The study included 20 male patients aged 18–49 (mean 27.25) years, who were hospitalized due to mandibular fractures at the Department of Maxillofacial and Plastic Surgery of the Medical University of Białystok. All patients were informed of the study methodology, after which they provided written consent to participate in the experiments. The study was approved by the Ethics Committee of the Medical University of Bialystok (R-I-002/394/2016). Among the patients, 15 (75%) suffered from mandibular body fracture, 1 (5%) had a fracture within the area of mandibular processes, and 4 (20%) had a fracture in the mandibular body and processes. Eight patients (35%) had a singular mandibular fracture, and 12 (60%) had double mandibular fractures.
The injuries were primarily caused by beating (13 patients, 65%) and falls (5 patients, 25%). One person (5%) was injured in a car accident, while in 1 (5%), the fracture cause could not be identified.
All patients (100%) included in the study were otherwise healthy (Table 1). Eleven (55%) declared that they smoked cigarettes on a regular basis. All the patients were subjected to examinations such as pantomographic x-ray (Figure 2, 3, 4, 7), computed tomography (Figure 5, 6), mandible x-ray in posterior–anterior projection (Figure 8), blood morphology and coagulation testing, analysis of electrolyte levels (Table 1), and electrocardiography. One day before the procedure, they were administered an antibiotic (Biofazolin, 2×1.0 g iv) and a proton pump inhibitor with stomach-protective function (Esomeprazol, 1×40 mg iv). All the patients were operated about 4 days from the injury under general endotracheal anesthesia. As a part of premedication, they were administered 7.5 mg Dormicum approximately 40 minutes before the procedure. Mandibular fractures were dressed through open reposition and osteosynthesis of the fragments via miniplates and titanium screws. Eleven patients (55%) had teeth remaining in the fracture gap. On the day of the procedure, the antibiotic and gastroprotective drug were again administered to the patients. In addition, an analgesic was introduced (Ketonal, 3×100 mg iv). A day after the procedure, the patients received 1 dose of Dexaven 4 mg iv, and 2 days after the procedure, 1 dose of Dexaven 2 mg iv. The antibiotic, gastroprotective drug, and analgesic (vitamin C, 3×300 mg) were continued to be administered to the patients throughout their stay at the hospital. All the patients had intermaxillary tractions installed on the IMF (Intermaxillary fixation) screws during the procedure. The tractions were kept for a period of about 2 weeks.
After hospitalization, the patients were recommended to ingest resorption drugs (calcium, vitamin C), and if needed, an analgesic (Ibuprofen or Paracetamol). In addition, the following were recommended: cold compresses to relieve swelling, Altacet compresses for 2–3 days, high-protein diet for 6 weeks, Eludril mouthwash thrice a day for 2 weeks, and dental check-ups. They were also advised to avoid excessive physical effort.
The control group comprised 15 healthy individuals (men), who were voluntary blood donors, aged 20–30 years, and had no systemic diseases and bone fractures within the last 5 years. Blood was collected from them after obtaining written approval for blood donation.
Blood sampling
Patients with facial fractures and healthy control subjects were recruited into the study after obtaining their informed consent. Six milliliters of blood were collected by venous arm puncture under aseptic conditions into test-tubes with clot activator. Blood was collected at four time points: before surgery (A), at 24 hours after surgery (B), at 2 weeks (C) and 6 weeks (D) after surgery. Serum was obtained by centrifugation at 2000 rpm for 10 min of blood samples and freeze at -80˚C until the date of analysis.
Determination of total NO concentration in serum
Nitrite (NO2-) and nitrate (NO3-) are stable final products of NO metabolism and may be used as indirect markers of NO. Total NO concentration was determined as sum of NO2- and NO3- concentrations in indirect method based on measurement of NO2- concentration in serum according to Griess reaction. In the presence of cadmium (Sigma-Aldrich, Steinheim, Germany) NO3- were reduced to NO2-, which are able to react in colorful reaction with Griess’s reagent (Sigma-Aldrich, Stainheim, Germany). Nitrite concentrations were determined by spectrophotometric analysis at 540 nm (UVN-340 ASYS Hitech GmbH microplate reader; Biogenet, Eugendorf, Austria) with reference to a standard curve. NO products were expressed as mM.
Determination of malonodialdehyde (MDA) concentration in serum
The concentration of malonodialdehyde (MDA) in serum was assessed by quantitative competitive enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Human Malonodialdehyde (MDA) ELISA kit; MyBioSource, San Diego, USA). Prior to the experiment, all provided reagents were held in room temperature for 30 min. Standards (100 µL), controls (100 µL) and serum samples (100 µL) were pipetted to anti-MDA antibody-coated wells. Then horseradish peroxidase (HRP)-conjugate (50 µL) was added and 96-well plate was incubated for 60 min at 37˚C. After 5 times wash, substrates for HRP enzymes were added (substrate A and B; 50 µL each). After 20 min incubation at 37˚C, stop solution was added to terminate the reaction, which turned color from blue to yellow. The intensity of color was measured spectrophotometrically at 450 nm with a ELx800 Absorbance Reader (BioTek Instruments, United States). The MDA concentration was evaluated based on the standard curve. The obtained results were expressed as ng/ml.
Determination of nitrotyrosine concentration in serum
The concentration of nitrotyrosine in serum was assessed by quantitative sandwich enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Nitrotyrosin ELISA Kit; Immunodiagnostic AG, Bensheim, Germany). Prior to the experiment, all provided reagents were held in room temperature for 30 min. Standards (100 µL), controls (100 µL) and serum samples (diluted 1:60 in assay buffer; 100 µL) were added to polyclonal goat anti-nitrotyrosine antibody-coated wells. Plate was incubated for 60 min at 37˚C and then 5 times washed. Then a peroxidase-conjugated polyclonal goat anti-human serum proteins antibody (100 µL) to wells and plate was incubated for 60 min in microplate shaker. After 5 times wash, substrate (tetramethylbenzidine, 100 µL) was added. Next, plate was incubated in dark for 15 min and finally, an acidic stop solution (100 µL) was added to terminate the reaction. Absorbance of colorful product was measured at 450 nm with a UVN-340 ASYS Hitech GmbH microplate reader (Biogenet, Eugendorf, Austria). The nitrotyrosine concentration was calculated from the standard curve of the absorbance vs standard concentration. The obtained results were expressed as nM.
Determination of asymmetrical dimethylarginine (ADMA) concentration in serum
The concentration of asymmetrical dimethylarginine (ADMA) in serum was assessed by quantitative double-sandwich enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Human Asymmetrical Dimethylarginine (ADMA) ELISA Kit; MyBioSource, San Diego, USA). Prior to the experiment, all provided reagents were held in room temperature for 30 min. Standards (50 µL), serum samples (50 µL) and sample diluent (50 µL) were added into a human ADMA monoclonal antibody-coated 96-well wells. Then the horseradish peroxidase (HRP)-conjugate reagent was added to each well and plate were incubated for 60 min at 37˚C. After washing 4 times, chromogen solutions A and B (50 µL each) were added to each well, then plate was incubated in dark for 15 min. Addition of stop solution (50 µL) terminate the reaction and changed sample color from blue to yellow. Absorbance was read at 450 nm with a UVN-340 ASYS Hitech GmbH microplate reader (Biogenet, Eugendorf, Austria). The ADMA concentration was calculated from the standard curve. The obtained results were expressed as ng/mL.
Statistical evaluation
Data were analyzed using STATISTICA version 13.1 program (StatSoft, Inc., Tulsa, OK). and results were express as Mean ± standard deviation (SD). The categorical data from repeated measurements were analyzed by variance analysis. To assess the distribution of total nitric oxide, malonyldialdehyde, nitrotyrosine and asymmetric dimethylarginine data Shapiro-Wilk's test of normality and visual inspection of Q-Q plots was used. Data revealed a normal distribution, hence we used ANOVA with Tukey’s post hoc test and Student t-test for pairwise comparisons. The relationship between total nitric oxide and its activity indicators was analyzed by Spearman’s rank correlation test. The results were considered significant if p-values were 0.05 or less. The data were plotted using STATISTICA version 13.1.