OPEN
`
`received: 29 June 2016
`accepted: 13 February 2017
`Published: 15 March 2017
`
`Crystallization and hardening of
`poly(ethylene-co-vinyl acetate)
`mouthguards during routine use
`
`Ryoko Kuwahara1, Ryotaro Tomita1, Natsumi Ogawa1, Kazunori Nakajima2,
`Tomotaka Takeda2, Hiroki Uehara1 & Takeshi Yamanobe1
`
`Mouthguards (MGs) made from poly(ethylene-co-vinyl acetate) (EVA) are widely used in contact sports
`to prevent injuries such as breaking teeth and lip lacerations and to reduce brain concussion. However,
`the changes in morphology and the molecular mobility of EVA, which can affect its physical properties
`during practical usage, have not been precisely examined. Therefore, we attempted to determine the
`main factors which lead to changes in MG performance after one season of practical use by high school
`rugby players. Solid-state nuclear magnetic resonance (NMR) and pulse NMR measurements showed
`the hardening of MGs, which was associated with an increased crystallinity of the EVA resulting from
`prolonged usage. Furthermore, our data indicated that the increase in the relative amount of the
`crystalline phase may be primarily attributed to temperature fluctuations and repeated changes in
`pressure, which could cause the hardening of EVA and eventually diminish the protective ability of MGs.
`
`Mouthguards (MGs) can prevent sports-related oral injuries and reduce concussions, therefore, they are
`recently being employed for various sports1–12. Materials employed for MGs are very limited, and only
`poly(ethylene-co-vinyl acetate) (EVA), olefin-based thermoplastic elastomers and styrene-based thermoplastic
`elastomers have been certified in Japan13–18. EVA is primarily used because of its low cost and facile processabil-
`ity—the melting point of EVA (ca. 30–80 °C) is particularly low, for example19. Indeed, EVA sheets can easily be
`treated with commercially available small MG manufacturing machines; therefore, EVA MGs can be prepared not
`only at dental clinics but also at sporting grounds.
`MGs are prepared by melting EVA sheets and subsequent molding with dental casts. MGs are detached from
`the molds after the temperature reaches room temperature. Following this, MGs undergo final occlusal checking
`by dentists before they are supplied to users. After MGs are used for long periods of time, some users report
`discomfort; in particular, they feel that their MGs are becoming hard. If hardening occurs, along with increased
`brittleness and reduced energy absorption capability, deterioration in the protective ability of the MG will occur
`concomitantly. To ensure that MGs are providing adequate safety levels to users, the guidelines for renewing MGs
`should preferably be based on scientific indicators.
`Because EVA is a macromolecule with entangled polymer chains comprising crystalline and amorphous
`phases20,21, we presumed that the discomfort relating to the fit of MGs would mainly be derived not from chem-
`ical degradation but from the state of those phases, which may be influenced by temperature fluctuations and/or
`repeated changes in pressure. In this study, we precisely analysed routinely used MGs and EVA films using dif-
`ferential scanning calorimetry (DSC), solid-state NMR and pulse NMR measurements22–27 to identify the factors
`that affect MG morphology and molecular mobility.
`Results and Discussion
`NMR spectroscopic analyses of MGs after one season of use. The solid-state cross-polarization
`magic-angle spinning (CP/MAS) 13C NMR spectra of a piece from MGϕ 1 and another from MG1 that contact on
`tooth#16, which is one of the most compressed occlusion parts of the MG, are displayed in Fig. 1. The notation
`MG1 represents a MG that was routinely used by user 1 for one season (10 months), whereas MGϕ 1 represents
`the excess portions of the MG material, which were obtained after the lamination and subsequent trimming of
`
`1Division of Molecular Science, Graduate School of Science and Technology, Gunma University, Kiryu, Gunma, Japan.
`2Department of Oral Health and Clinical Science, Division of Sports Dentistry, Tokyo Dental College, Chiyoda-ku,
`Tokyo, Japan. Correspondence and requests for materials should be addressed to T.Y. (email: yamanobe@gunma-u.
`ac.jp)
`
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`Figure 1. Solid-state CP/MAS 13C NMR spectra of (a) a piece from MGϕ 1 and (b) the piece of MG1 that
`contacts on tooth#16.
`
`MG2 MG3 MG4 MG5 MG6 MG7 MG8
`MG1
`42.5
`37.9
`39.7
`41.6
`42.1
`36.9
`41.2
`44.1
`MGϕ 1 MGϕ 2 MGϕ 3 MGϕ 4 MGϕ 5 MGϕ 6 MGϕ 7 MGϕ 8
`33.0
`35.0
`34.6
`32.7
`34.4
`38.0
`33.5
`35.9
`
`Table 1. Crystalline ratio (%) determined by solid-state CP/MAS 13C NMR spectroscopy.
`
`MG2 MG3 MG4 MG5 MG6 MG7 MG8
`MG1
`27.0
`26.5
`25.5
`24.5
`26.8
`24.9
`24.5
`30.6
`MGϕ 1 MGϕ 2 MGϕ 3 MGϕ 4 MGϕ 5 MGϕ 6 MGϕ 7 MGϕ 8
`22.7
`22.1
`23.7
`24.0
`23.1
`22.4
`22.7
`23.5
`
`Table 2. Rigid component (%) of MG determined using pulse NMR spectroscopy.
`
`MG1 and preserved at room temperature for one season. Peaks at approximately 33 and 31 ppm are known to
`result from CH2 units in the ethylene groups of the crystalline and amorphous phases, respectively, whereas the
`peak corresponding to CH3 in the methyl group of acetate appears at 22 ppm20,21. As shown in Fig. 1a, the inten-
`sity of the peak corresponding to the amorphous phase was higher than that corresponding to the crystalline
`phase. Conversely, in Fig. 1b, the intensity of the peak corresponding to the crystalline phase was higher than
`that corresponding to the amorphous phase. Such phenomena were observed in all the MGs examined in this
`study (Table 1). These results indicate that the usage of MGs can increase the ratio of the crystalline phase present
`in EVA. Incidentally, the solution 13C NMR spectra of used MGs remained unchanged compared with those of
`unused MGs (data not shown), indicating that no chemical decomposition of EVA (e.g., hydrolysis of the acetate
`group)28 occurred during the usage period.
`In solid-state CP/MAS 13C NMR, the efficiency of the cross-polarization from 1H to 13C in the crystalline
`phase is known to be higher than that in the amorphous phase. Therefore, in Fig. 1a, the ratio of the amount of
`the crystalline phase to that of the amorphous phase is, in fact, not as large as that indicated by the ratio of the
`two corresponding peaks. However, the change in the ratio of the peaks between unused and used MGs can be
`compared, and accordingly, the difference between the spectra (Fig. 1a,b) could be attributed to the crystallization
`during routine use.
`We then assumed that the increase in the crystalline ratio may increase the fraction of restricted components
`in MGs. Pulse NMR measurements were therefore performed using the above-mentioned pieces of MGϕ 1–8 and
`MG1–8 to evaluate molecular mobility29,30. The observed data were fit to a hybrid of exponential and Gaussian
`functions to obtain the fraction ratios for the rigid, intermediate, and mobile components (Figure S1). As shown
`in Table 2, which summarizes the fractions of the rigid component for MGϕ 1–8 and MG1–8, the magnetization
`fractions demonstrate that MG usage increases the rigid component instead of decreasing the intermediate and
`mobile components, indicating a reduction in molecular mobility.
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`Figure 2. DSC curves of the heating process of (a) EVA9, (b) EVA14, and (c) EVA28. Samples: unannealed
`(black), annealed at 60 °C (red), annealed at 80 °C (green), and annealed at 100 °C (blue). New endothermic
`peaks were shown with red arrows.
`
`In summary, the CP/MAS 13C NMR and pulse NMR measurements show that the usage of EVA MG increases
`the crystalline fraction, which restricts molecular mobility and leads to the eventual hardening of the MG.
`
`Verification of the effect of temperature fluctuations by DSC. We attempted to verify the effects
`of temperature fluctuations on the crystallization behaviour of EVA films (0.030 cm thickness) with different
`vinyl acetate (VA) contents of 9%, 14% and 28%, for EVA9, EVA14 and EVA28, respectively, using DSC analysis.
`According to the solution 13C NMR spectra, the VA content of EVA28 was nearly equal to that of the EVA sheet
`(Drufosoft®) used to prepare the examined MGs.
`Melting of unannealed EVA9 started at approximately 30 °C and ended after the stark endotherm at approx-
`imately 95 °C, as shown in the DSC curves of the heating process (Fig. 2a). In the cases of EVA14 and EVA28,
`melting started at approximately 30 °C; the former ended with a strong endotherm at approximately 90 °C, while
`the latter ended with a gently sloping approximately peak at approximately 70 °C (Fig. 2b,c). The decrease in the
`maximum intensity and temperature of the endothermic peaks depended on VA content, reflecting the thick-
`ness of lamellae31,32 mainly comprising ethylene units. Higher ethylene content, i.e. lower VA content, tended to
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`Figure 3. DSC curves of the heating process of Drufosoft® films with or without treatment by thermal cycles
`(100 times) executed using a protocol with repeated temperature fluctuations (a) between 25 °C and 37 °C or
`(b) between 6 °C and 22 °C. Samples: thermal-treated (red) and as-prepared (black). The new endothermic peak
`is shown with a red arrow.
`
`provide thicker lamellae and enhance the crystallinity of EVA, although the broad melting ranges indicated that
`the lengths of the polyethylene strands between the VA units were widely distributed. Meanwhile, it can be stated
`that thin lamellae are produced regardless of the VA content because all films started melting at a low temperature
`(approximately 30 °C). This suggested that the crystallization of MG could progress in the mouth, i.e. at body
`temperature, during routine use.
`Annealing of EVA9, EVA14, and EVA 28 resulted in a new gentle endothermic peak at approximately 70 °C
`when the films were annealed at 60 °C (Fig. 2a–c), and annealing of EVA9 and EVA14 at 80 °C resulted in a sharp
`endothermic peak at approximately 90 °C (Fig. 2a,b). Although the new endothermic peaks are attributed to the
`melting of the crystalline phase by the annealing treatments, it was commonly observed that annealing of the EVA
`films at the crystallization temperature (Tc)19 or the closest temperature above Tc resulted in a relatively clear peak
`maximum for the melting temperature (Tm), i.e. Tc = 80, 71 and 48 °C for EVA9, EVA14 and EVA28, respectively
`(Figs 2 and S2). Thus, these results indicate that the crystallinity of EVA MGs greatly depends on the status of the
`ethylene moieties and is significantly influenced by temperature fluctuations at ranges closer to Tc.
`Furthermore, we analysed the effect of temperature fluctuations using Drufosoft® films (thickness = 0.30 cm,
`which is approximately equal to that of typical MGs) with or without treatment of repeated thermal cycles (100
`times). Two different protocols of thermal cycling were employed; one was shuttling between 25 °C and 37 °C
`and the other was shuttling between 6 °C and 22 °C as a reference condition (Fig. 3). Intriguingly, in the former
`condition, a new endothermic peak appeared at approximately 45 °C (Fig. 3a), but, in contrast, the latter condition
`caused little changes in the DSC thermogram (Fig. 3b). These results show that repeated temperature fluctuations
`even between close temperatures, i.e., ambient and body temperatures, can affect the crystallinity of EVA MGs.
`
`Verification of the effect of temperature fluctuations using solid-state CP/MAS 13C NMR. The
`tendency towards crystallinity discovered in the DSC measurements was also confirmed by solid-state CP/MAS
`13C NMR analyses. As shown in Fig. 4a, although the intensity of the peak corresponding to the crystalline phase
`(at 33 ppm) was higher than that corresponding to the amorphous phase (at 31 ppm), the highest difference
`was observed in the spectrum of EVA9 annealed at 80 °C, which is equal to Tc. Similarly, the highest differences
`were observed in EVA14 annealed at 80 °C (Tc = 71 °C) and in EVA28 annealed at 60 °C (Tc = 48 °C), respectively
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`Figure 4. Solid-state CP/MAS 13C NMR spectra of (a) EVA9, (b) EVA14, and (c) EVA28. Samples: unannealed
`(black), annealed at 60 °C (red), annealed at 80 °C (green), and annealed at 100 °C (blue).
`
`(Fig. 4b,c). Furthermore, intriguingly, when the spectra of unannealed EVA films were compared, the ease of
`crystallization obviously reflected the ethylene content.
`
`Verification of the effect of temperature fluctuations using pulse NMR. The molecular mobility
`in EVA films (0.030 cm thickness) was analysed using pulse NMR measurements. As compared with the unan-
`nealed EVA9 film, EVA9 films annealed at either 60 °C or 80 °C exhibited increased fraction ratios and decreased
`spin-spin relaxation times (T2 values) for the rigid component (Table S1). EVA9 annealed at 100 °C provided
`similar values to unannealed EVA9. Among the fraction ratios and T2 values obtained for the rigid component in
`all EVA9 films examined, EVA9 annealed at 80 °C showed maximum and minimum values (36.9% and 8.90 μ s),
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`Figure 5. Solid-state CP/MAS 13C NMR spectra of (a) EVA28_1 and (b) EVA28_2 before (black) and after (red)
`repeated compression cycles (10,000 times at 6.0 MPa).
`
`respectively. In the case of EVA14, the maximum fraction ratio (30.6%) and the minimum T2 value (9.90 μ s) of the
`rigid component were observed in films annealed at 80 °C. Furthermore, the rigid component of EVA28 annealed
`at 60 °C provided the minimum T2 value (11.0 μ s), although the fraction ratio was equal to or somewhat smaller
`than the other EVA28 films. These results, showing the effects of temperature fluctuation on the hardening of EVA
`films, indicate that their hardening is well correlated with the crystallinity observed using DSC and solid-state
`CP/MAS 13C NMR measurements.
`
`Effects of repeated pressure on crystallization and compressive deformation behaviour. To
`study the changes in crystallinity resulting from repeated changes in pressure, EVA28 films (EVA28_1 and
`EVA28_2) were analysed by solid-state CP/MAS 13C NMR before and after repeated compression cycles (10,000
`times at 6.0 MPa). As shown in Fig. 5a, the crystalline component of EVA28_1, corresponding to the signal at
`33 ppm, seemed to increase slightly after compression (the ratios of the crystalline component in EVA28_1 before
`and after repeated compression cycles were 29.9% and 30.5%, respectively). This film was immediately subjected
`to cooling in ice cold water during its preparation. In contrast, an increase in the crystalline component after
`compression was clearly observed in EVA28_2, which was gradually cooled down to 25 °C during its preparation
`(the ratios of the crystalline component in EVA28_2 before and after repeated compression cycles were 32.0% and
`35.5%, respectively) (Fig. 5b). Although both results indicate that repeated mechanical compression can induce
`the crystallization of MGs, the difference may be because EVA-28_2 originally contained a higher ratio of the
`crystalline component than EVA-28_1, as shown in the NMR spectra of the uncompressed films. In particular,
`the difference indicates that, in addition to repeated mechanical compressions, the presence of seed crystals could
`further accelerate crystallization within EVA MGs.
`Finally, we analysed compressive deformation behaviour using Drufosoft® films (thickness = 0.30 cm) before
`and after repeated compression cycles (5,000 times at 6.0 MPa). As shown in Fig. 6, compressive stress–strain
`curves clearly showed that the repeated compression resulted in a decrease in compressive strain versus compres-
`sive stress, indicating the decreased capacity of MG to withstand mechanical compressions.
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`Figure 6. Compressive stress–strain curves of Drufosoft® films (thickness = 0.30 cm) before (black) and
`after (red) repeated compression cycles (5,000 times at 6.0 MPa).
`
`Conclusions
`We focused on temperature fluctuations and repeated pressure changes as possible causes of deterioration in EVA
`MGs. To this end, we precisely analysed used MGs, Drufosoft® and EVA films with different VA contents. The
`solid-state CP/MAS 13C NMR measurements showed significant increases in the crystalline components present
`in eight MGs after 10 months of routine use. These changes could generally be reproduced by heat annealing at
`temperatures around Tc. Furthermore, pulse NMR measurements indicated that increased crystallinity results
`to the hardening of EVA materials, which may lead to a loss of the MGs’ protective ability. Generally, MGs are
`used at around body temperature (37 °C), which is lower than the Tc of EVA28 (48 °C); however, repeated cycles
`of temperature fluctuations between ambient and body temperatures could result in increased crystallinity and
`hardening because melting of this material starts at around 30 °C. In addition, EVA MGs should not be stored
`around or above Tc, even for a short period of time.
`Repeated mechanical compression (6.0 MPa, 10,000 times) also increased the crystallinity of EVA28 films,
`although the effect was not as large as the effect observed in the solid-state 13C NMR spectra of annealed samples.
`Finally, we verified the effect of repeated pressure changes using used EVA material, i.e. Drufosoft® (6.0 MPa,
`5,000 times) and observed decreased compressive strain versus compressive stress in the compressed film.
`Our experimental data indicate that both temperature fluctuations and repeated pressure changes may affect
`the protective ability of EVA MGs, and we therefore suggest that changes in the crystallinity of EVA during
`routine use become one of the key scientific indicators of MG deterioration. Ideally, these changes should be
`measured by non-destructive methods, for e.g., using magnetic resonance imaging (MRI) apparatus, while the
`routinely used MGs were cut into pieces and analysed. However, MRI cannot be applied till now owing to techni-
`cal limitations in resolution and signal/noise ratios33. Further development of NMR apparatus and/or application
`of other spectroscopic methodologies, such as terahertz (THz) imaging34,35, will enable non-destructive detection
`of the crystallinity of EVA MGs.
`Experimental Procedure
`Materials. Clear-transparent Drufosoft® Type SQ EVA with a 3 mm thickness (Dreve-Dentamid GmbH,
`Unna, Germany) was used as the MG material. EVA pellets with various vinyl acetate (VA) contents, viz., 9, 14
`and 28% VA, were purchased from Scientific Polymer Products, Inc. (NY, USA). In this study, films prepared from
`those pellets were named EVA9, EVA14 and EVA28, respectively.
`
`Preparations of MGs and EVA films. Using a dental pressure laminate machine, Drufomat SQ
`(Dreve-Dentamid GmbH, Unna, Germany), eight MGs were prepared from respective dental casts, which were
`formed from eight high school rugby players (also referred to as ‘users’). The excess portions of the respective MG
`materials (MGϕ x), which were obtained after the lamination and subsequent trimming of MGs, were preserved
`at room temperature as controls, whereas the prepared MGs (MGx) were used for one season (10 months), where
`x = user number (Tables 1 and 2). Methods were conducted according to the relevant guidelines and regulations.
`Informed consent was obtained from all the participants. The implementation plan of this study was approved by
`the Ethics Committee of Tokyo Dental College (Ethical Clearance No. 437).
`EVA films, i.e. EVA9, EVA14 and EVA28, were prepared from the EVA pellets using a compression-molding
`machine, Table-type-test press SA-303-I-S (Tester Sangyo Co Ltd., Saitama, Japan). The pellets underwent com-
`pression molding at 230 °C and 45 MPa for 10 min followed by quick quenching in ice-cold water to yield the
`unannealed EVA films with a thickness of 0.30 or 0.030 cm. Annealed EVA films were prepared by reheating the
`unannealed films at 60, 80 or 100 °C for 60 min and subsequent quick quenching in ice-cold water. To confirm
`reproducibility, preparation and measurement of EVA films were performed in three independent experiments.
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`13C NMR measurements. The solution 13C NMR and the solid-state CP/MAS 13C NMR spectra were
`recorded on a Bruker AVANCE III spectrometer (Bruker BioSpin K.K., Kanagawa, Japan). The solution 13C NMR
`spectra were recorded at 75 MHz after samples were dissolved by soaking in chloroform overnight. Once the sam-
`ples are dissolved, the crystalline phase is no longer retained. Therefore, the spectra can only reflect the changes
`in chemical structure but not those in polymer morphologies. For the solid-state CP/MAS 13C NMR, all samples
`were packed in a zirconia rotor with a 4 mm diameter and spun at 4 kHz in the instrument. The contact and rep-
`etition times were set at 2 ms and 5 s, respectively.
`
`Pulse NMR measurements. Pulse NMR measurements were made on a JEOL MU-25 spectrometer (JEOL
`Ltd., Tokyo, Japan). A solid-echo pulse sequence provided free induction decay curves, which were fitted using a
`hybrid of exponential and Gaussian functions (Figure S1)29, and in this way fraction ratios and T2 values could be
`determined (Tables 2 and S1).
`
`DSC measurements. DSC measurements were conducted on a Diamond DSC instrument (PerkinElmer
`Japan Co., Ltd., Kanagawa, Japan) calibrated with indium and tin standards36,37. The DSC scans were performed
`under a nitrogen atmosphere over a temperature range from 0 to 150 °C at a heating rate of 10 °C/min.
`Repeated thermal cycle experiments. Drufosoft® films (thickness = 0.30 cm) were obtained by cutting
`the top face of box-type MG imitations (Figure S4), which were prepared as for the MGs using Drufomat SQ with
`a rectangular plaster cast instead of a cast constructed from a user. DSC curves were measured for the Drufosoft®
`films with or without treatment by repeated thermal cycles (100 times) using a TC-312 thermal cycler (Techne
`Ltd., Cambridge, UK)38–40 (Fig. 3). Some samples were set into the thermal cycler (Figure S5) followed by thermal
`treatment with two different protocols (repeated temperature fluctuations between 25 °C for 60 min and 37 °C for
`60 min, and between 6 °C for 60 min and 22 °C for 60 min) (Figure S3) prior to DSC measurements; the others
`were analysed by DSC without the thermal treatment.
`
`Repeated compression experiments. Two types of unannealed EVA28 films (length, width and thick-
`ness = 1.2 cm, 1.2 cm and 0.30 cm, respectively) underwent 10,000 repeated cycles of compression and release
`with 864 N of force (i.e. 6.0 MPa) using a Strograph E3-L (Toyo Seiki Seisaku-sho, Ltd., Tokyo, Japan) at room
`temperature. The first type of EVA film was prepared in compliance with the above-mentioned procedure to yield
`the EVA28_1 film. The second film type was prepared as follows: EVA28 pellets underwent compression molding
`at 230 °C and 45 MPa for 10 min followed by gradual overnight cooling to 25 °C in an incubator, M-230FN (Taitec
`Corporation, Saitama, Japan), to yield the EVA28_2 film. All samples were analysed by solid-state CP/MAS 13C
`NMR measurements.
`
`Compressive stress–strain measurements. Using a Strograph E3-L, compressive stress–strain curves
`were measured for Drufosoft® films (length, width and thickness = 0.5 cm, 0.5 cm and 0.30 cm, respectively)
`before and after repeated cycles (5,000 times) of compression and release at 150 N (i.e. 6.0 MPa) and at room
`temperature. Drufosoft® films were prepared as for the MGs using Drufomat SQ but using a rectangular plaster
`cast instead of a cast formed from a user.
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`Acknowledgements
`Financial supports from the Japan Society for the Promotion of Science (Grants 25463024, 25463025 and
`26288098) are gratefully acknowledged. The authors thank Katsushi Katano, DDS; and Fumio Yarita, DDS for
`their generous assistance in dental clinical works.
`Author Contributions
`R.K. performed most of the analytical experiments, processed the data, and drafted the manuscript. R.T. and N.O.
`contributed to the collection and assembly of the data and H.U. contributed to the analysis and interpretation of
`the data. K.N. and T.T. implemented the dental clinical work and data interpretation. T.Y. set up the project and
`led the research process. All authors have discussed the results and approved the manuscript.
`Additional Information
`Supplementary information accompanies this paper at http://www.nature.com/srep
`Competing Interests: The authors declare no competing financial interests.
`How to cite this article: Kuwahara, R. et al. Crystallization and hardening of poly(ethylene-co-