Influence of sport mouth-formed type mouthguards on saliva secretion and composition during jaw activity: a pilot study

Abstract

Background

We hypothesized that wearing a mouthguard may affect salivary flow and composition. Therefore, we investigated the impact of mouthguard use on saliva secretion rate and some salivary constituents under different jaw activities.

Methods

This was a randomized crossover experimental study including 13 volunteers. Saliva samples of the volunteers were collected and analysed in four different experimental conditions: without mouthguard with relaxed jaw (RJ), without mouthguard after repetitive jaw contractions (JC), with mouthguard with relaxed jaw (MG-RJ) and with mouthguard after repetitive jaw contractions (MG-JC).

Results

Saliva secretion rate was significantly higher in the condition MG-JC compared to all other conditions (P < 0.01). Total protein concentration and Mucin 5B (MUC5B) levels in saliva did not differ significantly between the four experimental conditions. Immunoglobulin A (IgA) concentrations were significantly lower in the condition MG-JC compared to all other conditions (P < 0.01).

Conclusion

Mouthguards increase saliva flow rate, compared to not wearing a mouthguard, and jaw contractions enhance this effect compared to a relaxed jaw. Wearing a mouthguard during active jaw activity reduced the concentration of salivary IgA.

Introduction

Saliva is a complex fluid produced by three pairs of major salivary glands, the parotid, the submandibular and sublingual gland, and by hundreds of minor salivary glands in the oral cavity. Saliva is composed of approximately 99% of water and 1% of organic and inorganic constituents [1]. Even at low concentrations, a wide array of organic constituents can be quantified, including amylase, albumin, mucin, creatinine, lactate, immunoglobulins, and hormones such as testosterone and cortisol [2].

Physical exercise has been consistently associated with changes in salivary biomarkers such as cortisol, α-amylase, and lactate, reflecting activation of the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system. Both moderate training and high-intensity isokinetic exercise have been shown to significantly increase salivary cortisol levels [3, 4]. Additionally, exercise-induced changes in salivary α-amylase showed a strong correlation with blood lactate levels, thereby supporting the potential use as a non-invasive marker of sympathetic activity during physical exercise [5]. Collectively, these studies emphasize the value of salivary biomarkers in the evaluation of physiological responses to exercise. Consequently, saliva has become a widely used, non-invasive medium for monitoring exercise-induced physiological changes, and its systematic assessment during training and competition could provide sport coaches with valuable insights [2, 6–8].

One major pathway to stimulate the salivary glands is by the stomatognathic system [9]. This functional unit, comprising the jaws, teeth, periodontal tissues, temporomandibular joints, masticatory muscles, and their neurovascular supply, facilitates mastication and is activated during jaw clenching.

Mouthguards are primarily used in various combat and ball sports to reduce the incidence and severity of dental injury during sport participation [10–13]. In addition to their primary role in injury prevention, both mouthguards and physical exercise have been shown to influence the stomatognathic system, by altering the distribution of bite force, changing the neuromuscular balance of the masseter muscles, and altering occlusal load [14]. However, it remains unclear whether the mouthguard-induced changes in the stomatognathic system, independent of physical activity, also contribute to alterations in salivary composition, especially since physical activity is known to affect salivary profiles [2, 15, 16].

It is hypothesized that wearing a mouthguard, even at rest, increases salivary flow rate and increases stress-related salivary biomarkers compared with not wearing a mouthguard [17, 18]. By examining the complex interplay between sport mouthguards and some salivary constituents, this study aims to contribute to a broader understanding of oral-systemic interactions and their implications for health outcomes in athletes. Therefore, we investigated the impact of mouthguard use on saliva secretion rate and some salivary constituents under different jaw activities.

Material & method

Participants and sample collection

We included 13 volunteers. Inclusion criteria were an age ≥ 18 years old and enough teeth to be able to support the mouthguard. Volunteers wearing dental protheses or using bimaxillary oral appliances (like sleep disorders appliances etc.) were excluded, as well as volunteers with a medical history of saliva-related conditions and/or use of medication that potentially affects saliva secretion. Each participant was asked if they had worn a sports mouthguard before.

Each participant underwent two separate sessions in the morning between 10.00 and 12.00 h AM, with and without wearing a mouthguard, with at least 90 min interval to eliminate carry-over effects. Participants were assigned merely in alternating order, based on the sequence of their registration and time available, to begin the session either with a mouthguard of without one. To eliminate changes in salivary flow rate and composition by physical exercise, participants had to sit during the experiment.

In this study, we used maxillary sport mouth-formed type mouthguards made of transparent thermoplastic Ethylene Vinyl Acetate (EVA) (Decathlon Netherlands B.V.). Mouthguards were formed according to the manufacturer’s instructions by the participants, while wearing gloves to avoid contamination. Although different types and designs of sports mouthguards are available, this type of mouthguard is most commonly used [19], and therefore most suitable for this pilot study.

Saliva samples were collected into a sterile container after each session without and with mouthguard during several jaw activities, resulting into four experimental conditions: without mouthguard with relaxed jaw (RJ), without mouthguard after repetitive jaw contractions (JC), with mouthguard with relaxed jaw (MG-RJ) and with mouthguard after repetitive jaw contractions (MG-JC). Saliva was collected after a relaxed jaw for 5 min and after 5 sets of 60 repetitive jaw contractions, with 15 s rest between sets, using a metronome at 1 Hz, total time of 6 min. To ensure complete collection of saliva accumulated when using a mouthguard, the saliva was first aspirated directly from the mouthguard while it remained in the participant’s mouth. The mouthguard was then removed, and any remaining saliva in the oral cavity was gently expectorated into a sterile collection container.

Salivary parameters

Whole saliva samples were collected in a 25 ml tube (Eppendorf, Hamburg, Germany) placed on ice. The salivary flow rates (in ml/min) were determined gravimetrically by reweighting the pre-weighed tubes, assuming (1 g = 1 ml), and dividing the volume by the number of minutes saliva was collected. Subsequently the saliva samples were clarified by centrifugation (10,000 g at 4 °C for 10 min) to remove epithelial cell debris and bacteria. The saliva supernatants were aliquoted and stored at -20 °C until further use. The total amount of proteins in samples were measured using the Pierce BCA Protein Assay Kit (Thermo Scientific) according to manufacturer’s protocol. The amylase activity was determined as described previously [20]. Mucin 5B (MUC5B) levels were determined by an enzyme-linked immunosorbent assay (ELISA), using the MUC5B-specific monoclonal antibody F2 as described previously [21].

IgA concentrations (in µg/ml) were measured using a monoclonal rabbit anti- (human secretory IgA) (Sigma-Aldrich). Microplates were coated with this antibody at room temperature overnight, and then with the saliva samples and a standard of purified human secretory IgA (Nordic-MUbio, Susteren, the Netherlands) for 2 h at 37 °C. Captured secretory IgA was detected with HRP-conjugated goat anti-(human IgA) (Sigma-Aldrich) [22].

Statistical analysis

Statistical analyses were performed using SPSS version 28.0 (IBM Corp, Armonk, USA). The Kolmogorov-Smirnov test was performed to test the normality of various parameters. All parameters were normally distributed. Therefore, mean and standard deviation were used in the descriptive analysis.

ANOVA repeated measures was conducted to examine differences between the four experimental conditions. Post-hoc comparisons were conducted using Bonferroni test when appropriate. Statistical significance was set at p < 0.05.

Results

In total, 13 voluntary participants were included, 8 women and 5 men, with a mean age of 38 years (SD 10). 38% of the volunteers reported that they had worn a sport mouthguard before.

Mean saliva secretion rate and composition for the four different conditions are presented in Table 1. The saliva secretion rate was significantly higher in the condition with mouthguard after jaw contractions (MG-JC) compared to all other conditions (RJ, JC, MG-RJ) (p < 0.01). Mean secretion rate was significantly different for both relaxed jaw and jaw contractions between without and with mouthguard (respectively p = 0.02 and p < 0.01).

Table 1.

Saliva secretion rate and composition expressed as mean values (SD) for four experimental conditions: RJ, JC, MG-RJ and MG-JC

	RJ	JC	MG-RJ	MG-JC	ANOVA
Secretion rate (ml/min)	0.45 (SD 0.25)ab	0.41 (SD 0.23)cd	0.77 (SD 0.48)ace	1.1 (SD 0.63)bde	p < 0.01
Total protein concentration (mg/ml)	2.36 (SD 0.93)	2.28 (SD 0.89)	2.01 (SD 0.96)	2.01 (SD 1.21)	p = 0.32
Mucin 5B concentration (µg/ml)	48.29 (SD 32.27)	57.78 (SD 38.73)	44.05 (SD 30.32)	39.79 (SD 28.27)	p = 0.39
IgA concentration (µg/ml)	136.21 (SD 43.22)b	172.84 (SD 131.48)d	109.56 (SD 51.52)e	68.51 (SD 39.28)bde	p < 0.01
IgA secretation rate (µg/min)	56.12 (SD 22.27)	58.36 (SD 32.50)	74.64 (SD 38.69)	63.10 (SD 31.31	P = 0.29

Bonferroni test < 0.05: ^a = RJ vs. MG-RJ ^b = RJ vs. MG-JC, ^c = JC vs. MG-RJ ^d = JC vs. MG-JC, ^e = MG-RJ vs. MG-JC

Mean salivary concentrations of total proteins and Mucin 5B (MUC5B) did not differ significantly between the four experimental conditions.

Mean immunoglobulin A (IgA) concentrations were significantly lower after MG-JC compared to all other conditions (RJ, JC, MG-RJ) (p < 0.01). Additionally, no significant difference of IgA concentration was found with relaxed jaw between with and without mouthguard (RJ vs. MG-RJ). Calculated mean IgA secretion rate did not differ significantly differ between conditions as presented in Table 1.

Amylase activity in saliva (expressed as AU/ml) did not differ significantly between the four conditions: RJ 1.05 (SD 0.58), JC 0.80 (SD 0.51), MG-RJ 0.91 (SD 0.64) and MG-JC 0.72 (SD 0.39). No significance was found between conditions.

Discussion

This study shows that wearing a mouthguard increased saliva secretion rate, compared to not wearing a mouthguard, and jaw contractions further reinforce this effect compared to a relaxed jaw. Increased salivary flow rate is often associated with enhanced protective effects for the oral cavity, which are crucial for maintaining oral health and preventing diseases like dental caries, periodontal disease, and mucosal infections [23, 24].

Total amount of proteins, active amylase, and MUC5B concentrations were unaffected in saliva by either jaw position or mouthguard usage. Only IgA concentrations were significantly different as mouthguard use with active jaw contractions was associated with lower IgA concentrations compared to without mouthguard and during mouthguard use in rest jaw condition. However, when corrected for the changes in salivary flow rate, the IgA secretion rate did not differ significantly between the four experimental conditions. This suggests that the observed decrease in IgA concentration during mouthguard use with active jaw contractions is due to a dilution effect resulting from an increased salivary secretion rate. The clinical impact of decreased salivary IgA concentrations remains unclear.

IgA serves as a critical component of the mucosal immune system and is produced by the salivary glands and serving as the first line of defence against oral pathogens by preventing their adhesion to mucosal surfaces and neutralizing toxins and viruses. A reduction in salivary IgA concentrations may compromise the capacity of saliva to inhibit adhesion and colonization of cariogenic bacteria such as Streptococcus mutans [25].

Exercise is known to influence IgA concentrations, however this largely depends on exercise intensity and duration. Moderate exercise may enhance immune function, potentially increasing IgA secretion, whereas prolonged and high-intensity exercise tends to supress IgA concentrations. The relationship between physical activity and the suppression of the immune system is not fully understood [26].

This study also has some limitations. Firstly, the sample size was limited to 13 participants, categorising this examination as a pilot study. Although preliminary findings suggest potential trends, the generalisability of these results is restricted. Consequently, a larger-scale study incorporating a more substantial number of participants is warranted to derive more reliable conclusions.

Additionally, the analysis was restricted to a limited number of salivary constituents. Expanding future research with inclusion of salivary cortisol and lactate levels, both at rest and post-exercise, could provide valuable insights, as existing literature suggests their relevance in athletic performance monitoring.

Finally, this study examined the salivary composition based on a single type of mouthguard, which may not be representative of the variety of designs available. Variations in mouthguard thickness and fit could influence jaw activity, and therefore salivary composition. The data presented in this study suggest that mouth guards do not have a direct effect on the concentrations of specific saliva components, but indirectly affect these through the amount of saliva secreted. Because saliva secretion rate is related to physical stimulation caused by wearing the device, it is important to focus future experiments on saliva secretion volume and the possible role of mouthguard material and shape.

Conclusion

The use of mouthguards significantly enhances salivary flow compared to not wearing a mouthguard, with repetitive jaw contractions further amplifying this effect relative to a relaxed jaw position. This increase in salivary flow is generally considered beneficial for maintaining oral health. Because saliva secretion rate is related to physical stimulation caused by wearing the device, it is important to focus future experiments on saliva secretion volume and the possible role of mouthguard material and shape.

Authors’ contributions

KV and HB conceived and designed the study. KV and AS performed the study. KV analyzed the data. HB, JL and FL critically revised the manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.

Declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the Academic Centre for Dentistry Amsterdam (ACTA) (#2020276) and was performed in accordance with the ethical principles as stated in the Declaration of Helsinki. Signed informed consent of the volunteers was obtained before they participated.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.