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Journal of Orthodontics, Vol. 41, 2014, 30–37
Comparison of residual monomer loss from cold-cure orthodontic acrylic resins processed by different polymerization techniques Tahereh Hosseinzadeh Nik1, Atefe Saffar Shahroudi2, Zeinab Eraghihzadeh3 and Farzaneh Aghajani4 1
Department of Orthodontics, School of Dentistry, Tehran University of Medical Sciences, Tehran, Iran; 2Department of Orthodontics, School of Dentistry, Lorestan University of Medical Sciences, Khoram-abad, Iran; 3Private Practice; 4Department of Dental Biomaterials, Faculty of Dentistry & Dental Research Center, Tehran University of Medical Sciences, Tehran, Iran
Introduction: This investigation aimed to assess and compare the amount of residual monomer (RM) released from removable orthodontic appliances constructed by sprinkle-on and dough techniques. Materials and methods: One hundred and twenty acrylic samples were prepared from orthodontic autopolymerized acrylic resins and divided into three groups, according to the processing method: sprinkle-on with polyclave, sprinkle-on without polyclave and dough technique. After polymerization, the specimens of each group were immersed in distilled water for 24 h, 48 h, 72 h and 1 week. High-performances liquid chromatography (HPLC) was utilized to measure residual monomer content. Results: Maximum observed RM was 1284.91¡129.07 ppm measured for sprinkle-on technique without polyclave after 24 h of water immersion. At this time, the level of RM was significantly different among the three applied techniques (P,0.05). In all soaking time groups, sprinkle-on technique with polyclave released the least amount of RM. Within each group, the maximum monomer releasing was observed after the first 24 h and decreases were observed in subsequent time groups. The reduction over the time was not significant in the polyclave groups (P.0.05). Conclusion: The sprinkle-on technique with polyclave and longer water immersion reduced residual monomer released from acrylic orthodontic appliances. Key words: Acrylic resins, cold-cure, polymerization technique, residual monomer Received 23 April 2013; accepted 6 July 2013
Introduction Acrylic resins (ARs) have been applied in dentistry since 1946, mostly as a denture base material and commonly consisting of methacrylates, especially methyl methacrylate (MMA) (Price, 1994). Owing to their acceptable physiological and chemical characteristics as well as easy handling, good aesthetic and low cost, these resins have been considered as a suitable material to use in the oral environment and have been widely applied (Faltermeier et al., 2007). Poly methyl methacrylate (PMMA) resins have been used widely in orthodontics for the construction of removable appliances; such as retainers and functional appliances or auxiliary fixed appliances. They polymerize by a free radical addition polymerization mechanism than involves the stages of activation, initiation, propagation and termination. Activation may be by one of the heatcure, cold-cure, light-cure or microwave polymerized methods (Jagger, 1978). With a few exceptions, resins applied in orthodontics are cold-cure which are manipulated by one of two different techniques: the dough (mass Address for correspondence: F. Aghajani Department of Dental Biomaterials, Faculty of Dentistry & Dental Research Center, Tehran University of Medical Sciences, Tehran, Iran. Email: [email protected] # 2014 British Orthodontic Society
technique) and sprinkle-on technique (additional technique). Comparatively, the former method is sometimes more preferable since in additional technique, control of the acrylic thickness is compromised (Faltermeier et al., 2007). Generally, the polymerization reaction is incomplete in polymers which is influenced by polymerization properties and conditions. The degree of conversion in cold-cure acrylic resins is lower than heat-cure ones, which means a higher unreacted monomer content is found in cold-cure resins (Kalipc¸ilar et al., 1991; Vallittu et al., 1998; Bartoloni et al., 2000). The uncombined residual monomers (RMs) alter the polymer molecular weight and exert deleterious effects on the mechanical and biological properties of the final product. They act as plasticizers and diminish compressive strength of the material (Dogan et al., 1995; Arab et al., 1989), and also reduce biocompatibility leading to an increase in the probability of an allergic reaction (Auzerie et al., 2001; Mikai et al., 2006), including erythema, oedema, urticaria, burning sensation and pain
DOI 10.1179/1465313313Y.0000000078
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(Lunder, 2000; Koutis and Freeman, 2001). Necrosis and hypersalivation have also been reported (Stungis and Fink, 1969; Goncalvers et al., 2006). The cytotoxicity of MMA has been proved (Tsuchiya et al., 1994; Kedjarune et al., 1999; Huang et al., 2001) and its genotoxicity is also open to dispute (Gigola et al., 2001). Kedjarune et al. (1999) and Tsuchiya et al. (1994) found MMA released into saliva to be toxic in human oral fibroblast cell culture. In an in vitro study, Huang et al. (2001) reported that the eluates from denture base resins were cytotoxic to human oral epithelial cell line and primary human oral fibroblasts. The standard level of RM of auto-polymerized AR was lower than 3.5% (Harrison and Huggett, 1992). The British Standard Specification for Orthodontic Resins (BS 6747:1987) specifies an upper limit not more than 3.5% by mass of methyl methacrylate monomer (Harrison and Huggett, 1992). However, since none of the applied techniques could meet this, 5% was established by ISO 207952:2010. However, it was reported that RM as low as 0.5% could cause hypersensitivity of clinicians’ hands and dermatitis due to prolonged skin contact (Estlander et al., 1984). In orthodontics, applying a cold-cure method with a higher RM level may put patients at higher risk of developing biologic adverse effects. More importantly, the majority of orthodontic patients are children or adolescents and the time of contact with the acrylic appliances is long since they should be worn nocturnally too. The long duration of wearing removable appliances and the low age range of patients who wear these exacerbate the complications of PMMA cytotoxicity in orthodontic patients (Rose et al., 2000; Goncalvers et al., 2006). Nevertheless, little clinical data on the orthodontic acryl are available. To reduce RM and consequently cytotoxicity of the of AR, some methodological modifications have been suggested, such as processing the acrylic in hot water under high pressure using a polyclave (Lee et al., 2002; Bayraktar et al., 2006) and microwave irradiation (Patil et al., 2009). Gonc¸aves et al. (2008) have also concluded that mechanical polishing was associated with lower levels of RM. Although the comparison of cold-cure and heatcure acrylic resins has been widely investigated in the literature, the differences between various auto-polymerization techniques have not received much attention. Thus, the objective of this investigation was to assess the RM from removable orthodontic appliances constructed by two conventional techniques (sprinkle-on and dough technique) using high-performance liquid chromatography (HPLC). In addition, the effects of soaking processed acrylic resin in distilled water on reduction of RM level were evaluated.
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Material and methods In this study, one brand of orthodontic cold-cure acrylic resin (Orthocryl, Dentaurum, Germany) was tested in vitro. The powder was mainly consisted of PMMA and the liquid contained MMA monomer. In order to obtain an appropriate acrylic specimen size, representative of clinical practice the mean area and thickness of five removable orthodontic retainers, which were randomly selected, were measured and stainless steel moulds of the same size were prepared (3064062.5 mm3). Forty specimens were made by the dough technique and another 80 by the sprinkle-on method, following the manufacturer’s instructions. For the dough technique, powder and liquid were mixed in a glass cup with a ratio of 2.5 g powder to 1 ml liquid as recommended by the manufacturer. After reaching the dough stage (about 4– 5 min), the specimens were packed in the moulds at 20¡2uC temperature. For the sprinkle-on technique, powder was sprinkled into the mould to form a thin uniform layer. Liquid was gradually added using a syringe to soak the powder entirely. Since in some orthodontic labs, polyclaves are not used routinely for the sprinkle-on technique, half of the specimens produced by this method (n540) were not placed in a polyclave, letting them polymerize in the room environment for about 15 min. The other 40 specimens were subsequently placed in a poyclave (1 l Bu¨chiglasuster polyclave reactor) and kept under a pressure of 2.2 bar in 50uC water for 25 min. Following removed from the mould each specimen was polished according to the manufacturer’s instructions and was immersed immediately in 20 ml of distilled water. In this way, three study groups were formed as summarized in Table 1. The residual monomer eluted into the distilled water was measured after 24 h, 48 h, 72 h and 1 week of storage, respectively. The water was exchanged after each time interval with the distilled water containing the unreacted monomers released from the acrylic specimens being collected into a 15 ml Falcon tube. In order to measure the released monomers, HPLC (channel: 1) was used. For the active phase, a solution of acetonitrile–water (50 : 50 volume ratio) at a flow rate of 1 ml/min was used. C18 or RP 18 columns with a length of 25 cm length and a particle size of 1 mm were used as the stationary phase. Residual monomer concentration was determined by measuring the absorbance at 254 nm using calibration standard solutions. These solutions were at 50, 100, 200, 400, 600,800, 1000, 1250 and 1500 ppm residual monomer concentrations. Each specimen was injected three times (100 ml each time) into the chromatographer and the mean concentration was recorded in ppm as the final monomer concentration of that specimen.
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Figure 1 Standard calibration curve
Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS for Windows, version 17) software. This comprised one-way ANOVA and Tamhane multiple comparison. The level of significance was set at P50.05.
Results Calibration curves and the regression equations representing the relation between the curve area and monomer concentration are depicted in Figures 1 and 2. Figure 3 demonstrates a sample chromatogram of a specimen which is representative of the chromatograms prepared and used in this study. Mean and standard deviations of the residual monomer concentrations of sprinkle-on without polyclave (first
method), sprinkle-on with polyclave (second method) and dough technique (third method), in different time intervals, are presented in Table 2. Overall, the maximum residual monomer was 1284.91¡129.07 ppm, which belonged to the sprinkle-on technique without polyclave after 24 h of distilled water immersion, while the sprinkleon technique with polyclave after 1 week in similar condition, released the least amount of unreacted monomer (49.73¡22.57 ppm) (Table 2). After the first 24 h, the level of RM was significantly different among all three applied technique (P,0.05). In the second follow-up (after 2 days), the amount of RM in sprinkle-on technique with polyclave was not significantly different from dough technique (P50.819). However, sprinkle-on technique without polyclave exhibited dramatically higher RM than the other two (P,0.05) (Table 3).
Table 1 Description of study groups Group name
Processing method
n
Duration of water soaking (follow-ups)
A
Sprinkle-on technique without polyclave
40
B
Sprinkle-on technique with polyclave
40
C
Dough technique
40
24 h 48 h 72 h 1 week 24 h 48 h 72 h 1 week 24 h 48 h 72 h 1 week
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Figure 2
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Residual monomer loss from cold-cure acrylic resin
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Typical chromatogram of standard monomer concentration 1000 ppm
Being immersed in distilled water for 72 h, the dough technique did not differ from the first method (P50.249), while the level of residual monomer of the second method (54.71¡19.99) was notably lower than the other two. At the end of the first week, no significant differences were observed among the three studied polymerization technique (P.0.05) (Table 3). Regarding the alteration of the unreacted monomer content with time within each study group, sprinkle-on technique without polyclave after 24 h was not greatly different from 48 h (P.0.05), but the difference among other follow-ups was significant (Table 4). The maximum
monomer releasing was observed after the first 24 h and a dramatic decrease ensued in the next 6 days of the week. No significant differences existed between any time interval results (after 24, 48 and 72 h and 1 week) for sprinkle-on technique with polyclave (P.0.05). For the dough technique, the only significant difference was reported between the first and the last follow-ups (P,0.05) (Table 4). Similar to the first method, the maximum monomer releasing was after being 24-h soaking in distilled water and during the following week, its reduction was noticeable (Table 2). A graph depicting the amount of RM released from
Figure 3 A sample chromatogram pertained to acrylic specimens prepared by dough technique after being soaked for 72 h in distilled water
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Table 2 Mean residual monomer concentration in ppm of different processing methods in various time intervals Duration of water soaking
Processing method
Mean residual monomer concentration (ppm)
Standard deviation
24 h
A B C
1284.91 97.00 450.13
129.07 58.69 180.53
48 h
A B C
927.15 265.87 360.67
287.47 247.08 198.98
72 h
A B C
547.15 54.71 411.33
103.42 19.99 180.08
1 week
A B C
161.01 49.73 74.86
60.77 22.57 22.44
A: sprinkle-on technique without polyclave; B: sprinkle-on technique with polyclave; C: dough technique.
study acrylic specimens over the time is shown in Figure 4.
Discussion The presence of unreacted monomers is the most unfavourable characteristic of polymers since they can act as a plasticizer and diminish the polymer’s strength, as well as a potential tissue irritant (Arab et al., 1989; Dogan et al., 1995). Allergic reactions which have been ascribed to releasing formaldehyde, methyl methacrylate, methacrylate acid and benzoic acid are reported too (Mikai et al., 2006). In a recent study by Kopperud et al. (2011) formaldehyde was found to leach from powder-and-liquid methacrylate-based orthodontic base-plate materials. Regarding the higher level of the aforementioned substances in cold-cure AR rather than
other types, and the wide applications of these resins in orthodontics (Goncalves et al., 2006), concern aroused about the potential hazards of unreacted monomer has fostered a desire to minimize its amount. Accordingly, this study was conducted to compare three different cold-cure processing techniques with regard to the unreacted monomer. HPLC was applied to measure the residual monomers. The effectiveness of this method in measuring RM of methacrylate has been confirmed by previous investigations (Urban et al., 2006). Statistical analysis of the results revealed that the amount of RM eluted from the samples processed by sprinkle-on or dough technique and polymerized by polyclave was significantly less than sprinkle-on technique without polyclave after 24 and 48 h of storage in water. These results were concurrent with those of Gonc¸ alves et al. (2008) who concluded
Table 3 The comparison of different cold-cure acrylic resin processing method in various time intervals, regarding the amount of residual monomer Duration of water soaking
Processing method
Processing method
Mean difference
24 h
A
B C C
1187.91* 834.78* 2353.12*
50.13 78.46 67.11
,0.001 ,0.001 0.002
B C C
661.28* 566.48* 294.79 492.43*
138.02 123.60 116.91
0.001 0.002 0.819
B 48 h
A B
72 h
A B
1 week
A B
Standard deviation
P-value
B C C
135.82 2356.61*
37.24 73.42 64.05
,0.001 0.249 0.002
B C C
111.28 86.15 225.13
24.31 24.30 11.25
0.312 0.919 0.122
*Significant difference (P,0.05). A: sprinkle-on technique without polyclave, B: sprinkle-on technique with polyclave, C: dough technique.
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Residual monomer loss from cold-cure acrylic resin
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Figure 4 The amount of RM released from study acrylic specimens in ppm (vertical axis) over the time. A1: sprinkle-on technique without polyclave after 24 h water soaking; A2: sprinkle-on technique without polyclave after 48 hr water soaking; A3: sprinkle-on technique without polyclave after 72-h water soaking; A4: sprinkle-on technique without polyclave after 1-week water soaking; B1: sprinkle-on technique with polyclave after 24-h water soaking; B2: sprinkle-on technique with polyclave after 48-h water soaking; B3: sprinkle-on technique with polyclave after 72-h water soaking; B4: sprinkle-on technique with polyclave after 1-week water soaking; C1: dough technique after 24-h water soaking; C2: dough technique after 48-h water soaking; C3: dough technique after 72-h water soaking; C4: dough technique after 1-week water soaking
less RM was found where an additional technique rather than a mass one was used to prepare the specimens. However, longer follow-ups were not carried out in that study. Table 4
Effect of temperature and pressure on the degree of polymerization There have been several reports in the literature regarding the effects of temperature and pressure on
Comparison of the residual monomer content in different time intervals within each study groups
Study groups
Duration of water soaking
Duration of water soaking
Mean difference
Standard deviation
P-value
A
24 h
48 h 72 h 1 week
357.756 737.758* 1123.89*
111.41 58.47 51.89
0.057 ,0.001 ,0.001
48 h
72 h 1 week
380.00* 766.14*
108.01 104.20
0.040 0.001
B
C
72 h
1 week
43.18
,0.001
24 h
48 h 72 h 1 week
2168.87 42.28 48.27
386.13*
95.66 21.92 22.23
0.547 0.422 0.320
48 h
72 h 1 week
211.15 216.14
93.65 93.72
0.330 0.310
72 h
1 week
4.98
10.66
0.988
24 h
48 h 72 h 1 week
94.98 90.15 64.31
0.933 0.999 0.043
48 h
72 h 1 week
250.66 285.81
94.88 70.79
0.996 0.281
72 h
1 week
336.47
54.16
0.699
89.45 38.79 375.26*
*Significant difference (P,0.05). A: sprinkle-on technique without polyclave; B: sprinkle-on technique with polyclave; C: dough technique.
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the polymerization degree of acrylic resins. Lee et al. (2002) concluded that curing in temperatures higher than 50uC would impart an 80% reduction in RM and a 50% increase in surface hardness of auto-polymerized AR. Raising polymerization temperature from 30 to 60uC resulted in a 1.3% decrease in RM concentration (Vallittu et al., 1998). Similarly, in the current study, the applied pressure and heat produced by polyclave significantly decreased the level of eluted RM (1284.91¡ 129 ppm in sprinkle-on technique without polyclave compared to 97.00¡59 ppm in sprinkle-on technique with polyclave) (Table 2). The cold-cure AR strength is also influenced by heat. Transverse strength and transverse module in 60–80uC has been reported to increase to twice the amount at 32uC (Ogawa et al., 2000). Overall, temperature and pressure greatly influence the amount of RM and mechanical properties of AR. However, according to Lee et al. (2002), in spite of a strong relationship between heat and RM, pressure alone is not effective by itself. The maximum amount of detected RM was reported after 24 h of storage in water ensued by a progressive reduction until the first week on which no significant difference existed among study groups. According to Stafford and Brooks(1985), this fall in the levels of residual monomer of the orthodontic resins in time occurs by two mechanisms: (1) the continued polymerization of the monomer and (2) the leaching out of the monomer. The leaching-out mechanism has little contribution and is probably the effect of surface monomer being released. Subsequent leaching out would be a very slow process (Stafford and Brooks, 1985). Effect of water and air on polymerization Being exposed to air, polymerization of AR would be inhibited by oxygen, which in turn manifested as no reduction in RM even after 40 days. On the other hand, water inhibits O2 absorption resulting in a gradual reduction of RM with time. Accordingly, it is suggested to store acrylic orthodontic appliances in water during the polymerization process and also to soak them in water overnight before being fitted, which causes most of the leachable monomer to get released (Stafford and Brooks, 1985; Urban et al., 2009). In the sprinkle-on technique with polyclave, the amounts of RM were not significantly different at 24, 48, 72 h and 1 week, although it was observed that the result at 48 h differed from others regarding the mean concentration of RM (Figure 4). It seems that the high level of standard deviation is the reason of this disparity. General variations of AR and lack of sufficient control on the amount of monomer in sprinkle-on technique may have contributed to this high standard deviation. On the other hand, when
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the polyclave was not applied, the differences between all subsequent results were significant. This means that the longer a specimen was stored in water, the less monomer was detectable. However, even after a week of water immersion, the released RM (161.01 ppm) was still higher than the first follow-up of sprinkle-on technique with polyclave (97.00 ppm) (Table 2). Therefore, it can be concluded that storing the acrylic appliances in water, no matter how long, will not compensate for drawbacks of avoiding polyclave. With the dough technique, the only significant difference exists between 24 h and 1 week follow-ups. High level of standard deviation was a contributing factor. Moreover, practical errors during measurement of RM could adversely affect the results. The level of RM leached into water was lower in sprinkle-on technique with polyclave than dough technique in all follow-ups, especially in 24 and 72 h. Consequently, it is preferable to apply sprinkle-on technique rather than dough technique in constructing acrylic orthodontic appliances to minimize RM. However, in case that polyclave is not available, one had better apply dough technique rather than sprinkle-on (Table 2). Kleinsasser et al. (2006) suggested that RM leaches into saliva and distributes in pulp, mucosa and salivary garlands. They also claimed tumorigenic characteristics for methacrylates. In the study of Rose et al. (2000), coldcure orthodontic AR (with 1034.9 mg/cm3 or with 930.9 mg/cm3 RM) was considered slightly cytotoxic, while heat-cure acryl (with 45.4¡3.4 mg/cm3 RM) was considered as nontoxic. Another study reported RM as low as 933 mg/cm3 as cytotoxic and genotoxic (Yang et al., 2003). In the current study, the minimum detected RM observed after 1 week, was 74.86 ppm for dough technique and 49.73 and 161.06 ppm in sprinkle-on technique with and without polyclave, respectively. However, application of AR in spite of the high RM is inevitable. Therefore, efforts should be made to minimize it as much as possible. Along with the aforementioned methods, other considerations such as appropriate proportioning of liquid and powder and additional polymerization cycles can be beneficial. It has also been stated that mechanical polishing of acrylic appliances before application can significantly decrease RM as it reduces its leaching (Gonc¸alves et al., 2008). By contrast, high values of residual monomers have been reported with chemical polishing (Nunes de Mello et al., 2003). Not to be lost in this discussion, some fluctuations were observed between samples of this study, which may be due to inherent variations in preparation technique. Comparison of different commercially available AR regarding the amount of RM or comparing mechanical properties of the aforementioned techniques is suggested
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for further studies. And since this study was conducted in vitro, designing studies in oral environment can lead to more realistic results. Conclusions 1. All of the applied methods (sprinkle-on technique with polyclave, sprinkle-on technique without polyclave and dough technique) showed relatively high levels of residual monomer. 2. Among the three applied techniques, sprinkle-on technique with polyclave demonstrated the lowest level of residual monomer. 3. The percentages of residual monomer were reduced after 24 h of water storage, which was more noticeable for those methods with higher initial unreacted monomer content.
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Journal of Orthodontics, Vol. 41, 2014, 30–37
Comparison of residual monomer loss from cold-cure orthodontic acrylic resins processed by different polymerization techniques Tahereh Hosseinzadeh Nik1, Atefe Saffar Shahroudi2, Zeinab Eraghihzadeh3 and Farzaneh Aghajani4 1
Department of Orthodontics, School of Dentistry, Tehran University of Medical Sciences, Tehran, Iran; 2Department of Orthodontics, School of Dentistry, Lorestan University of Medical Sciences, Khoram-abad, Iran; 3Private Practice; 4Department of Dental Biomaterials, Faculty of Dentistry & Dental Research Center, Tehran University of Medical Sciences, Tehran, Iran
Introduction: This investigation aimed to assess and compare the amount of residual monomer (RM) released from removable orthodontic appliances constructed by sprinkle-on and dough techniques. Materials and methods: One hundred and twenty acrylic samples were prepared from orthodontic autopolymerized acrylic resins and divided into three groups, according to the processing method: sprinkle-on with polyclave, sprinkle-on without polyclave and dough technique. After polymerization, the specimens of each group were immersed in distilled water for 24 h, 48 h, 72 h and 1 week. High-performances liquid chromatography (HPLC) was utilized to measure residual monomer content. Results: Maximum observed RM was 1284.91¡129.07 ppm measured for sprinkle-on technique without polyclave after 24 h of water immersion. At this time, the level of RM was significantly different among the three applied techniques (P,0.05). In all soaking time groups, sprinkle-on technique with polyclave released the least amount of RM. Within each group, the maximum monomer releasing was observed after the first 24 h and decreases were observed in subsequent time groups. The reduction over the time was not significant in the polyclave groups (P.0.05). Conclusion: The sprinkle-on technique with polyclave and longer water immersion reduced residual monomer released from acrylic orthodontic appliances. Key words: Acrylic resins, cold-cure, polymerization technique, residual monomer Received 23 April 2013; accepted 6 July 2013
Introduction Acrylic resins (ARs) have been applied in dentistry since 1946, mostly as a denture base material and commonly consisting of methacrylates, especially methyl methacrylate (MMA) (Price, 1994). Owing to their acceptable physiological and chemical characteristics as well as easy handling, good aesthetic and low cost, these resins have been considered as a suitable material to use in the oral environment and have been widely applied (Faltermeier et al., 2007). Poly methyl methacrylate (PMMA) resins have been used widely in orthodontics for the construction of removable appliances; such as retainers and functional appliances or auxiliary fixed appliances. They polymerize by a free radical addition polymerization mechanism than involves the stages of activation, initiation, propagation and termination. Activation may be by one of the heatcure, cold-cure, light-cure or microwave polymerized methods (Jagger, 1978). With a few exceptions, resins applied in orthodontics are cold-cure which are manipulated by one of two different techniques: the dough (mass Address for correspondence: F. Aghajani Department of Dental Biomaterials, Faculty of Dentistry & Dental Research Center, Tehran University of Medical Sciences, Tehran, Iran. Email: [email protected] # 2014 British Orthodontic Society
technique) and sprinkle-on technique (additional technique). Comparatively, the former method is sometimes more preferable since in additional technique, control of the acrylic thickness is compromised (Faltermeier et al., 2007). Generally, the polymerization reaction is incomplete in polymers which is influenced by polymerization properties and conditions. The degree of conversion in cold-cure acrylic resins is lower than heat-cure ones, which means a higher unreacted monomer content is found in cold-cure resins (Kalipc¸ilar et al., 1991; Vallittu et al., 1998; Bartoloni et al., 2000). The uncombined residual monomers (RMs) alter the polymer molecular weight and exert deleterious effects on the mechanical and biological properties of the final product. They act as plasticizers and diminish compressive strength of the material (Dogan et al., 1995; Arab et al., 1989), and also reduce biocompatibility leading to an increase in the probability of an allergic reaction (Auzerie et al., 2001; Mikai et al., 2006), including erythema, oedema, urticaria, burning sensation and pain
DOI 10.1179/1465313313Y.0000000078
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(Lunder, 2000; Koutis and Freeman, 2001). Necrosis and hypersalivation have also been reported (Stungis and Fink, 1969; Goncalvers et al., 2006). The cytotoxicity of MMA has been proved (Tsuchiya et al., 1994; Kedjarune et al., 1999; Huang et al., 2001) and its genotoxicity is also open to dispute (Gigola et al., 2001). Kedjarune et al. (1999) and Tsuchiya et al. (1994) found MMA released into saliva to be toxic in human oral fibroblast cell culture. In an in vitro study, Huang et al. (2001) reported that the eluates from denture base resins were cytotoxic to human oral epithelial cell line and primary human oral fibroblasts. The standard level of RM of auto-polymerized AR was lower than 3.5% (Harrison and Huggett, 1992). The British Standard Specification for Orthodontic Resins (BS 6747:1987) specifies an upper limit not more than 3.5% by mass of methyl methacrylate monomer (Harrison and Huggett, 1992). However, since none of the applied techniques could meet this, 5% was established by ISO 207952:2010. However, it was reported that RM as low as 0.5% could cause hypersensitivity of clinicians’ hands and dermatitis due to prolonged skin contact (Estlander et al., 1984). In orthodontics, applying a cold-cure method with a higher RM level may put patients at higher risk of developing biologic adverse effects. More importantly, the majority of orthodontic patients are children or adolescents and the time of contact with the acrylic appliances is long since they should be worn nocturnally too. The long duration of wearing removable appliances and the low age range of patients who wear these exacerbate the complications of PMMA cytotoxicity in orthodontic patients (Rose et al., 2000; Goncalvers et al., 2006). Nevertheless, little clinical data on the orthodontic acryl are available. To reduce RM and consequently cytotoxicity of the of AR, some methodological modifications have been suggested, such as processing the acrylic in hot water under high pressure using a polyclave (Lee et al., 2002; Bayraktar et al., 2006) and microwave irradiation (Patil et al., 2009). Gonc¸aves et al. (2008) have also concluded that mechanical polishing was associated with lower levels of RM. Although the comparison of cold-cure and heatcure acrylic resins has been widely investigated in the literature, the differences between various auto-polymerization techniques have not received much attention. Thus, the objective of this investigation was to assess the RM from removable orthodontic appliances constructed by two conventional techniques (sprinkle-on and dough technique) using high-performance liquid chromatography (HPLC). In addition, the effects of soaking processed acrylic resin in distilled water on reduction of RM level were evaluated.
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Material and methods In this study, one brand of orthodontic cold-cure acrylic resin (Orthocryl, Dentaurum, Germany) was tested in vitro. The powder was mainly consisted of PMMA and the liquid contained MMA monomer. In order to obtain an appropriate acrylic specimen size, representative of clinical practice the mean area and thickness of five removable orthodontic retainers, which were randomly selected, were measured and stainless steel moulds of the same size were prepared (3064062.5 mm3). Forty specimens were made by the dough technique and another 80 by the sprinkle-on method, following the manufacturer’s instructions. For the dough technique, powder and liquid were mixed in a glass cup with a ratio of 2.5 g powder to 1 ml liquid as recommended by the manufacturer. After reaching the dough stage (about 4– 5 min), the specimens were packed in the moulds at 20¡2uC temperature. For the sprinkle-on technique, powder was sprinkled into the mould to form a thin uniform layer. Liquid was gradually added using a syringe to soak the powder entirely. Since in some orthodontic labs, polyclaves are not used routinely for the sprinkle-on technique, half of the specimens produced by this method (n540) were not placed in a polyclave, letting them polymerize in the room environment for about 15 min. The other 40 specimens were subsequently placed in a poyclave (1 l Bu¨chiglasuster polyclave reactor) and kept under a pressure of 2.2 bar in 50uC water for 25 min. Following removed from the mould each specimen was polished according to the manufacturer’s instructions and was immersed immediately in 20 ml of distilled water. In this way, three study groups were formed as summarized in Table 1. The residual monomer eluted into the distilled water was measured after 24 h, 48 h, 72 h and 1 week of storage, respectively. The water was exchanged after each time interval with the distilled water containing the unreacted monomers released from the acrylic specimens being collected into a 15 ml Falcon tube. In order to measure the released monomers, HPLC (channel: 1) was used. For the active phase, a solution of acetonitrile–water (50 : 50 volume ratio) at a flow rate of 1 ml/min was used. C18 or RP 18 columns with a length of 25 cm length and a particle size of 1 mm were used as the stationary phase. Residual monomer concentration was determined by measuring the absorbance at 254 nm using calibration standard solutions. These solutions were at 50, 100, 200, 400, 600,800, 1000, 1250 and 1500 ppm residual monomer concentrations. Each specimen was injected three times (100 ml each time) into the chromatographer and the mean concentration was recorded in ppm as the final monomer concentration of that specimen.
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Figure 1 Standard calibration curve
Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS for Windows, version 17) software. This comprised one-way ANOVA and Tamhane multiple comparison. The level of significance was set at P50.05.
Results Calibration curves and the regression equations representing the relation between the curve area and monomer concentration are depicted in Figures 1 and 2. Figure 3 demonstrates a sample chromatogram of a specimen which is representative of the chromatograms prepared and used in this study. Mean and standard deviations of the residual monomer concentrations of sprinkle-on without polyclave (first
method), sprinkle-on with polyclave (second method) and dough technique (third method), in different time intervals, are presented in Table 2. Overall, the maximum residual monomer was 1284.91¡129.07 ppm, which belonged to the sprinkle-on technique without polyclave after 24 h of distilled water immersion, while the sprinkleon technique with polyclave after 1 week in similar condition, released the least amount of unreacted monomer (49.73¡22.57 ppm) (Table 2). After the first 24 h, the level of RM was significantly different among all three applied technique (P,0.05). In the second follow-up (after 2 days), the amount of RM in sprinkle-on technique with polyclave was not significantly different from dough technique (P50.819). However, sprinkle-on technique without polyclave exhibited dramatically higher RM than the other two (P,0.05) (Table 3).
Table 1 Description of study groups Group name
Processing method
n
Duration of water soaking (follow-ups)
A
Sprinkle-on technique without polyclave
40
B
Sprinkle-on technique with polyclave
40
C
Dough technique
40
24 h 48 h 72 h 1 week 24 h 48 h 72 h 1 week 24 h 48 h 72 h 1 week
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Figure 2
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Residual monomer loss from cold-cure acrylic resin
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Typical chromatogram of standard monomer concentration 1000 ppm
Being immersed in distilled water for 72 h, the dough technique did not differ from the first method (P50.249), while the level of residual monomer of the second method (54.71¡19.99) was notably lower than the other two. At the end of the first week, no significant differences were observed among the three studied polymerization technique (P.0.05) (Table 3). Regarding the alteration of the unreacted monomer content with time within each study group, sprinkle-on technique without polyclave after 24 h was not greatly different from 48 h (P.0.05), but the difference among other follow-ups was significant (Table 4). The maximum
monomer releasing was observed after the first 24 h and a dramatic decrease ensued in the next 6 days of the week. No significant differences existed between any time interval results (after 24, 48 and 72 h and 1 week) for sprinkle-on technique with polyclave (P.0.05). For the dough technique, the only significant difference was reported between the first and the last follow-ups (P,0.05) (Table 4). Similar to the first method, the maximum monomer releasing was after being 24-h soaking in distilled water and during the following week, its reduction was noticeable (Table 2). A graph depicting the amount of RM released from
Figure 3 A sample chromatogram pertained to acrylic specimens prepared by dough technique after being soaked for 72 h in distilled water
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Table 2 Mean residual monomer concentration in ppm of different processing methods in various time intervals Duration of water soaking
Processing method
Mean residual monomer concentration (ppm)
Standard deviation
24 h
A B C
1284.91 97.00 450.13
129.07 58.69 180.53
48 h
A B C
927.15 265.87 360.67
287.47 247.08 198.98
72 h
A B C
547.15 54.71 411.33
103.42 19.99 180.08
1 week
A B C
161.01 49.73 74.86
60.77 22.57 22.44
A: sprinkle-on technique without polyclave; B: sprinkle-on technique with polyclave; C: dough technique.
study acrylic specimens over the time is shown in Figure 4.
Discussion The presence of unreacted monomers is the most unfavourable characteristic of polymers since they can act as a plasticizer and diminish the polymer’s strength, as well as a potential tissue irritant (Arab et al., 1989; Dogan et al., 1995). Allergic reactions which have been ascribed to releasing formaldehyde, methyl methacrylate, methacrylate acid and benzoic acid are reported too (Mikai et al., 2006). In a recent study by Kopperud et al. (2011) formaldehyde was found to leach from powder-and-liquid methacrylate-based orthodontic base-plate materials. Regarding the higher level of the aforementioned substances in cold-cure AR rather than
other types, and the wide applications of these resins in orthodontics (Goncalves et al., 2006), concern aroused about the potential hazards of unreacted monomer has fostered a desire to minimize its amount. Accordingly, this study was conducted to compare three different cold-cure processing techniques with regard to the unreacted monomer. HPLC was applied to measure the residual monomers. The effectiveness of this method in measuring RM of methacrylate has been confirmed by previous investigations (Urban et al., 2006). Statistical analysis of the results revealed that the amount of RM eluted from the samples processed by sprinkle-on or dough technique and polymerized by polyclave was significantly less than sprinkle-on technique without polyclave after 24 and 48 h of storage in water. These results were concurrent with those of Gonc¸ alves et al. (2008) who concluded
Table 3 The comparison of different cold-cure acrylic resin processing method in various time intervals, regarding the amount of residual monomer Duration of water soaking
Processing method
Processing method
Mean difference
24 h
A
B C C
1187.91* 834.78* 2353.12*
50.13 78.46 67.11
,0.001 ,0.001 0.002
B C C
661.28* 566.48* 294.79 492.43*
138.02 123.60 116.91
0.001 0.002 0.819
B 48 h
A B
72 h
A B
1 week
A B
Standard deviation
P-value
B C C
135.82 2356.61*
37.24 73.42 64.05
,0.001 0.249 0.002
B C C
111.28 86.15 225.13
24.31 24.30 11.25
0.312 0.919 0.122
*Significant difference (P,0.05). A: sprinkle-on technique without polyclave, B: sprinkle-on technique with polyclave, C: dough technique.
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Residual monomer loss from cold-cure acrylic resin
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Figure 4 The amount of RM released from study acrylic specimens in ppm (vertical axis) over the time. A1: sprinkle-on technique without polyclave after 24 h water soaking; A2: sprinkle-on technique without polyclave after 48 hr water soaking; A3: sprinkle-on technique without polyclave after 72-h water soaking; A4: sprinkle-on technique without polyclave after 1-week water soaking; B1: sprinkle-on technique with polyclave after 24-h water soaking; B2: sprinkle-on technique with polyclave after 48-h water soaking; B3: sprinkle-on technique with polyclave after 72-h water soaking; B4: sprinkle-on technique with polyclave after 1-week water soaking; C1: dough technique after 24-h water soaking; C2: dough technique after 48-h water soaking; C3: dough technique after 72-h water soaking; C4: dough technique after 1-week water soaking
less RM was found where an additional technique rather than a mass one was used to prepare the specimens. However, longer follow-ups were not carried out in that study. Table 4
Effect of temperature and pressure on the degree of polymerization There have been several reports in the literature regarding the effects of temperature and pressure on
Comparison of the residual monomer content in different time intervals within each study groups
Study groups
Duration of water soaking
Duration of water soaking
Mean difference
Standard deviation
P-value
A
24 h
48 h 72 h 1 week
357.756 737.758* 1123.89*
111.41 58.47 51.89
0.057 ,0.001 ,0.001
48 h
72 h 1 week
380.00* 766.14*
108.01 104.20
0.040 0.001
B
C
72 h
1 week
43.18
,0.001
24 h
48 h 72 h 1 week
2168.87 42.28 48.27
386.13*
95.66 21.92 22.23
0.547 0.422 0.320
48 h
72 h 1 week
211.15 216.14
93.65 93.72
0.330 0.310
72 h
1 week
4.98
10.66
0.988
24 h
48 h 72 h 1 week
94.98 90.15 64.31
0.933 0.999 0.043
48 h
72 h 1 week
250.66 285.81
94.88 70.79
0.996 0.281
72 h
1 week
336.47
54.16
0.699
89.45 38.79 375.26*
*Significant difference (P,0.05). A: sprinkle-on technique without polyclave; B: sprinkle-on technique with polyclave; C: dough technique.
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the polymerization degree of acrylic resins. Lee et al. (2002) concluded that curing in temperatures higher than 50uC would impart an 80% reduction in RM and a 50% increase in surface hardness of auto-polymerized AR. Raising polymerization temperature from 30 to 60uC resulted in a 1.3% decrease in RM concentration (Vallittu et al., 1998). Similarly, in the current study, the applied pressure and heat produced by polyclave significantly decreased the level of eluted RM (1284.91¡ 129 ppm in sprinkle-on technique without polyclave compared to 97.00¡59 ppm in sprinkle-on technique with polyclave) (Table 2). The cold-cure AR strength is also influenced by heat. Transverse strength and transverse module in 60–80uC has been reported to increase to twice the amount at 32uC (Ogawa et al., 2000). Overall, temperature and pressure greatly influence the amount of RM and mechanical properties of AR. However, according to Lee et al. (2002), in spite of a strong relationship between heat and RM, pressure alone is not effective by itself. The maximum amount of detected RM was reported after 24 h of storage in water ensued by a progressive reduction until the first week on which no significant difference existed among study groups. According to Stafford and Brooks(1985), this fall in the levels of residual monomer of the orthodontic resins in time occurs by two mechanisms: (1) the continued polymerization of the monomer and (2) the leaching out of the monomer. The leaching-out mechanism has little contribution and is probably the effect of surface monomer being released. Subsequent leaching out would be a very slow process (Stafford and Brooks, 1985). Effect of water and air on polymerization Being exposed to air, polymerization of AR would be inhibited by oxygen, which in turn manifested as no reduction in RM even after 40 days. On the other hand, water inhibits O2 absorption resulting in a gradual reduction of RM with time. Accordingly, it is suggested to store acrylic orthodontic appliances in water during the polymerization process and also to soak them in water overnight before being fitted, which causes most of the leachable monomer to get released (Stafford and Brooks, 1985; Urban et al., 2009). In the sprinkle-on technique with polyclave, the amounts of RM were not significantly different at 24, 48, 72 h and 1 week, although it was observed that the result at 48 h differed from others regarding the mean concentration of RM (Figure 4). It seems that the high level of standard deviation is the reason of this disparity. General variations of AR and lack of sufficient control on the amount of monomer in sprinkle-on technique may have contributed to this high standard deviation. On the other hand, when
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the polyclave was not applied, the differences between all subsequent results were significant. This means that the longer a specimen was stored in water, the less monomer was detectable. However, even after a week of water immersion, the released RM (161.01 ppm) was still higher than the first follow-up of sprinkle-on technique with polyclave (97.00 ppm) (Table 2). Therefore, it can be concluded that storing the acrylic appliances in water, no matter how long, will not compensate for drawbacks of avoiding polyclave. With the dough technique, the only significant difference exists between 24 h and 1 week follow-ups. High level of standard deviation was a contributing factor. Moreover, practical errors during measurement of RM could adversely affect the results. The level of RM leached into water was lower in sprinkle-on technique with polyclave than dough technique in all follow-ups, especially in 24 and 72 h. Consequently, it is preferable to apply sprinkle-on technique rather than dough technique in constructing acrylic orthodontic appliances to minimize RM. However, in case that polyclave is not available, one had better apply dough technique rather than sprinkle-on (Table 2). Kleinsasser et al. (2006) suggested that RM leaches into saliva and distributes in pulp, mucosa and salivary garlands. They also claimed tumorigenic characteristics for methacrylates. In the study of Rose et al. (2000), coldcure orthodontic AR (with 1034.9 mg/cm3 or with 930.9 mg/cm3 RM) was considered slightly cytotoxic, while heat-cure acryl (with 45.4¡3.4 mg/cm3 RM) was considered as nontoxic. Another study reported RM as low as 933 mg/cm3 as cytotoxic and genotoxic (Yang et al., 2003). In the current study, the minimum detected RM observed after 1 week, was 74.86 ppm for dough technique and 49.73 and 161.06 ppm in sprinkle-on technique with and without polyclave, respectively. However, application of AR in spite of the high RM is inevitable. Therefore, efforts should be made to minimize it as much as possible. Along with the aforementioned methods, other considerations such as appropriate proportioning of liquid and powder and additional polymerization cycles can be beneficial. It has also been stated that mechanical polishing of acrylic appliances before application can significantly decrease RM as it reduces its leaching (Gonc¸alves et al., 2008). By contrast, high values of residual monomers have been reported with chemical polishing (Nunes de Mello et al., 2003). Not to be lost in this discussion, some fluctuations were observed between samples of this study, which may be due to inherent variations in preparation technique. Comparison of different commercially available AR regarding the amount of RM or comparing mechanical properties of the aforementioned techniques is suggested
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for further studies. And since this study was conducted in vitro, designing studies in oral environment can lead to more realistic results. Conclusions 1. All of the applied methods (sprinkle-on technique with polyclave, sprinkle-on technique without polyclave and dough technique) showed relatively high levels of residual monomer. 2. Among the three applied techniques, sprinkle-on technique with polyclave demonstrated the lowest level of residual monomer. 3. The percentages of residual monomer were reduced after 24 h of water storage, which was more noticeable for those methods with higher initial unreacted monomer content.
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