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 alpha-amylase. 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 alpha-amylase 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 also sometimes observed. The postoperative recovery process generally takes around 7 days, with inflammation being the primary side effect during the healing period.
Multiple factors can influence patient discomfort, including the complexity and duration of the surgery, the surgeon’s 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. Postoperative symptom management has improved considerably over the past few years due to a better understanding of not only the 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.
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. Human saliva contains a variety of proteins, hormones, antibodies, drugs, and cytokines 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. As the composition of saliva can be influenced by systemic changes, specific biomarkers could help identify certain disease conditions. 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.
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. Biomarkers from oral fluids have been used to evaluate the response to therapies such as periodontal surgery combined with matrix metalloproteinase inhibition. 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.
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. 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 receptor-mediated mechanism. Secretory IgA levels in the saliva have also been proposed as a potentially useful immunological marker of stress. Finally, human saliva also contains clinically relevant proteins, many of which come from the blood and may also prove useful in clinical applications.
After surgery, there is a systemic reaction that encompasses endocrine, immunological, and haematological changes. 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. 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 alpha-amylase, 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 .
| 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 |
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 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 time-points: before surgery, immediately after surgery, 2 h later, and 1 week later. Samples were collected in sterile containers and kept at −80 °C 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, 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 μg/ml) and was used to prepare serial dilution standards (1500, 1000, 500, 250, 25, 15, and 5 μg/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 μl 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 °C, 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 μl of each sample in duplicate, the standards and blank were added to the corresponding wells. Next, 50 μl 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 μl of 3,3′,5,5′-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 μl 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 μg/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 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 non-smokers. 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 (μg/ml) throughout the four different stages of the study ( Table 2 ).
| Before surgery | Post-surgery | After 2 h | After 7 days | |
|---|---|---|---|---|
| Alpha-amylase (nmol/ml) | 100.55 ± 3.1 * | 105.46 ± 9.0 | 114.15 ± 17.1 * , ** | 102.13 ± 3.0 ** |
| IgA (ng/ml) | 4662.43 ± 1492.6 | 3686.18 ± 2015.7 | 4348.58 ± 2383.2 | 4562.23 ± 1495.1 |
| Total proteins μg/ml | 689.15 ± 424.8 | 926.63 ± 367.4 | 1104.55 ± 587.9 | 1050.98 ± 356.5 |
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 ).
