G” surface EMG electrodes (Covidien, Dublin). Bipolar electrodes captured the action potentials from the muscles studied, and the surface electrodes were positioned parallel to the muscle fibres. Their position is obligatory with numerical code of the electrodes: 1 right temporal, 2 left temporal, 3 right masseter, 4 left masseter (Figure 1).
The clenching test was performed on the cotton rolls, necessary for standardizing the values: the ratio between basal values (clenching without cotton rollers) and those referring to the frame on the cotton rolls, identified the influence of dental occlusion on neuromuscular balance. The n˚ 3 cotton rolls were positioned parallel to the dental arch, on the occlusal surface behind the first premolars, in order to avoid dental contact. After the clenching tests performed on the cotton rolls and in maximum intercuspation, the procedures previously described were also followed for the custom made mouthguards. The tests were performed in the same way and each for the duration of 5 sec, and the athletes were invited to clench as hard as possible, and to maintain the same level of contraction for all the recording. During tests performance, the atlhetes were verbally encouraged to perform at their best.
In the electromyographic analysis of the present study, were used the symmetry indexes proposed by Ferrario et al. and widely used in scientific literature   .
The indexes considered were the following:
・ GLOBAL INDEX: parameter that summarizes the main occlusal indexes;
・ POC TA: percentage overlapping coefficient for anterior temporalis;
・ POC MM: percentage overlapping coefficient for masseter;
・ BAR: index of the occlusal barycentre;
・ TORS: mandibular torsion index;
・ IMPACT: standardized activity index;
・ ASIM: index of asymmetry in absolute value.
These indexes expressed as a percentage as indicated by Ferrario et al.  .
2.5. Cardiopulmonary Test Procedure
The cardiopulmonary test performed in this study was of maximal incremental type, with the ramp method that allowed to increase the work load in a more physiological way. The first fundamental step consisted in the calibration of the instruments, after which the electrodes were applied to the patient for the monitoring of all 12 electrocardiographic derivations and the mask was positioned on the face to convey all the respiratory flows inside the ergospirometer. It was important at this stage to check that there were no air leaks between the mask and the skin and reminded the patient to breathe exclusively with the mouth (Figure 2).
Figure 1. The athlete during electromyography analysis with mouthguard: the surface electrodes were positioned parallel to the muscle fibres of masseters and temporalis.
Figure 2. The athlete during cardiopulmonary test.
At this point, the athlete was positioned on the mobile platform, and basal values acquired. The test began with a preheating with workloads of lower intensity (25 Watts). During the stress test, the number of cycles per min to maintain increased (70 - 80 cycles per min) and the loads were greater (increases of 70 Watts every 2 min), until the maximum tolerability of the individual was reached, or rather, when they were no longer able to continue running. Generally, the incremental test lasted between 8 and 10 min, while the next phase of recovery (with the subject at rest) occured in a variable time between 2 and 5 min. The data of the cardiopulmonary parameters acquired and monitored graphically in real time throughout the test, were finally processed by software.
Environmental conditions necessary:
・ temperature between 18˚C - 22˚C;
・ relative humidity not higher than 55%;
・ well-ventilated environment;
・ calm and silence around the subject.
The parameters analyzed by the cardiopulmonary test are:
・ maximum oxygen uptake (VO2max);
・ minute ventilation (VE);
・ breathing reserve (BR).
2.6. Statistic Analysis
The data are expressed as mean values ± standard deviation. Statistical significance was accepted at P < 0.05.
Data collected from dental examination on athletes are show in Figure 3. The athletes demonstrated a very poor oral health, as confirmed by high values of clinical indexes: DMFT 7 ± 4.45, PL + 41.18% and BOP + 29.41%. Athletes with a previous history of dental trauma were 52.94%, but only the 23.53% of players used boil & bite mouthguard.
29.41% of athletes reported temporomandibular joint disorders, 37.50% history of orthodontic appliances, 50% presence of oral habits and 23.08% were affected by bruxism (Figure 3).
Table 1 shows the results collected by electromyographic analysis on the 26 athletes analysed in the present study.
By comparing the mean values (“A” and “B”) for all the considered indexes, can be observed that there was an improvement in the (B) condition with mouthguard. In particular improvements statistically significant were registered in the following indexes: the GLOBAL INDEX (85.15 ± 5.55 without mouthguard, 88.92 ± 1.02 with mouthguard, p < 0.05); the BAR (82.79 ± 9.34, for A; 89.75 ± 2.12 for B, p < 0.05); the IMPACT (99.12 ± 49.02 for A, 126.35 ± 46.40 for B, p < 0.05); the ASIM (5.67 ± 4.52 for A, 3.93 ± 2.59 for B, p < 0.05).
Table 2 shows the results relating to respiratory parameters, obtained from the cardiopulmonary test. By analysing the results, can be observed slight differences between the condition “A” (without mouthguard) and condition “B” (with mouthguard).
Table 1. Electromyographic results revealed in athletes in different conditions: without mouthguard (A) and with mouthguard (B).
§ S p < 0.05.
Table 2. Respiratory parameters detected in athletes in conditions without mouthguard (A) and with mouthguard (B).
Figure 3. Prevalence (%) of clinical characteristics of the studied populations.
The present study demonstrated that a correctly performed individual mouthguard, in addition to trauma prevention, offered a better neuromuscular balance of the masticatory muscles.
The electromyography analysis showed that an improvement occured for several analysed indexes, in the condition with mouthguard. In particular, four parameters significantly improved: GLOBAL INDEX, BAR, IMPACT and ASIM. This involved that individual mouthguard symmetrized the work of masseter and temporal muscles to the point of better balancing the distribution of occlusal loads both in the anterior-posterior direction and in the lateral direction and offered the possibility of producing more muscular work. Moreover, it also resulted in a better comfort for the athlete, as well as a series of possible improvements on the activation of muscle chains, with positive repercussions on posture, balance and inevitably, on athletic performance.
Raquel et al.  described a study concerning the electromyographic analysis of masticatory muscles in subjects who used individual mouthguards.
The authors compared the electromyographic activity of the masticatory muscles before and after training, with and without the use of a mouthguard by different indexes in respect to present study.
They observed that the electromyographic parameters remained unchanged; in this way, stable muscle activity was allowed during training  . These results corroborated the hypothesis of the present study: the custom made mouthguard must be well calibrated and not altered the masticatory muscle activity. The muscle activity must be stable or as reported in this study, improved.
Moreover, in the present study the athletes wearing an individual mouthguard were performed by means of cardiopulmonary test.
The results highlighted slight differences between the condition without and with mouthguard.
These data encouraged the use of the individual mouthguard because it should not interfere with the athlete’s respiratory function.
These results confirmed what is reported in the literature   : wearing a custom made mouthguard well calibrated during sporting activities did not alter the athlete’s main respiratory physiological parameters. These results provided further support to encouraging athletes to wear the mouthguard during sport activities.
The individual mouthguards were distinguished by their ability to offer the patient adequate protection, comfort in use and adequate aesthetics given by soft tissue support, as well as the possibility of customizing the device.
The relationship between mouthguard and ecological changes in the oral cavity is still under debate. Instead of this, various strategies can be taken to avoid ecological changes in the oral cavity due to use of mouthguards, and different substances can be applied inside the mouthguard, e.g. chlorhexidine which is commonly used as an active ingredient to prevent the formation of plaque and to reduce its pathogenicity  .
Moreover, the application of casein, within custom-made EVA mouthguards, positively influenced salivary flow, increased pH values, the amount of stimulated saliva and the buffering capacity of the athlete, improving their state of oral health, which was negatively affected by the use of common mouthguards  .
The present study demonstrated that electromyographic analysis, cardiopulmonary test associated with previously reported tests as microbial markers, immune status and sporting characteristics  were important for establishing guidelines for management of training load in order to minimize physical stress and the risk of oral infection.
The lack of literature highlights the need for further study on a very current problem in the field of sport dentistry.
The individual mouthguard significantly improved the neuromuscular balance of the masticatory muscles, symmetrizing the work of the masseter and temporal muscles to the point of better balancing the distribution of occlusal loads both in the anterior-posterior direction and in the lateral direction and offered the possibility of producing more muscular work. Moreover, it did not constitute an impediment that significantly disturbs the athlete’s breathing during physical exertion.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.