Fig. 2. Concentrations of IgA (left; ng/ml) and total protein (right; mg/ml) measured throughout the perioperative process (before surgery to 7 days after surgery).
people have better circulation and elasticity, and the jaw bone is more resilient than in older patients, making bone removal less complicated and resulting in less postoperative pain and inflammation, with greater healing potential of the jaw.1,33 Nevertheless, current publications fail to agree on the link between the difficulty of extraction and postoperative complications. Moreover, different studies have linked many factors to the varying consequences of the surgery. This is mainly attributable to the use of different diagnostic criteria to identify postoperative inflammation (most of which are evaluated through clinical observation). Thus, objective procedures to evaluate inflammation, such as measuring the concentration of salivary components, have already been published. Due to the importance and prevalence of this type of surgical intervention, there was the need for a clinical study of patients requiring third molar extraction with a view to comparing the concentrations of salivary biomarkers during the period of post-surgical oral inflammation. Referring to current evidence, the results indicate that inflammation is high after surgery due to an increase in the production of inflammatory mediators.1 Today, saliva is widely recognized as a biological fluid useful for the diagnosis of oral diseases such as caries and periodontitis. Inflammatory biomarkers in the oral cavity are biomolecules such as proteins and derivatives that can be measured to evaluate the inflammation in oral processes. Studies in which the levels of alphaamylase, IgA, and total protein in the saliva have been related to dental anxiety have been published.29 However, it has not been determined whether the levels of these biomarkers are influenced by the acute inflammatory process post extraction. Studies have shown that the salivary levels of total protein, IgA, and alphaamylase are altered when inflammation occurs as a result of infectious processes within the oral cavity. For this reason, the patients in the present study were given drug treatment at the highest level used within routine clinical practice following the extraction of their lower third molars: antibiotic and anti-inflammatory treatment for 5–7 days . This was done to avoid any possible infectious complications that could have altered the levels of total protein, IgA, and alpha-amylase; i.e., it was attempted to ensure that changes in the values of the biomarkers measured in this study were due to the acute inflammatory process post extraction under the usual clinical conditions.
Please cite this article in press as: Gutie´rrez-Corrales A, et al. Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.10.005
YIJOM-3532; No of Pages 7
Salivary biomarkers and postoperative swelling The main objective of this study was to identify differences in the salivary concentrations of these biomarkers before and after surgery and to identify the inflammatory biomarkers that are affected by the processes occurring in the surgical area during the postoperative period. In this study, it was sought to analyze inflammation after third molar extraction objectively through the measurement of salivary biomarkers that have been studied previously for this type of surgery. Therefore, total protein, IgA, and alpha-amylase levels were recorded throughout the perioperative period. It was found that alphaamylase was the only biomarker that showed distinctly different salivary concentrations in the preoperative and postoperative periods. Based on these results, data from studies on the use of these biomarkers to assess, for example, dental anxiety,29 should be viewed with caution (in the case of alphaamylase), and future studies should prioritize the use of biomarkers that are not affected by the acute inflammatory process post extraction (e.g. IgA or total protein) . In recent years, more has been learned about the genetic and biochemical properties of total protein in the saliva. Proteins and glycoproteins, which form part of the biofilm within the oral cavity, help maintain moisture and act as a protection mechanism for the teeth and surrounding area. The saliva also contains defence proteins that form an effective molecular defence network within the oral cavity, although some of these proteins are found in very low concentrations. Additionally, the levels of these proteins on the oral surfaces, such as the periodontal groove, buccal wounds, or ulcers, can be greater in response to immune system reactions and inflammatory processes.34 With regard to the currently available evidence, only a few studies have investigated salivary proteins linked to oral cavity processes. In a recent study performed by Bulut et al., acute phase protein levels were identified as significant indicators of postoperative infection after mandibular molar extraction in patients who were prescribed antibiotics.35 Vibhakar et al. attempted to establish a correlation between the concentration of salivary total protein and dental caries.36 A total of 39 patients participated in that study, in which saliva was collected by the spitting method and was analyzed following the relevant protocol. They found a significant correlation between caries and the concentration of total protein. In another study, Hong et al. used salivary proteins as a parameter to
evaluate the lipoteichoic acid of Streptococcus mutans (a representative oral pathogen that causes dental caries and pulpal inflammation) in humans with and without caries.37 Regarding IgA, several publications have focused on understanding the process by which IgA concentrations change in saliva, but there is still no clear consensus . Within the immune system, salivary fluid components such as salivary amylase, lysozymes, cystatins, peroxidases, and many others are the products of innate and acquired immunity to different processes. The flow of saliva and its components is indicative of factors such as the subject’s overall state of health, sex, and age.38 IgA is an immunoglobulin-type protein that functions as a biomarker for immunological activity taking place in the sero-mucinous fluids as the primary defence against pathogens. The level of this protein increases significantly in cases of infectious disease, as do the levels of immunoglobulin G (IgG) and immunoglobulin M (IgM) in periodontitis, showing a positive correlation between the severity of periodontal and gingival damage and the concentration of IgA. The concentrations of immunoglobulins are crucial in antibacterial, antifungal, and antiviral processes.39,40 In periodontitis, in which both the soft and hard tissues are involved, the immune system response protects these tissues and also mediates the injury using immunoglobulins present in the inflamed gingiva; measuring these will help to better understand them. With regard to total protein and its correlation with postsurgical inflammation, no articles linking salivary IgA concentrations to inflammation in the oral cavity appear to have been published. Nevertheless, some studies have found a link between salivary IgA concentrations and periodontal disease. Brandtzaeg demonstrated a difference between saliva samples from healthy and inflamed gingiva using an immunofluorescence method to observe the increase in number of plasma cells containing IgA.41 A study performed by Gross et al. reported that immunoglobulin concentrations, mainly IgG and IgA, were higher in inflamed gingiva (due to periodontitis) than in normal tissue.42 These results were statistically significant and in agreement with those of other studies that had used the same procedure, published by Byers et al.43 and Gu¨ven and De Visscher.40 Kugler et al. performed a study to investigate factors such as age, saliva flow, and mood states and their influence on the normal range of salivary IgA, and found that patients with anxiety
5
or stress tended to have higher salivary IgA concentrations than others.44 No relevant differences were found when controlling for sex (increased secretions of salivary IgA were reported only for pregnant women). The protein most frequently found in saliva is salivary alpha-amylase, an enzyme of polysaccharide degradation that is used in the treatment and evaluation of inflammatory diseases and is also crucial to the metabolism of bacteria during plaque formation.45 In its enzymatic role, salivary alpha-amylase catalyzes the a-1- and 4-glycosidic links of carbohydrates . It is also closely linked to Streptococcus in the oral cavity.34 Alpha-amylase is produced in and secreted from the salivary glands under the control of the autonomic nervous system; thus, this protein has also been analyzed to help understand the mechanisms of the autonomic nervous system . These processes have been directly correlated with anxiety, stress, pain, and other conditions.23,46 The concentration levels of endogenous substances of human serum in the saliva can be very useful in the diagnosis of pathologies like oral or breast cancer, periodontal disease, and Sjo¨gren syndrome. Clinical trials have been performed to investigate the relationships between varying salivary alpha-amylase concentrations and other variables.47 Sadi et al. investigated the correlation between this biomarker and dental anxiety, as well as age, sex, race, pain, and any traumatic experience that might affect the patient’s mood.48 Their study was the first to evaluate the relationship between dental anxiety and the level of alpha-amylase in salivary fluid, but the results did not reveal any correlation and there were no differences between patients of different age, sex, or race. In another study, Nater et al. attempted to identify a link between salivary alpha-amylase and markers of stress.49 They found significant differences in salivary alpha-amylase responses under conditions of stress; thus, it can be concluded that salivary alpha-amylase is a valid marker of stress. The overall effects of stress on the initial levels of alpha-amylase, as shown in the present study, should be studied further . It was found that the alpha-amylase concentration measured at 2 h after surgery was higher than the preoperative value. The high level of this salivary biomarker at 2 h after extraction represents an immune system reaction and response to the inflammatory processes . All extractions were performed on impacted molars; therefore, the extent of the osteotomy required and the migration of all
Please cite this article in press as: Gutie´rrez-Corrales A, et al. Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.10.005
YIJOM-3532; No of Pages 7
6
Gutie´rrez-Corrales et al.
inflammatory mediators may account for this increase. Additionally, many studies have shown that the swelling increases in intensity at 24 h after surgery. This inflammation usually begins within hours of the surgery and may last several days, depending on different factors, and may explain the association between variables at 7 days after the surgery. It is widely known that swelling and pain persist for several days after surgery in the majority of cases, which is why patients with more inflammation also report higher levels of pain. In conclusion, the results of this study showed that the concentration of salivary alpha-amylase is related to the surgery and the inflammatory processes occurring after the extraction of impacted lower third molars. However, a similar correlation was not seen for IgA and total protein. Thus, the study of dental anxiety using salivary biomarkers should be based primarily on IgA and total protein, since these are not affected by the surgical procedure. The use of alpha-amylase for the same purpose should be done with caution, since the interaction of alphaamylase with the post-extraction recovery process must be controlled for . The results of this study indicate the need for further research on this protein to identify those factors that are linked to inflammation after lower third molar extraction and to better understand how they affect the concentration of salivary alphaamylase during the postoperative period. It would also be of interest to study the behaviour of biomarkers in the saliva other than those analyzed in this study that may be linked more strongly to the inflammation occurring during the process of recovery after the extraction of a retained lower third molar. Funding
None. Competing interests
None. Ethical approval
This study was approved by the Ethics Committee of Virgen del Rocı´o Hospital in Seville, Spain. Patient consent
Written informed consent forms were signed by all patients who agreed to participate in the study.
References 1. de Santana-Santos T, de Souza-Santos AA, Martins-Filho PR, da Silva LC, de Oliveira E, Silva ED, et al. Prediction of postoperative facial swelling, pain and trismus following third molar surgery based on preoperative variables. Med Oral Patol Oral Cir Bucal 2013;18:e65–70. 2. Laskin DM, Abubaker AO, Strauss RA. Accuracy of predicting the duration of a surgical operation. J Oral Maxillofac Surg 2013;71:446–7. 3. Torres-Lagares D, Recio-Lora C, CastilloDali G, Ruiz-de-Leon-Hernandez G, HitaIglesias P, Serrera-Figallo M, et al. Influence of state anxiety and trate anxiety in postoperative in oral surgery. Med Oral Patol Oral Cir Bucal 2014;19:e403–8. 4. Seymour R, Meechan JG, Blair GS. An investigation into post-operative pain after third molar surgery under local analgesia. Br J Oral Maxillofac Surg 1985;23:410–8. 5. Hu S, Loo JA, Wong DT. Human body fluid proteome analysis. Proteomics 2006;6: 6326–53. 6. Agha-Hosseini F, Shirzad N, Moosavi MS. Evaluation of xerostomia and salivary flow rate in Hashimoto’s thyroiditis. Med Oral Patol Oral Cir Bucal 2016;21:e1–5. 7. Skrinjar I, Brailo V, Vidovic-Juras D, Vucicevic-Boras V, Milenovic A. Evaluation of pretreatment serum interleukin-6 and tumour necrosis factor alpha as a potential biomarker for recurrence in patients with oral squamous cell carcinoma. Med Oral Patol Oral Cir Bucal 2015;20:e402–7. 8. Wei P, Li C, Qiang L, Li Z, Hua H. Role of salivary anti-SSA/B antibodies for diagnosing primary Sjo¨gren’s syndrome. Med Oral Patol Oral Cir Bucal 2015;20:e156–60. 9. Pereira DL, Vilela VS, Dos Santos TC, Pires FR. Clinical and laboratorial profile and histological features on minor salivary glands from patients under investigation for Sjo¨gren’s syndrome. Med Oral Patol Oral Cir Bucal 2014;19:e237–41. 10. Bachmeier E, Mazzeo MA, Lo´pez MM, Linares JA, Jarchum G, Wietz FM, et al. Mucositis and salivary antioxidants in patients undergoing bone marrow transplantation (BMT). Med Oral Patol Oral Cir Bucal 2014;19:e444–50. 11. Rahim MA, Rahim ZH, Ahmad WA, Hashim OH. Can saliva proteins be used to predict the onset of acute myocardial infarction among high-risk patients. Int J Med Sci 2015;12:329–35. 12. Powell J, DiLeo T, Roberge R, Coca A, Kim JH. Salivary and serum cortisol levels during recovery from intense exercise and prolonged, moderate exercise. Biol Sport 2015;32:91–5. 13. Delacroix DL, Dive C, Rambaud JC, Vaerman JP. IgA subclasses in various secretions and in serum. Immunology 1982;47: 383–5.
14. Stephen KW, McCrossan J, Mackenzie D, Macfarlane CB, Speirs CF. Factors determining the passage of drugs from blood into saliva. Br J Clin Pharmacol 1980;9:51–5. 15. Krishnan R, Thayalan DK, Padmanaban R, Ramadas R, Annasamy RK, Anandan N. Association of serum and salivary tumor necrosis factor-a with histological grading in oral cancer and its role in differentiating premalignant and malignant oral disease. Asian Pac J Cancer Prev 2014;15:7141–8. 16. Kuehl MN, Rodriguez H, Burkhardt BR, Alman AC. Tumor necrosis factor-a, matrix-metalloproteinases 8 and 9 levels in the saliva are associated with increased hemoglobin A1c in type 1 diabetes subjects. PLOS ONE 2015;10:e0125320. 17. Pfaffe T, Cooper-White J, Beyerlein P, Kostner K, Punyadeera C. Diagnostic potential of saliva: current state and future applications. Clin Chem 2011;57:675–87. 18. Yoon AJ, Cheng B, Philipone E, Turner R, Lamster IB. Inflammatory biomarkers in saliva: assessing the strength of association of diabetes mellitus and periodontal status with the oral inflammatory burden. J Clin Periodontol 2012;39:434–40. 19. Prasad S, Tyagi AK, Aggarwal BB. Detection of inflammatory biomarkers in saliva and urine: potential in diagnosis, prevention, and treatment for chronic diseases. Exp Biol Med (Maywood) 2016;241:783–99. 20. Zhang L, Henson BS, Camargo PM, Wong DT. The clinical value of salivary biomarkers for periodontal disease. Periodontol 2000 2009;51:25–37. 21. Gapski R, Hasturk H, Van Dyke TE, Oringer RJ, Wang S, Braun TM, et al. Systemic MMP inhibition for periodontal wound repair: results of a multi-centre randomized-controlled clinical trial. J Clin Periodontol 2009;36:149–56. 22. Kinney JS, Morelli T, Braun T, Ramseier CA, Herr AE, Sugai JV, et al. Saliva/pathogen biomarker signatures and periodontal progression. J Dent Res disease 2011;90:752–8. 23. Nater UM, Rohleder N. Salivary alpha-amylase as a non-invasive biomarker for the sympathetic nervous system: current state of research. Psychoneuroendocrinology 2009;34:486–96. 24. Brandtzaeg P. Do salivary antibodies reliably reflect both mucosal and systemic immunity. Ann N Y Acad Sci 2007;1098:288–311. 25. Tsujita S, Morimoto K. Secretory IgA in saliva can be a useful stress marker. Environ Health Prev Med 1999;4:1–8. 26. Pink R, Simek J, Vondrakova J, Faber E, Michl P, Pazdera J, et al. Saliva as a diagnostic medium. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2009;153: 103–10. 27. Fa´bia´n TK, Hermann P, Beck A, Feje´rdy P, Fa´bia´n G. Salivary defense proteins: their network and role in innate and acquired oral immunity. Int J Mol Sci 2012;13:4295–320.
Please cite this article in press as: Gutie´rrez-Corrales A, et al. Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.10.005
YIJOM-3532; No of Pages 7
Salivary biomarkers and postoperative swelling 28. Desborough JP. The stress response to trauma and surgery. Br J Anaesth 2000;85: 109–17. 29. Robles TF, Sharma R, Park KS, Harrell L, Yamaguchi M, Shetty V. Utility of a salivary biosensor for objective assessment of surgery-related stress. J Oral Maxillofac Surg 2012;70:2256–63. 30. Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72: 248–54. 31. Akadiri OA, Obiechina AE. Assessment of difficulty in third molar surgery – a systematic review. J Oral Maxillofac Surg 2009;67: 771–4. 32. Carvalho RW, do Egito Vasconcelos BC. Assessment of factors associated with surgical difficulty during removal of impacted lower third molars. J Oral Maxillofac Surg 2011;69:2714–21. 33. Haug RH, Abdul-Majid J, Blakey GH, White RP. Evidenced-based decision making: the third molar. Dent Clin N Am 2009;53:77–96. 34. Fa´bia´n TK, Hermann P, Beck A, Feje´rdy P, Feje´rdy P, Fa´bia´n G. Salivary defense proteins: their network and role in innate and acquired oral immunity. Int J Mol Sci 2012;13:4295–320. 35. Bulut E, Bulut S, Etikan I, Koseoglu O. The value of routine antibiotic prophylaxis in mandibular third molar surgery: acute-phase protein levels as indicators of infection. J Oral Sci 2001;43:117–22. 36. Vibhakar PA, Patankar SR, Yadav MR, Vibhakar P. Salivary total protein levels
37.
38.
39.
40.
41.
42.
43.
44.
and their correlation to dental caries. Int J Oral Maxillofac Pathol 2013;4:13–6. Hong SW, Seo DG, Baik JE, Cho K, Yun CH, Han SH. Differential profiles of salivary proteins with affinity to Streptococcus mutans lipoteichoic acid in caries-free and caries-positive human subjects. Mol Oral Microbiol 2014;29:208–18. Kim KD, Zhao J, Auh S, Yang X, Du P, Tang H, et al. Adaptive immune cells temper initial innate responses. Nat Med 2007;13: 1248–52. Favre L, Spertini F, Corthe´sy B. Secretory IgA possesses intrinsic modulatory properties stimulating mucosal and systemic immune responses. J Immunol 2005;175: 2793–800. Gu¨ven O, De Visscher JG. Salivary IgA in periodontal disease. J Periodontol 1982;53: 334–5. Brandtzaeg P. Human secretory immunoglobulins. 3. Immunochemical and physicochemical studies of secretory IgA and free secretory piece. Acta Pathol Microbiol Scand B Microbiol Immunol 1971;79:165– 88. Gross A, Setterstrom JA, D’Alessandro SM, Van Swol RL. Immunoglobulins in periodontal tissues. I. Concentrations of immunoglobulins in normal and inflamed gingiva. J Periodontol 1979;50:581–5. Byers CW, Toto PD, Gargiulo AW. Levels of immunoglobulins IgG, IgA, and IgM in the human inflamed gingiva. J Periodontol 1975;46:387–90. Kugler J, Hess M, Haake D. Secretion of salivary immunoglobulin A in relation to age, saliva flow, mood states, secretion of
45.
46.
47.
48.
49.
7
albumin, cortisol, and catecholamines in saliva. J Clin Immunol 1992;12:45–9. Lamby-Tovar CP, Gomez-Gonzalez OL, Jaramillo-Gomez LM. Salivary a-Amilase: Relation with Dental Caries and Health in General. Univ Odontol 2013;32:93–101. Strahler J, Mueller A, Rosenloecher F, Kirschbaum C, Rohleder N. Salivary alpha-amylase stress reactivity across different age groups. Psychophysiology 2010;47: 587–95. Filaire E, Dreux B, Massart A, Nourrit B, Rama LM, Teixeira A. Salivary alpha-amylase, cortisol and chromogranin A responses to a lecture: impact of sex. Eur J Appl Physiol 2009;106:71–7. Sadi H, Finkelman M, Rosenberg M. Salivary cortisol, salivary alpha amylase, and the dental anxiety scale. Anesth Prog 2013;60: 46–53. Nater UM, La Marca R, Florin L, Moses A, Langhans W, Koller MM, et al. Stress-induced changes in human salivary alphaamylase activity – associations with adrenergic activity. Psychoneuroendocrinology 2006;31:49–58.
Address: Daniel Torres-Lagares Department of Stomatology School of Dentistry University of Seville C/ Avicena s/n 41009 Seville Spain. Fax: +34 95 448 11 29 E-mail: [email protected]
Please cite this article in press as: Gutie´rrez-Corrales A, et al. Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.10.005
Int. J. Oral Maxillofac. Surg. 2016; xxx: xxx–xxx http://dx.doi.org/10.1016/j.ijom.2016.10.005, available online at http://www.sciencedirect.com
Clinical Paper Oral Surgery
Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars
A. Gutie´rrez-Corrales, E. Campano-Cuevas, G. Castillo-Dalı´, ´ . Serrera-Figallo, M.-A D. Torres-Lagares, J.-L. Gutie´rrez-Pe´rez Department of Stomatology, School of Dentistry, University of Seville, Seville, Spain
A. Gutie´rrez-Corrales, E. Campano-Cuevas, G. Castillo-Dalı´, M.-A´. SerreraFigallo, D. Torres-Lagares, J.-L. Gutie´rrez-Pe´rez: Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars. Int. J. Oral Maxillofac. Surg. 2016; xxx: xxx–xxx. # 2016 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Abstract. Many authors have studied various different parameters in relation to postoperative anxiety after the extraction of third molars. However, the effect that the acute inflammatory process occurring post extraction could have on these parameters has not been studied. Certain salivary biomarkers, although not specifically inflammatory, may be affected by the acute inflammatory process occurring following the extraction of a retained lower third molar. Three biomarkers were assessed in this study: total protein, immunoglobulin A (IgA), and alphaamylase. A total of 15 patients were recruited. Four samples of saliva were taken from each patient: before extraction, immediately after extraction, at 2 h after extraction, and at 7 days after extraction. The concentrations of the proteins in the saliva were measured. The average values of each marker were compared across the different stages of the study. Statistical analysis revealed that of the three salivary biomarkers, only alpha-amylase was associated with an inflammatory response to the surgery (P < 0.05). These results suggest the possibility that salivary alphaamylase levels may be affected by the acute inflammation occurring post extraction; therefore, this would not be an appropriate marker to use in the study of other situations, unless this interference is controlled for.
The extraction of impacted lower third molars is one of the most common surgical techniques performed in the oral cavity. This procedure may result in postoperative pain, inflammation, and/or trismus. The inflammatory process is usually characterized by soft tissue swelling and subsequent facial deformity, and a degree of trismus is 0901-5027/000001+07
also sometimes observed. The postoperative recovery process generally takes around 7 days, with inflammation being the primary side effect during the healing period.1,2 Multiple factors can influence patient discomfort, including the complexity and duration of the surgery, the surgeon’s
Key words: oral swelling; third molar surgery; alpha-amylase; immunoglobulin A; total proteins. Accepted for publication 13 October 2016
technique, iatrogenic complications, etc. Minimizing these factors increases satisfaction with the treatment, improves the patient’s quality of life, and reduces the fear of surgical interventions.3 Postoperative symptom management has improved considerably over the past few years due to a better understanding of not only the
# 2016 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Gutie´rrez-Corrales A, et al. Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.10.005
YIJOM-3532; No of Pages 7
2
Gutie´rrez-Corrales et al.
pathophysiological causes of pain and inflammation, but also the mechanisms of action and pharmacodynamics of the analgesics and anti-inflammatories used to treat them. Therefore, greater emphasis is now placed on the importance of preventing both pain and inflammation. Pain and inflammation are typically brief and peak in intensity during the early postoperative period, within the first 24 h post extraction; swelling generally appears within 48–72 h after the surgery.4 Human saliva is a biological fluid with enormous diagnostic potential. As it can be collected non-invasively, it presents a viable alternative to blood, serum, or plasma.5–10 Human saliva contains a variety of proteins,11 hormones,12 antibodies,13 drugs,14 and cytokines15 that enter the saliva through the blood, so most compounds found in the blood are also present in saliva. Many of these arrays of proteins are useful for the detection and treatment of oral and systemic diseases.5 As the composition of saliva can be influenced by systemic changes, specific biomarkers could help identify certain disease conditions.11 Recent studies have demonstrated how saliva can aid in the diagnosis of cardiovascular disease, systemic and local inflammation, hepatic damage, autoimmune disease, and insulin resistance.11,16–19 Current clinical diagnostic methods are unable to detect the onset of periodontal inflammation or to identify those patients at greatest risk of periodontal disease progression.20 Biomarkers from oral fluids have been used to evaluate the response to therapies such as periodontal surgery combined with matrix metalloproteinase inhibition.21 The analysis of recent data supports the use of salivary biomarkers associated with matrix destruction, inflammation, the host response, and bone turnover in the diagnosis of periodontal disease progression.22 Salivary alpha-amylase has been proposed as an important biomarker of stress in terms of autonomic dysregulation, as it increases in response to both physical and psychological stress via interactions with the autonomic nervous system.23 On the other hand, one of the major antibodies present in saliva is immunoglobulin A (IgA), which is generally synthesized by plasma cells in the salivary glands and then exported by an epithelial receptormediated mechanism.24 Secretory IgA levels in the saliva have also been proposed as a potentially useful immunological marker of stress.25 Finally, human saliva also contains clinically relevant proteins, many of which come from the
blood and may also prove useful in clinical applications.26,27 After surgery, there is a systemic reaction that encompasses endocrine, immunological, and haematological changes.28 Certain studies have investigated the value of IgA, alpha-amylase, and the total protein in saliva as indicators of dental anxiety during the extraction of retained lower third molars.23,25,29 However, the effect that the acute inflammatory process occurring post extraction could have on the values of these biomarkers has not been studied. In view of this possible relationship, the results of these previous studies should be interpreted with caution, as this possible interference was not controlled for . The present study was designed to evaluate the levels of salivary alphaamylase, IgA, and total protein in the saliva as biomarkers for inflammation after lower third molar surgery. Patients and methods
A total of 20 patients requiring impacted third molar extractions were initially considered for inclusion. The final study sample comprised 15 subjects between 20 and 37 years of age. The other individuals did not complete the study due to a lack of compliance with the protocol. The surgical extractions took place in the oral and maxillofacial clinic of a hospital in Seville, Spain. Third-year residents of a master’s degree programme in oral surgery performed the procedures. Each patient underwent an exhaustive radiographic study (a panoramic radiograph was used to classify the lower third molars based on Winter’s classification: mesioangular, distoangular, vertical, and horizontal impactions) and diagnosis to ensure that the surgery was as simple as possible. In addition, the purpose of the clinical study was explained to each patient to make them aware of the importance of their collaboration. Complete case histories were collected: age, sex, and smoking status (smoker or non-smoker), among others. Written informed consent forms were signed by all patients who agreed to participate in the study. The inclusion criteria for the study were the following: patient with impacted lower third molars with a moderate degree of difficulty (5–7 Koerner classification); no relevant systemic pathology (ASA I as per the American Society of Anesthesiologists classification). A complete periodontal examination was also included; it was imperative that the patient did not have periodontitis or any oral infection prior to the surgery. The study was
approved by the institutional ethics committee. Variables and data measurement
One researcher was responsible for collecting data on the following variables: age, sex, smoking status, depth of impaction using the Pell and Gregory classification, position using Winter’s classification, and whether the patient was taking any medication. One tooth extraction was performed per session, with an interval of 15 days between interventions to allow the tissues to heal and recover from the first extraction. The first specimen of saliva was collected before the third molar was removed. The patient was asked to refrain from smoking and vigorous exercise for 2 h prior to the collection of saliva. Saliva was allowed to flow in the floor of mouth and 1 ml was then collected using an auto-aspiration pipette tip. After this, the patient received local anaesthetic (articaine 4% and epinephrine 1:100,000). Due to the position and localization of the third molar, all cases required an osteotomy, which was performed using a 20,000-rpm hand piece under irrigation . Some cases required tooth sectioning . At the end of the intervention a 3–0 suture was used to facilitate soft tissue healing and avoid infections in the area. The patient demographic data and the Koerner index for the extracted teeth are shown in Table 1. The time taken to perform the extraction ranged from 7 to 25 min (if it was longer than 30 min, the patient was excluded from the study). A second saliva sample was taken immediately after extraction, trying to avoid any blood. The patient then remained in the waiting room for 2 h without eating or drinking anything, after which a third saliva sample was taken. The final saliva sample was taken 7 days later, before the stitches were removed. Ibuprofen (600 mg every 8 h for 7 days) and amoxicillin/clavulanate (875 mg/ 125 mg every 8 h for 5 days) were Table 1. Clinical and demographic patient data. Sex Male 8 patients Female 7 patients Age range 20–37 years Smoker Yes 7 patients No 8 patients Koerner index 5 6 teeth 6 15 teeth 7 9 teeth
Please cite this article in press as: Gutie´rrez-Corrales A, et al. Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.10.005
YIJOM-3532; No of Pages 7
Salivary biomarkers and postoperative swelling prescribed as anti-inflammatory agents during the postsurgical period. Each patient received an explanation of hygiene techniques and recommendations for the postoperative period, and received a booklet in which to record pain, inflammation, complications, and fulfilment of the prescription for the 7 days of follow-up. Sample collection and analysis
As mentioned above, unstimulated whole saliva was collected at four different timepoints: before surgery, immediately after surgery, 2 h later, and 1 week later. Samples were collected in sterile containers and kept at 80 8C in a vertical deep freezer (ULT-1786-V Model, Value Series; REVCO, Marietta, Ohio, United States of America) until testing. The biochemical analyses were performed by the Biology Service of the University of Seville Centre for Research, Technology and Innovation (CITIUS). Total protein was evaluated as per the Bradford method,30 using the Coomassie Plus (Bradford) Assay Kit (Thermo Fisher Scientific, Marietta, Ohio, United States of America). The working standard 1-ml ampoule of albumin served as the most concentrated protein standard (2000 mg/ml) and was used to prepare serial dilution standards (1500, 1000, 500, 250, 25, 15, and 5 mg/ml) with distilled water as the diluent. A test tube with only 1 ml of distilled water served as the dilution blank. Ten microlitres of sample and standard were added to the corresponding well. Three hundred microlitres of Coomassie Plus Reagent was added to each well and incubated for 10 min at room temperature. After incubation, absorbance was measured at 595 nm using a Multi-detection Microplate Reader (Synergy HT Model; BioTek Instruments, Winooski, Vermont, United States of America) and the standard graph was plotted. This graph was then used to estimate the amount of protein present in a given sample. Alpha-amylase levels were estimated using the kinetic method with an amylase assay kit (Abcam, Cambridge, United Kingdom). Fifty microlitres of each saliva sample and standard dilutions were added to a 96-well plate. Then, 100 ml of Reaction Mix was added to each well and
mixed thoroughly. The optical density (OD) was measured immediately (T0) on the Synergy HT microplate reader at 405 nm. After incubation at 25 8C, the output for various times were measured (T1) at the same wavelength. Salivary IgA was measured using a competitive enzyme-linked immunosorbent assay method with the Human IgA Platinum ELISA kit (eBioscience, San Diego, California, United States of America). A 96-well plate coated with anti-IgA was used, and two-fold serial dilutions were prepared for the standard curve. With 20 ml of each sample in duplicate, the standards and blank were added to the corresponding wells. Next, 50 ml of diluted horseradish peroxidase (HRP)-conjugated antibody were added to every well and the plate incubated at room temperature for 1 h on a microplate shaker set at 400 rpm. After washing, 100 ml of 3,30 ,5,50 -tetramethylbenzidine (TMB) substrate solution was pipetted into each well and the plate incubated at room temperature for 30 min. The enzyme reaction was stopped by quickly pipetting 100 ml of Stop Solution into each well. The absorbance of each well was read immediately on the Synergy HT spectrophotometer at a wavelength of 450 nm. Average absorbance values were calculated for each set of duplicate standards and samples. The mean absorbance of each standard concentration was plotted to create a standard curve, which was then used to determine the human IgA concentration of each sample.
Statistical analysis
Data were entered into SPSS version 13.0 software (SPSS Inc., Chicago, IL, USA) and the Kolmogorov–Smirnov test was used to check the normality of the data. With the aim of detecting differences in averages of 10 nmol/ml (alpha-amylase), 400 ng/ml (IgA), and 100 mg/ml (total protein), with a variance of double this difference, an alpha error of 5%, and a beta error of 20%, it was determined that a sample size of 12 was required; a larger sample was recruited to allow for possible losses . Subsequently, a two-by-two comparison of the four values found for each
3
biomarker was performed using the Student t-test for paired samples. Results
A total of 15 patients fulfilled the study criteria; five other patients were ruled out, as they did not meet the inclusion criteria. A total of 30 mandibular molar extractions were performed. Eight of the patients were male (53%) and seven were female (47%); they ranged in age from 20 to 37 years. Only patients with a diagnosis of impacted lower third molars were selected for the study (100%). Seven of the patients were identified as smokers and eight as nonsmokers. The patients completed the proposed treatment . No complications were identified during the postoperative period . The data relating to the healing process and postoperative inflammation were fully compatible with clinical norms . The variables recorded were the salivary concentrations of alpha-amylase (nmol/ml), IgA (ng/ml), and total protein (mg/ml) throughout the four different stages of the study (Table 2). On statistical analysis, the only salivary biomarker that was found to be correlated with the inflammatory response to surgery was alpha-amylase; this was significant at 2 h and at 1-week after extraction (P < 0.05). Thus, the concentration of alpha-amylase after third molar removal was relevant (P < 0.05) (Fig. 1). No statistically significant results were obtained for IgA and total protein. There was no correlation between post-surgical inflammation and the concentration of these two salivary biomarkers. A peak in the values of IgA could be observed at 2 h after the surgical procedure, and values of total proteins increases during post-surgery period. No data linked them directly to postoperative inflammation (Fig. 2). Discussion
The surgical removal of third molars is considered one of the most common surgeries performed in the oral cavity. Impacted lower third molar extraction is usually accompanied by postoperative swelling, pain, restricted ability to open
Table 2. Concentrations of biomarkers at the different study time-points. Alpha-amylase (nmol/ml) IgA (ng/ml) Total proteins mg/ml *
Before surgery
Post-surgery
After 2 h
After 7 days
100.55 3.1* 4662.43 1492.6 689.15 424.8
105.46 9.0 3686.18 2015.7 926.63 367.4
114.15 17.1*,** 4348.58 2383.2 1104.55 587.9
102.13 3.0** 4562.23 1495.1 1050.98 356.5
P < 0.01. P < 0.005.
**
Please cite this article in press as: Gutie´rrez-Corrales A, et al. Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.10.005
YIJOM-3532; No of Pages 7
4
Gutie´rrez-Corrales et al.
Fig. 1. Concentration of alpha-amylase (nmol/ml) throughout the perioperative period (before surgery to 7 days after surgery).
the mouth, and, in rare cases, the possibility of complications such as alveolar osteitis, infection, nerve injury, abscesses, or mandible fracture. Regarding the surgical difficulty of extracting impacted third molars, Akadiri and Obiechina concluded in their literature review that the scientific community has reached a consensus on the assessment of difficulty, which has been classified into different variables.31 The most relevant radiographic factors in the assessment and prediction of surgical difficulty are the depth of impaction, molar angulation, and root morphology. Of course, other variables also influence the degree of surgical difficulty, with the current literature reporting surgeon experience and the type of intervention (duration of surgery) as having the greatest impact. In terms of demographic variables, patient age is the principal variable to consider, and although sex, weight, and ethnicity have also been mentioned in the scientific literature as factors influencing
the immediate reaction to third molar surgery, an objective assessment is lacking. Carvalho and do Egito Vasconcelos reported a prospective cohort study of 285 patients undergoing impacted lower third molar extraction, with a total of 473 surgeries performed.32 They concluded that only some variables of surgical difficulty must be taken into account when predicting complications. Root number and morphology, tooth position, periodontal space, and the relationship to the second molar were identified as the variables influencing surgical difficulty; meanwhile sex, body mass index, associated pathologies, and the relationship to the mandibular canal were not relevant predictors. The majority of patients undergoing third molar surgery in the present study were between 20 and 37 years old. Based on data concerning the age of patients undergoing third molar extractions, it is clear that the procedure is most frequently performed on patients between the ages of 18 and 30 years. This is because younger
Fig. 2. Concentrations of IgA (left; ng/ml) and total protein (right; mg/ml) measured throughout the perioperative process (before surgery to 7 days after surgery).
people have better circulation and elasticity, and the jaw bone is more resilient than in older patients, making bone removal less complicated and resulting in less postoperative pain and inflammation, with greater healing potential of the jaw.1,33 Nevertheless, current publications fail to agree on the link between the difficulty of extraction and postoperative complications. Moreover, different studies have linked many factors to the varying consequences of the surgery. This is mainly attributable to the use of different diagnostic criteria to identify postoperative inflammation (most of which are evaluated through clinical observation). Thus, objective procedures to evaluate inflammation, such as measuring the concentration of salivary components, have already been published. Due to the importance and prevalence of this type of surgical intervention, there was the need for a clinical study of patients requiring third molar extraction with a view to comparing the concentrations of salivary biomarkers during the period of post-surgical oral inflammation. Referring to current evidence, the results indicate that inflammation is high after surgery due to an increase in the production of inflammatory mediators.1 Today, saliva is widely recognized as a biological fluid useful for the diagnosis of oral diseases such as caries and periodontitis. Inflammatory biomarkers in the oral cavity are biomolecules such as proteins and derivatives that can be measured to evaluate the inflammation in oral processes. Studies in which the levels of alphaamylase, IgA, and total protein in the saliva have been related to dental anxiety have been published.29 However, it has not been determined whether the levels of these biomarkers are influenced by the acute inflammatory process post extraction. Studies have shown that the salivary levels of total protein, IgA, and alphaamylase are altered when inflammation occurs as a result of infectious processes within the oral cavity. For this reason, the patients in the present study were given drug treatment at the highest level used within routine clinical practice following the extraction of their lower third molars: antibiotic and anti-inflammatory treatment for 5–7 days . This was done to avoid any possible infectious complications that could have altered the levels of total protein, IgA, and alpha-amylase; i.e., it was attempted to ensure that changes in the values of the biomarkers measured in this study were due to the acute inflammatory process post extraction under the usual clinical conditions.
Please cite this article in press as: Gutie´rrez-Corrales A, et al. Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.10.005
YIJOM-3532; No of Pages 7
Salivary biomarkers and postoperative swelling The main objective of this study was to identify differences in the salivary concentrations of these biomarkers before and after surgery and to identify the inflammatory biomarkers that are affected by the processes occurring in the surgical area during the postoperative period. In this study, it was sought to analyze inflammation after third molar extraction objectively through the measurement of salivary biomarkers that have been studied previously for this type of surgery. Therefore, total protein, IgA, and alpha-amylase levels were recorded throughout the perioperative period. It was found that alphaamylase was the only biomarker that showed distinctly different salivary concentrations in the preoperative and postoperative periods. Based on these results, data from studies on the use of these biomarkers to assess, for example, dental anxiety,29 should be viewed with caution (in the case of alphaamylase), and future studies should prioritize the use of biomarkers that are not affected by the acute inflammatory process post extraction (e.g. IgA or total protein) . In recent years, more has been learned about the genetic and biochemical properties of total protein in the saliva. Proteins and glycoproteins, which form part of the biofilm within the oral cavity, help maintain moisture and act as a protection mechanism for the teeth and surrounding area. The saliva also contains defence proteins that form an effective molecular defence network within the oral cavity, although some of these proteins are found in very low concentrations. Additionally, the levels of these proteins on the oral surfaces, such as the periodontal groove, buccal wounds, or ulcers, can be greater in response to immune system reactions and inflammatory processes.34 With regard to the currently available evidence, only a few studies have investigated salivary proteins linked to oral cavity processes. In a recent study performed by Bulut et al., acute phase protein levels were identified as significant indicators of postoperative infection after mandibular molar extraction in patients who were prescribed antibiotics.35 Vibhakar et al. attempted to establish a correlation between the concentration of salivary total protein and dental caries.36 A total of 39 patients participated in that study, in which saliva was collected by the spitting method and was analyzed following the relevant protocol. They found a significant correlation between caries and the concentration of total protein. In another study, Hong et al. used salivary proteins as a parameter to
evaluate the lipoteichoic acid of Streptococcus mutans (a representative oral pathogen that causes dental caries and pulpal inflammation) in humans with and without caries.37 Regarding IgA, several publications have focused on understanding the process by which IgA concentrations change in saliva, but there is still no clear consensus . Within the immune system, salivary fluid components such as salivary amylase, lysozymes, cystatins, peroxidases, and many others are the products of innate and acquired immunity to different processes. The flow of saliva and its components is indicative of factors such as the subject’s overall state of health, sex, and age.38 IgA is an immunoglobulin-type protein that functions as a biomarker for immunological activity taking place in the sero-mucinous fluids as the primary defence against pathogens. The level of this protein increases significantly in cases of infectious disease, as do the levels of immunoglobulin G (IgG) and immunoglobulin M (IgM) in periodontitis, showing a positive correlation between the severity of periodontal and gingival damage and the concentration of IgA. The concentrations of immunoglobulins are crucial in antibacterial, antifungal, and antiviral processes.39,40 In periodontitis, in which both the soft and hard tissues are involved, the immune system response protects these tissues and also mediates the injury using immunoglobulins present in the inflamed gingiva; measuring these will help to better understand them. With regard to total protein and its correlation with postsurgical inflammation, no articles linking salivary IgA concentrations to inflammation in the oral cavity appear to have been published. Nevertheless, some studies have found a link between salivary IgA concentrations and periodontal disease. Brandtzaeg demonstrated a difference between saliva samples from healthy and inflamed gingiva using an immunofluorescence method to observe the increase in number of plasma cells containing IgA.41 A study performed by Gross et al. reported that immunoglobulin concentrations, mainly IgG and IgA, were higher in inflamed gingiva (due to periodontitis) than in normal tissue.42 These results were statistically significant and in agreement with those of other studies that had used the same procedure, published by Byers et al.43 and Gu¨ven and De Visscher.40 Kugler et al. performed a study to investigate factors such as age, saliva flow, and mood states and their influence on the normal range of salivary IgA, and found that patients with anxiety
5
or stress tended to have higher salivary IgA concentrations than others.44 No relevant differences were found when controlling for sex (increased secretions of salivary IgA were reported only for pregnant women). The protein most frequently found in saliva is salivary alpha-amylase, an enzyme of polysaccharide degradation that is used in the treatment and evaluation of inflammatory diseases and is also crucial to the metabolism of bacteria during plaque formation.45 In its enzymatic role, salivary alpha-amylase catalyzes the a-1- and 4-glycosidic links of carbohydrates . It is also closely linked to Streptococcus in the oral cavity.34 Alpha-amylase is produced in and secreted from the salivary glands under the control of the autonomic nervous system; thus, this protein has also been analyzed to help understand the mechanisms of the autonomic nervous system . These processes have been directly correlated with anxiety, stress, pain, and other conditions.23,46 The concentration levels of endogenous substances of human serum in the saliva can be very useful in the diagnosis of pathologies like oral or breast cancer, periodontal disease, and Sjo¨gren syndrome. Clinical trials have been performed to investigate the relationships between varying salivary alpha-amylase concentrations and other variables.47 Sadi et al. investigated the correlation between this biomarker and dental anxiety, as well as age, sex, race, pain, and any traumatic experience that might affect the patient’s mood.48 Their study was the first to evaluate the relationship between dental anxiety and the level of alpha-amylase in salivary fluid, but the results did not reveal any correlation and there were no differences between patients of different age, sex, or race. In another study, Nater et al. attempted to identify a link between salivary alpha-amylase and markers of stress.49 They found significant differences in salivary alpha-amylase responses under conditions of stress; thus, it can be concluded that salivary alpha-amylase is a valid marker of stress. The overall effects of stress on the initial levels of alpha-amylase, as shown in the present study, should be studied further . It was found that the alpha-amylase concentration measured at 2 h after surgery was higher than the preoperative value. The high level of this salivary biomarker at 2 h after extraction represents an immune system reaction and response to the inflammatory processes . All extractions were performed on impacted molars; therefore, the extent of the osteotomy required and the migration of all
Please cite this article in press as: Gutie´rrez-Corrales A, et al. Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.10.005
YIJOM-3532; No of Pages 7
6
Gutie´rrez-Corrales et al.
inflammatory mediators may account for this increase. Additionally, many studies have shown that the swelling increases in intensity at 24 h after surgery. This inflammation usually begins within hours of the surgery and may last several days, depending on different factors, and may explain the association between variables at 7 days after the surgery. It is widely known that swelling and pain persist for several days after surgery in the majority of cases, which is why patients with more inflammation also report higher levels of pain. In conclusion, the results of this study showed that the concentration of salivary alpha-amylase is related to the surgery and the inflammatory processes occurring after the extraction of impacted lower third molars. However, a similar correlation was not seen for IgA and total protein. Thus, the study of dental anxiety using salivary biomarkers should be based primarily on IgA and total protein, since these are not affected by the surgical procedure. The use of alpha-amylase for the same purpose should be done with caution, since the interaction of alphaamylase with the post-extraction recovery process must be controlled for . The results of this study indicate the need for further research on this protein to identify those factors that are linked to inflammation after lower third molar extraction and to better understand how they affect the concentration of salivary alphaamylase during the postoperative period. It would also be of interest to study the behaviour of biomarkers in the saliva other than those analyzed in this study that may be linked more strongly to the inflammation occurring during the process of recovery after the extraction of a retained lower third molar. Funding
None. Competing interests
None. Ethical approval
This study was approved by the Ethics Committee of Virgen del Rocı´o Hospital in Seville, Spain. Patient consent
Written informed consent forms were signed by all patients who agreed to participate in the study.
References 1. de Santana-Santos T, de Souza-Santos AA, Martins-Filho PR, da Silva LC, de Oliveira E, Silva ED, et al. Prediction of postoperative facial swelling, pain and trismus following third molar surgery based on preoperative variables. Med Oral Patol Oral Cir Bucal 2013;18:e65–70. 2. Laskin DM, Abubaker AO, Strauss RA. Accuracy of predicting the duration of a surgical operation. J Oral Maxillofac Surg 2013;71:446–7. 3. Torres-Lagares D, Recio-Lora C, CastilloDali G, Ruiz-de-Leon-Hernandez G, HitaIglesias P, Serrera-Figallo M, et al. Influence of state anxiety and trate anxiety in postoperative in oral surgery. Med Oral Patol Oral Cir Bucal 2014;19:e403–8. 4. Seymour R, Meechan JG, Blair GS. An investigation into post-operative pain after third molar surgery under local analgesia. Br J Oral Maxillofac Surg 1985;23:410–8. 5. Hu S, Loo JA, Wong DT. Human body fluid proteome analysis. Proteomics 2006;6: 6326–53. 6. Agha-Hosseini F, Shirzad N, Moosavi MS. Evaluation of xerostomia and salivary flow rate in Hashimoto’s thyroiditis. Med Oral Patol Oral Cir Bucal 2016;21:e1–5. 7. Skrinjar I, Brailo V, Vidovic-Juras D, Vucicevic-Boras V, Milenovic A. Evaluation of pretreatment serum interleukin-6 and tumour necrosis factor alpha as a potential biomarker for recurrence in patients with oral squamous cell carcinoma. Med Oral Patol Oral Cir Bucal 2015;20:e402–7. 8. Wei P, Li C, Qiang L, Li Z, Hua H. Role of salivary anti-SSA/B antibodies for diagnosing primary Sjo¨gren’s syndrome. Med Oral Patol Oral Cir Bucal 2015;20:e156–60. 9. Pereira DL, Vilela VS, Dos Santos TC, Pires FR. Clinical and laboratorial profile and histological features on minor salivary glands from patients under investigation for Sjo¨gren’s syndrome. Med Oral Patol Oral Cir Bucal 2014;19:e237–41. 10. Bachmeier E, Mazzeo MA, Lo´pez MM, Linares JA, Jarchum G, Wietz FM, et al. Mucositis and salivary antioxidants in patients undergoing bone marrow transplantation (BMT). Med Oral Patol Oral Cir Bucal 2014;19:e444–50. 11. Rahim MA, Rahim ZH, Ahmad WA, Hashim OH. Can saliva proteins be used to predict the onset of acute myocardial infarction among high-risk patients. Int J Med Sci 2015;12:329–35. 12. Powell J, DiLeo T, Roberge R, Coca A, Kim JH. Salivary and serum cortisol levels during recovery from intense exercise and prolonged, moderate exercise. Biol Sport 2015;32:91–5. 13. Delacroix DL, Dive C, Rambaud JC, Vaerman JP. IgA subclasses in various secretions and in serum. Immunology 1982;47: 383–5.
14. Stephen KW, McCrossan J, Mackenzie D, Macfarlane CB, Speirs CF. Factors determining the passage of drugs from blood into saliva. Br J Clin Pharmacol 1980;9:51–5. 15. Krishnan R, Thayalan DK, Padmanaban R, Ramadas R, Annasamy RK, Anandan N. Association of serum and salivary tumor necrosis factor-a with histological grading in oral cancer and its role in differentiating premalignant and malignant oral disease. Asian Pac J Cancer Prev 2014;15:7141–8. 16. Kuehl MN, Rodriguez H, Burkhardt BR, Alman AC. Tumor necrosis factor-a, matrix-metalloproteinases 8 and 9 levels in the saliva are associated with increased hemoglobin A1c in type 1 diabetes subjects. PLOS ONE 2015;10:e0125320. 17. Pfaffe T, Cooper-White J, Beyerlein P, Kostner K, Punyadeera C. Diagnostic potential of saliva: current state and future applications. Clin Chem 2011;57:675–87. 18. Yoon AJ, Cheng B, Philipone E, Turner R, Lamster IB. Inflammatory biomarkers in saliva: assessing the strength of association of diabetes mellitus and periodontal status with the oral inflammatory burden. J Clin Periodontol 2012;39:434–40. 19. Prasad S, Tyagi AK, Aggarwal BB. Detection of inflammatory biomarkers in saliva and urine: potential in diagnosis, prevention, and treatment for chronic diseases. Exp Biol Med (Maywood) 2016;241:783–99. 20. Zhang L, Henson BS, Camargo PM, Wong DT. The clinical value of salivary biomarkers for periodontal disease. Periodontol 2000 2009;51:25–37. 21. Gapski R, Hasturk H, Van Dyke TE, Oringer RJ, Wang S, Braun TM, et al. Systemic MMP inhibition for periodontal wound repair: results of a multi-centre randomized-controlled clinical trial. J Clin Periodontol 2009;36:149–56. 22. Kinney JS, Morelli T, Braun T, Ramseier CA, Herr AE, Sugai JV, et al. Saliva/pathogen biomarker signatures and periodontal progression. J Dent Res disease 2011;90:752–8. 23. Nater UM, Rohleder N. Salivary alpha-amylase as a non-invasive biomarker for the sympathetic nervous system: current state of research. Psychoneuroendocrinology 2009;34:486–96. 24. Brandtzaeg P. Do salivary antibodies reliably reflect both mucosal and systemic immunity. Ann N Y Acad Sci 2007;1098:288–311. 25. Tsujita S, Morimoto K. Secretory IgA in saliva can be a useful stress marker. Environ Health Prev Med 1999;4:1–8. 26. Pink R, Simek J, Vondrakova J, Faber E, Michl P, Pazdera J, et al. Saliva as a diagnostic medium. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2009;153: 103–10. 27. Fa´bia´n TK, Hermann P, Beck A, Feje´rdy P, Fa´bia´n G. Salivary defense proteins: their network and role in innate and acquired oral immunity. Int J Mol Sci 2012;13:4295–320.
Please cite this article in press as: Gutie´rrez-Corrales A, et al. Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.10.005
YIJOM-3532; No of Pages 7
Salivary biomarkers and postoperative swelling 28. Desborough JP. The stress response to trauma and surgery. Br J Anaesth 2000;85: 109–17. 29. Robles TF, Sharma R, Park KS, Harrell L, Yamaguchi M, Shetty V. Utility of a salivary biosensor for objective assessment of surgery-related stress. J Oral Maxillofac Surg 2012;70:2256–63. 30. Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72: 248–54. 31. Akadiri OA, Obiechina AE. Assessment of difficulty in third molar surgery – a systematic review. J Oral Maxillofac Surg 2009;67: 771–4. 32. Carvalho RW, do Egito Vasconcelos BC. Assessment of factors associated with surgical difficulty during removal of impacted lower third molars. J Oral Maxillofac Surg 2011;69:2714–21. 33. Haug RH, Abdul-Majid J, Blakey GH, White RP. Evidenced-based decision making: the third molar. Dent Clin N Am 2009;53:77–96. 34. Fa´bia´n TK, Hermann P, Beck A, Feje´rdy P, Feje´rdy P, Fa´bia´n G. Salivary defense proteins: their network and role in innate and acquired oral immunity. Int J Mol Sci 2012;13:4295–320. 35. Bulut E, Bulut S, Etikan I, Koseoglu O. The value of routine antibiotic prophylaxis in mandibular third molar surgery: acute-phase protein levels as indicators of infection. J Oral Sci 2001;43:117–22. 36. Vibhakar PA, Patankar SR, Yadav MR, Vibhakar P. Salivary total protein levels
37.
38.
39.
40.
41.
42.
43.
44.
and their correlation to dental caries. Int J Oral Maxillofac Pathol 2013;4:13–6. Hong SW, Seo DG, Baik JE, Cho K, Yun CH, Han SH. Differential profiles of salivary proteins with affinity to Streptococcus mutans lipoteichoic acid in caries-free and caries-positive human subjects. Mol Oral Microbiol 2014;29:208–18. Kim KD, Zhao J, Auh S, Yang X, Du P, Tang H, et al. Adaptive immune cells temper initial innate responses. Nat Med 2007;13: 1248–52. Favre L, Spertini F, Corthe´sy B. Secretory IgA possesses intrinsic modulatory properties stimulating mucosal and systemic immune responses. J Immunol 2005;175: 2793–800. Gu¨ven O, De Visscher JG. Salivary IgA in periodontal disease. J Periodontol 1982;53: 334–5. Brandtzaeg P. Human secretory immunoglobulins. 3. Immunochemical and physicochemical studies of secretory IgA and free secretory piece. Acta Pathol Microbiol Scand B Microbiol Immunol 1971;79:165– 88. Gross A, Setterstrom JA, D’Alessandro SM, Van Swol RL. Immunoglobulins in periodontal tissues. I. Concentrations of immunoglobulins in normal and inflamed gingiva. J Periodontol 1979;50:581–5. Byers CW, Toto PD, Gargiulo AW. Levels of immunoglobulins IgG, IgA, and IgM in the human inflamed gingiva. J Periodontol 1975;46:387–90. Kugler J, Hess M, Haake D. Secretion of salivary immunoglobulin A in relation to age, saliva flow, mood states, secretion of
45.
46.
47.
48.
49.
7
albumin, cortisol, and catecholamines in saliva. J Clin Immunol 1992;12:45–9. Lamby-Tovar CP, Gomez-Gonzalez OL, Jaramillo-Gomez LM. Salivary a-Amilase: Relation with Dental Caries and Health in General. Univ Odontol 2013;32:93–101. Strahler J, Mueller A, Rosenloecher F, Kirschbaum C, Rohleder N. Salivary alpha-amylase stress reactivity across different age groups. Psychophysiology 2010;47: 587–95. Filaire E, Dreux B, Massart A, Nourrit B, Rama LM, Teixeira A. Salivary alpha-amylase, cortisol and chromogranin A responses to a lecture: impact of sex. Eur J Appl Physiol 2009;106:71–7. Sadi H, Finkelman M, Rosenberg M. Salivary cortisol, salivary alpha amylase, and the dental anxiety scale. Anesth Prog 2013;60: 46–53. Nater UM, La Marca R, Florin L, Moses A, Langhans W, Koller MM, et al. Stress-induced changes in human salivary alphaamylase activity – associations with adrenergic activity. Psychoneuroendocrinology 2006;31:49–58.
Address: Daniel Torres-Lagares Department of Stomatology School of Dentistry University of Seville C/ Avicena s/n 41009 Seville Spain. Fax: +34 95 448 11 29 E-mail: [email protected]
Please cite this article in press as: Gutie´rrez-Corrales A, et al. Relationship between salivary biomarkers and postoperative swelling after the extraction of impacted lower third molars, Int J Oral Maxillofac Surg (2016), http://dx.doi.org/10.1016/j.ijom.2016.10.005