Leachability of denture-base acrylic resins in artificial saliva T. Koda1. H. Tsuchiya2 M. YamauchP S. Ohtani2 N. Takagi 2 J. Kawano1
1First Department of Prosthodontics 2Department of Dental Pharmacology Asahi University School of Dentistry 1851 Hozumi, Motosu Gifu 501-02, Japan Received December 12, 1988 Accepted November 29, 1989 *Corresponding author This study was partly supported by a grant from Miyata Science Research Foundation. Dent Mater 6:13-16, January, 1990 Abstract-We studied the influence of
salivary acidity on leachability of denturebase acrylic resins with etiological interest in denture stomatitis because denture surfaces are frequently exposed to acidic conditions in the oral cavities. Auto-, heat-, and microwave-polymerized resins were immersed in artificial saliva with pH ranging from 4.0 to 6.8 at 37°C, and leachables were pursued quantitatively with time. Methyl methacrylate, methacrylic acid, and benzoic acid leached from all resins. Their concentrations in the saliva were markedly high for auto-polymerized resins, while leachability of heat- and microwave-polymerized resins was so low that quantitative analysis of leachables was impossible. Lower pH showed higher concentrations of methyl methacrylate, although no apparent association was confirmed between salivary acidity and its own leachability. The concentrations of methacrylic acid increased remarkably with an increase in pH, which was probably due to hydrolysis of methyl methacrylate. These results suggest that chemotoxic actions of auto-polymerized resins are potentially ascribable to methyl methacrylate under more acidic conditions and to methacrylic acid under less acidic conditions.
enture stomatitis caused by the wearing of acrylic dentures has been ascribed to poorly fitting dentures, unbalanced occlusion, and fungal infection with Candida strains (Budtz-JSrgensen, 1974; Olsen, 1975). In addition to these etiological factors, another possible factor is the chemotoxicity of substances present in the denture-base acrylic polymers (Bergman, 1977; Hensten-Pettersen and Wictorin, 1979; Weaver and Goebel, 1980). Since the toxic substances must leach into saliva to elicit inflammatory reactions on oral mucosa, leaching properties of the acrylic resins have been widely studied for estimation of their potential toxicity. Leachability of a residual monomer, methyl methacrylate (MMA), has been investigated on resins immersed in distilled water (Lamb et al., 1982; Stafford and Brooks, 1985; Szabo et al., 1986) and in aqueous solutions of organic solvents (Fujisawa and Masuhara, 1979; Koda et al., 1987). Dentures placed in oral cavities are continuously washed by saliva and frequently covered by plaque. Saliva and plaque are acidified by fermentative and dietary acids (Olsen and Birkeland, 1977; Imfeld et al., 1978; Harper et al., 1986), resulting in the exposure of denture surfaces to acidic environments. It is valuable, for presumption of toxic potency, for leaching experiments of the acrylic resins to be performed in immersion solutions of diverse acidity. In the p r e s e n t study, we immersed representative acrylic resins in artificial saliva of different pH (acidic to neutral) and quantitatively analyzed MMA and other leachables by high-performance liquid chromatography (HPLC). We examined whether their leaching concentrations differed in acidic and neutral saliva, and estimated an association between salivary acidity and leachability of the acrylic resins.
D
MATERIALS AND METHODS
Auto-polymerized resins (Rebaron No. 3, pink), heat-polymerized resins (Acron No. 8, live pink), and microwave-polymerized resins (AcronMC No. 8, live pink) were purchased from G-C Dental Industrial Corp. (Tokyo, Japan). They were polymerized by use of the monomer-liquid/ pre-polymer-powder ratios and the processing conditions described in the manufacturer's directions. For heat polymerization, the dough was heated at 70°C for 90 rain, and then at 100°C for 30 rain. For microwave polymerization, the dough was subjected to microwave radiation for three min. Resin disks (thickness of 2.0 mm _+ 0.2 mm and outer diameter of 8.5 mm _+ 0.2 ram) were prepared from the acrylic polymers (Koda et al., 1987). The disks were placed in Teflon ®capped vials (volume of 7.0 mL; Gasukuro Kogyo, Tokyo, Japan) containing 5.0 mL of artificial saliva prepared as described below. The vials were gently shaken at 37°C in a water-bath incubation. Aliquots of the immersion solutions were sampled through Teflone membranes of the vial caps by a micro-syringe at time intervals of one day. They were subjected to HPLC analysis immediately after sampling. Artificial saliva was prepared based on the method of Katz et al. (1986). The salivary pH was adjusted to 4.0, 5.0, 6.0, and 6.8 by the addition of an aqueous phosphoric acid solution (2.0 tool/L). MMA, methacrylic acid (M), benzoic acid (BA), and methyl acrylate (MA), used as an internal standard, were purchased from Tokyokasei (Tokyo, Japan). Their standard solutions were prepared daily by dissolution with the artificial saliva of pH 4.0. Water was distilled by all-glass apparatus after being purified by a Milli-Q water purification system (Nihon Millipore, Tokyo, Japan). All other chemicals were of analytical reagent grade.
Dental Materials/January 1990
13
The internal standard solution (20.0 or 100.0 nmoVmL, 50 ~L) was added to the immersion solution (100 ~L) sampled from the Teflon®-capped vials, and then the mixture (100 ~L) was loaded onto an HPLC column. The column (4.6 mm i.d. × 25 cm) was laboratory-packed with NC Gel C-18 (particle size, 5 ~m; Sakata, Tokyo, Japan) by the packing method of Tsuchiya et al. (1989). The separation was performed by an HPLC system similar to one used in our previous study (Koda et al., 1989). A mobile phase, 30% (v/v) acetonitrile in 0.05 mol/L sodium phosphate buffer (pH 3/0), was pumped at a flow rate of 1.0 mL/min and at a column t e m p e r a t u r e of 50°C. UV absorbance of eluates from the HPLC column Was detected at a wavelength of 210 nm. Leachables were determined based on calibration graphs, which we prepared by plotting peak height ratios of standard MMA, M, and BA to the internal standard vs. their concentrations. The calibration graphs for all substances showed good linearity in a range of from at least 0.5 to 1000.0 nmol/mL. When standard solutions and three-day immersion solutions (pH 6.8) of the auto-
A
peroxide added as an initiator (A1 Doori et al., 1988). When auto-polymerized resins were immersed in the saliva of different pH, MMA, M, and BA revealed the leaching profiles shown in Fig. 2. MMA leached into artificial saliva offered concentration ranges almost similar to those obtained from the leaching in distilled water (Koda et al., 1989). MMA concentrations increased with a decrease in the pH. At maximum leaching, however, the concentration at pH 4.0 was 1.5 times that at pH 6.8. MMA concentrations reached maximum at five to seven days after immersion, and then gradually decreased. Since the decreasing curves were steeper at the higher pH, the difference in MMA concentrations between acidic and neutral saliva became more remarkable by increases in the immersion time. M concentrations increased significantly with an increase in the pH. After 10 days of immersion, the concentration at pH 6.8 was over 10 times that at pH 4.0. However, no pH-dependent difference was observed in leaching concentrations of BA. A l t h o u g h MMA, M, and BA leached from heat- and microwave-
polymerized resins were analyzed, the coefficients of variation (n = 10) in peak height ratios to the internal standard were within 0.35% for all substances of both solutions. RESULTS AND DISCUSSION
A preliminary leaching experiment was carried out in mixed human saliva. However, insoluble substances produced during incubation of the saliva prevented the reliable HPLC analysis of leachables. Thus, the artificial saliva used by Katz et al. (1986) was used as the immersion solution after confirmation that it did not influence the HPLC analysis. Representative chromatograms of standards and auto-polymerized resins are shown in Fig. 1. Under optimized HPLC conditions, M, MA (an internal standard), BA, and MMA were separated (in that retention order) with good resolution. In addition to MMA derived from a residual monomer, peaks corresponding to M and BA were obtained from all resins. M is presumably ascribable to the hydrolytic p r o d u c t of MMA (Corkill et al., 1976), and BA to the decomposition product of benzoyl
B
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Fig. 1. High-performance liquid chromatograms obtained from a standard solution (A) and auto-potymerized acrylic resins (B-E). The standard solution [containing M (20.0 nmol/mL), BA (50.0 nmol/mL), and MMA (500.0 nmol/mL)] and the MA solution (100.0 nmol/mL) were used. The resins were immersed in artificial saliva of pH 4.0 Ior one (B) and six days (C), and pH 6.8 for one (D) and six days (E). Peaks: 1 = M, 2 = MA, 3 = BA, and 4 = MMA. The magnitude of detector attenuation is 1/8 for the 4th peak.
14
KODA et a l . / L E A C H A B I L I T Y OF A C R Y L I C R E S I N S I N S A L I V A
polymerized resins, their concentrations in the saliva were far less than those of auto-polymerized resins. Quantitative analysis of the leachability was not performable, because concentrations of all leachables were below the reliable determination range. However, chromatographic profiles indicated that MMA and M tended to leach from heat- and microwave-polymerized resins in saliv a r y - p H - and i m m e r s i o n - t i m e dependent manners similar to those observed in auto-polymerized resins. Representative results of heatpolymerized resins are shown in Fig. 3. Leaching profiles of auto-polymerized resins indicate that MMA is liable to maintain higher concentrations in the saliva under acidic conditions. The denture surfaces are frequently exposed to low pH by plaque accumulation, fermentation, and dietary consumption (Olsen and Birkeland, 1977; Imfeld et al., 1978; Harper et al., 1986). In such acidic environments of oral cavities, MMA potentially participates in the cause of mucosal damage by the wearing of acrylic dentures. After maximum leaching, MMA concentrations declined, depending on an increase of salivary pH. Disappearance of MMA in the saliva may
M M A
be ascribed to surface volatilization, oxidation, or hydrolysis. The Teflone-capped vials were used in the present study to prevent the disappearance of volatilized MMA. Surface volatilization is not accountable for a pH-increase-dependent decline in MMA concentrations. MMA is oxidized by dissolved oxygen and produces formaldehyde (Ruyter, 1980). After immersion of auto-polymerized resins in the saliva at pH 6.8, formaldehyde was analyzed by gas chromatography. However, the production of formaldehyde accountable for the disappearance of MMA was not confirmed. MMA is hydrolyzed to M and methanol (Corkill et al., 1976). MMA solutions were prepared with artificial saliva of different pH (4.0 to 8.0). T h e y w e r e allowed to stand at 37°C and were analyzed by HPLC at appropriate time intervals. When the pH increased, the peak height of MMA decreased with the appearance of the peak of M. There were relatively good correlations in concentration changes b e t w e e n " d i s a p p e a r e d " MMA and "appeared" M, and an apparent pH-increase-dependency in M production. Hydrolysis of MMA possibly results in a fall of MMA concentration (Baker et al., 1988). MMA concentrations influenced by acidity
pH 4.0
may be due to instability in the saliva rather than to pH-dependency in leachability itself. Whether M that was detected is derived from hydrolysis in the saliva of MMA leached from the resins or whether the leaching of M is present in the resins as the hydrolysate of MMA is inconclusive. Anyhow, it is apparent that M concentrations increase remarkably when salivary acidity decreases. M produces adverse effects similar to those of MMA (Mir et al., 1973). Our preliminary cell culture study using HeLa cells or fibroblasts showed that cytotoxicity of M was significant in the concentration ranges obtained from the present leaching experiments. That study also suggested that the toxic potency of M might be corresponding to or greater than that of MMA. M may participate in the occurrence of mucosal damage by acrylic resins, especially under neutral conditions. The present study does not necessarily allow for conclusions to be drawn on the possible association between salivary acidity and leachability itself. However, differences in acidity considerably influence concentrations of both MMA and M in immersion saliva, leading to higher MMA concentrations at acidic pH and higher M concentrations at neutral
B A
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Fig. Z Leachability of MMA, M, and BA from auto-polymerized acrylic resins immersed in artificial saliva of different pH. Each point and bar indicate mean value and SE.
Dental Materials/Januar?/1990 15
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Fig. 3. High-performance liquid chromatograms obtained from a standard solution (A) and heat-polymerized acrylic resins (B-E). The standard solution [containing M (5.0 nmol/mL), BA (10.0 nmol/mL), and MMA (10.0 nmol/mL)] and the MA solution (20.0 nmol/mL) were used. The resins were immersed in the artificial saliva of pH 4.0 for one (B) and six days (C), and pH 6.8 for one (D) and six days (E). Peak numbers as in Fig. 1.
pH. Variations in salivary acidity may modify adverse actions of autopolymerized resins by altering relative concentration ratios between cytotoxic MMA and M in saliva. REFERENCES
AL DOORI, D.; HUGGETT, R.; BATES, J.F.; and BROOKS, S.C. (1988): A Comparison of Denture Base Acrylic Resins Polymerized by Microwave Irradiation and by Conventional Water Bath Curing Systems, Dent Mater 4: 25-32. BAKER, S.; BROOKS,S.C.; and WALKER, D.M. (1988): The Release of Residual Monomeric Methyl Methacrylate from Acrylic Appliance in the Human Mouth: An Assay for Monomer in Saliva, J Dent Res 67: 1295-1299. BERGMAN, B. (1977): The Effects of Prosthodontic Materials on Oral Tissues, Oral Sci Rev 10: 75-93. BUDTZ-JORGENSEN, E. (1974): The Significance of Candida albicans in Denture Stomatitis, Scand J Dent Res 82: 151-190. CORKILL, J.A.; LLOYD,E.J.; HOYLE, P.; CROUT, D.H.G.; LING, R.S.M.; JAMES, M.L.; and PIPER, R.J. (1976): Toxicology of Methyl Methacrylate: The Rate of Disappearance of Methyl Methacrylate in Human Blood in vitro, Clin Chim Acta 68: 141-146. FUJISAWA, S. and MASUHARA,E. (1979): Analysis of Eluate Extracted from Dental Filling Resin by High Per-
formance Liquid Chromatography, J Jpn Soc Dent Apparat Mater 20: 121-
131. HARPER, D.S.; ABELSON, D.C.; and JENSEN, M.E. (1986): Human Plaque Acidity Models, J Dent Res 65: 15031510. HENSTEN-PE'ITERSEN,A. and WICTORIN, L. (1979): The Cytotoxic Effect of Denture Base Polymers, Acta Odontol Scand 39: 101-106. IMFELD, T.; HIRSCH, R.S.; and MUHLEMANN, H.R. (1978): Telemetric Recordings of Interdental Plaque pH during Different Meal Patterns, Br Dent J 144: 40-45. KATZ, S.; PARK, K.K.; STOOKEY, G.K.; and SCHEMEHORN,B.R. (1986): Development and Initial Testing of a Model for in vitro Formation of Pit and Fissure Caries, Caries Res 20: 424428. KODA, T.; TSUCHIYA,H.; YAMAUCHI,M.; HOSHINO, Y.; TAKAGI, N.; and KAWANO, J. (1987): High-Performance Liquid Chromatographic Analysis of Residual Monomer Eluted from Dental Acrylic Resins, J Gifu Dent Soc 14: 356-363. KODA, T.; TSUCHIYA,H.; YAMAUCHI,M.; HOSHINO, Y.; TAKAGI, N.; and KAWANO, J. (1989): High-Performance Liquid Chromatographic Estimation of Eluates from Denture Base Polymers, J Dent 17: 84-89. LAMB, D.J.; ELLIS, B.; and PRIESTLEY, D. (1982): Loss into Water of Residual Monomer from Autopolymerizing
16 KODA et al./LEACHABILITY OF ACRYLIC RESINS IN SALIVA
Dental Acrylic Resin, Biomater 3: 155159. SIR, G.N.; LAWRENCE, W.H.; and AUTIAN, J. (1973): Toxicological and Pharmacological Actions of Methacrylate Monomers I: Effects on Isolated, Perfused Rabbit Heart, J Pharm Sci 62: 778-782. OLSEN, I. (1975): Denture Stomatitis, Acta Odontol Scand 33: 41-46. OLSEN, I. and BIRKELAND,J.M. (1977): Denture Stomatitis-Yeast Occurrence and the pH of Saliva and Denture Plaque, Scand J Dent Res 85: 130134. RUYTER, I. E. (1980): Release of Formaldehyde from Denture Base Polymers, Acta Odontol Scand 38: 17-27. STAFFORD, G.D. and BROOKS, S.C. (1985): The Loss of Residual Monomer from Acrylic Orthodontic Resins, Dent Mater 1: 135-138. SZABO, G.; STAFFORD, G.D.; HUGGEVr, R.; and BROOKS,S.C. (1986): The Loss of Residual Monomer from Denture Base Polymers Coated with an Ultraviolet Light-Activated Polymer, Dent Mater 3: 64-66. TSUCHIYA, H.; HAYASHI,T.; 0HTANI, S.; and TAKAGI,N. (1989): HPLC Analysis of Time-Dependent Changes in Urinary Excretion of Indoleamines Following Tryptophan Administration, Biomed Chromatogr 3:157-160. WEAVER, R.E. and GOEBEL, W.M. (1980): Reactions to Acrylic Resin Dental Prostheses, J Prosthet Dent 43: 138-142.
1First Department of Prosthodontics 2Department of Dental Pharmacology Asahi University School of Dentistry 1851 Hozumi, Motosu Gifu 501-02, Japan Received December 12, 1988 Accepted November 29, 1989 *Corresponding author This study was partly supported by a grant from Miyata Science Research Foundation. Dent Mater 6:13-16, January, 1990 Abstract-We studied the influence of
salivary acidity on leachability of denturebase acrylic resins with etiological interest in denture stomatitis because denture surfaces are frequently exposed to acidic conditions in the oral cavities. Auto-, heat-, and microwave-polymerized resins were immersed in artificial saliva with pH ranging from 4.0 to 6.8 at 37°C, and leachables were pursued quantitatively with time. Methyl methacrylate, methacrylic acid, and benzoic acid leached from all resins. Their concentrations in the saliva were markedly high for auto-polymerized resins, while leachability of heat- and microwave-polymerized resins was so low that quantitative analysis of leachables was impossible. Lower pH showed higher concentrations of methyl methacrylate, although no apparent association was confirmed between salivary acidity and its own leachability. The concentrations of methacrylic acid increased remarkably with an increase in pH, which was probably due to hydrolysis of methyl methacrylate. These results suggest that chemotoxic actions of auto-polymerized resins are potentially ascribable to methyl methacrylate under more acidic conditions and to methacrylic acid under less acidic conditions.
enture stomatitis caused by the wearing of acrylic dentures has been ascribed to poorly fitting dentures, unbalanced occlusion, and fungal infection with Candida strains (Budtz-JSrgensen, 1974; Olsen, 1975). In addition to these etiological factors, another possible factor is the chemotoxicity of substances present in the denture-base acrylic polymers (Bergman, 1977; Hensten-Pettersen and Wictorin, 1979; Weaver and Goebel, 1980). Since the toxic substances must leach into saliva to elicit inflammatory reactions on oral mucosa, leaching properties of the acrylic resins have been widely studied for estimation of their potential toxicity. Leachability of a residual monomer, methyl methacrylate (MMA), has been investigated on resins immersed in distilled water (Lamb et al., 1982; Stafford and Brooks, 1985; Szabo et al., 1986) and in aqueous solutions of organic solvents (Fujisawa and Masuhara, 1979; Koda et al., 1987). Dentures placed in oral cavities are continuously washed by saliva and frequently covered by plaque. Saliva and plaque are acidified by fermentative and dietary acids (Olsen and Birkeland, 1977; Imfeld et al., 1978; Harper et al., 1986), resulting in the exposure of denture surfaces to acidic environments. It is valuable, for presumption of toxic potency, for leaching experiments of the acrylic resins to be performed in immersion solutions of diverse acidity. In the p r e s e n t study, we immersed representative acrylic resins in artificial saliva of different pH (acidic to neutral) and quantitatively analyzed MMA and other leachables by high-performance liquid chromatography (HPLC). We examined whether their leaching concentrations differed in acidic and neutral saliva, and estimated an association between salivary acidity and leachability of the acrylic resins.
D
MATERIALS AND METHODS
Auto-polymerized resins (Rebaron No. 3, pink), heat-polymerized resins (Acron No. 8, live pink), and microwave-polymerized resins (AcronMC No. 8, live pink) were purchased from G-C Dental Industrial Corp. (Tokyo, Japan). They were polymerized by use of the monomer-liquid/ pre-polymer-powder ratios and the processing conditions described in the manufacturer's directions. For heat polymerization, the dough was heated at 70°C for 90 rain, and then at 100°C for 30 rain. For microwave polymerization, the dough was subjected to microwave radiation for three min. Resin disks (thickness of 2.0 mm _+ 0.2 mm and outer diameter of 8.5 mm _+ 0.2 ram) were prepared from the acrylic polymers (Koda et al., 1987). The disks were placed in Teflon ®capped vials (volume of 7.0 mL; Gasukuro Kogyo, Tokyo, Japan) containing 5.0 mL of artificial saliva prepared as described below. The vials were gently shaken at 37°C in a water-bath incubation. Aliquots of the immersion solutions were sampled through Teflone membranes of the vial caps by a micro-syringe at time intervals of one day. They were subjected to HPLC analysis immediately after sampling. Artificial saliva was prepared based on the method of Katz et al. (1986). The salivary pH was adjusted to 4.0, 5.0, 6.0, and 6.8 by the addition of an aqueous phosphoric acid solution (2.0 tool/L). MMA, methacrylic acid (M), benzoic acid (BA), and methyl acrylate (MA), used as an internal standard, were purchased from Tokyokasei (Tokyo, Japan). Their standard solutions were prepared daily by dissolution with the artificial saliva of pH 4.0. Water was distilled by all-glass apparatus after being purified by a Milli-Q water purification system (Nihon Millipore, Tokyo, Japan). All other chemicals were of analytical reagent grade.
Dental Materials/January 1990
13
The internal standard solution (20.0 or 100.0 nmoVmL, 50 ~L) was added to the immersion solution (100 ~L) sampled from the Teflon®-capped vials, and then the mixture (100 ~L) was loaded onto an HPLC column. The column (4.6 mm i.d. × 25 cm) was laboratory-packed with NC Gel C-18 (particle size, 5 ~m; Sakata, Tokyo, Japan) by the packing method of Tsuchiya et al. (1989). The separation was performed by an HPLC system similar to one used in our previous study (Koda et al., 1989). A mobile phase, 30% (v/v) acetonitrile in 0.05 mol/L sodium phosphate buffer (pH 3/0), was pumped at a flow rate of 1.0 mL/min and at a column t e m p e r a t u r e of 50°C. UV absorbance of eluates from the HPLC column Was detected at a wavelength of 210 nm. Leachables were determined based on calibration graphs, which we prepared by plotting peak height ratios of standard MMA, M, and BA to the internal standard vs. their concentrations. The calibration graphs for all substances showed good linearity in a range of from at least 0.5 to 1000.0 nmol/mL. When standard solutions and three-day immersion solutions (pH 6.8) of the auto-
A
peroxide added as an initiator (A1 Doori et al., 1988). When auto-polymerized resins were immersed in the saliva of different pH, MMA, M, and BA revealed the leaching profiles shown in Fig. 2. MMA leached into artificial saliva offered concentration ranges almost similar to those obtained from the leaching in distilled water (Koda et al., 1989). MMA concentrations increased with a decrease in the pH. At maximum leaching, however, the concentration at pH 4.0 was 1.5 times that at pH 6.8. MMA concentrations reached maximum at five to seven days after immersion, and then gradually decreased. Since the decreasing curves were steeper at the higher pH, the difference in MMA concentrations between acidic and neutral saliva became more remarkable by increases in the immersion time. M concentrations increased significantly with an increase in the pH. After 10 days of immersion, the concentration at pH 6.8 was over 10 times that at pH 4.0. However, no pH-dependent difference was observed in leaching concentrations of BA. A l t h o u g h MMA, M, and BA leached from heat- and microwave-
polymerized resins were analyzed, the coefficients of variation (n = 10) in peak height ratios to the internal standard were within 0.35% for all substances of both solutions. RESULTS AND DISCUSSION
A preliminary leaching experiment was carried out in mixed human saliva. However, insoluble substances produced during incubation of the saliva prevented the reliable HPLC analysis of leachables. Thus, the artificial saliva used by Katz et al. (1986) was used as the immersion solution after confirmation that it did not influence the HPLC analysis. Representative chromatograms of standards and auto-polymerized resins are shown in Fig. 1. Under optimized HPLC conditions, M, MA (an internal standard), BA, and MMA were separated (in that retention order) with good resolution. In addition to MMA derived from a residual monomer, peaks corresponding to M and BA were obtained from all resins. M is presumably ascribable to the hydrolytic p r o d u c t of MMA (Corkill et al., 1976), and BA to the decomposition product of benzoyl
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Fig. 1. High-performance liquid chromatograms obtained from a standard solution (A) and auto-potymerized acrylic resins (B-E). The standard solution [containing M (20.0 nmol/mL), BA (50.0 nmol/mL), and MMA (500.0 nmol/mL)] and the MA solution (100.0 nmol/mL) were used. The resins were immersed in artificial saliva of pH 4.0 Ior one (B) and six days (C), and pH 6.8 for one (D) and six days (E). Peaks: 1 = M, 2 = MA, 3 = BA, and 4 = MMA. The magnitude of detector attenuation is 1/8 for the 4th peak.
14
KODA et a l . / L E A C H A B I L I T Y OF A C R Y L I C R E S I N S I N S A L I V A
polymerized resins, their concentrations in the saliva were far less than those of auto-polymerized resins. Quantitative analysis of the leachability was not performable, because concentrations of all leachables were below the reliable determination range. However, chromatographic profiles indicated that MMA and M tended to leach from heat- and microwave-polymerized resins in saliv a r y - p H - and i m m e r s i o n - t i m e dependent manners similar to those observed in auto-polymerized resins. Representative results of heatpolymerized resins are shown in Fig. 3. Leaching profiles of auto-polymerized resins indicate that MMA is liable to maintain higher concentrations in the saliva under acidic conditions. The denture surfaces are frequently exposed to low pH by plaque accumulation, fermentation, and dietary consumption (Olsen and Birkeland, 1977; Imfeld et al., 1978; Harper et al., 1986). In such acidic environments of oral cavities, MMA potentially participates in the cause of mucosal damage by the wearing of acrylic dentures. After maximum leaching, MMA concentrations declined, depending on an increase of salivary pH. Disappearance of MMA in the saliva may
M M A
be ascribed to surface volatilization, oxidation, or hydrolysis. The Teflone-capped vials were used in the present study to prevent the disappearance of volatilized MMA. Surface volatilization is not accountable for a pH-increase-dependent decline in MMA concentrations. MMA is oxidized by dissolved oxygen and produces formaldehyde (Ruyter, 1980). After immersion of auto-polymerized resins in the saliva at pH 6.8, formaldehyde was analyzed by gas chromatography. However, the production of formaldehyde accountable for the disappearance of MMA was not confirmed. MMA is hydrolyzed to M and methanol (Corkill et al., 1976). MMA solutions were prepared with artificial saliva of different pH (4.0 to 8.0). T h e y w e r e allowed to stand at 37°C and were analyzed by HPLC at appropriate time intervals. When the pH increased, the peak height of MMA decreased with the appearance of the peak of M. There were relatively good correlations in concentration changes b e t w e e n " d i s a p p e a r e d " MMA and "appeared" M, and an apparent pH-increase-dependency in M production. Hydrolysis of MMA possibly results in a fall of MMA concentration (Baker et al., 1988). MMA concentrations influenced by acidity
pH 4.0
may be due to instability in the saliva rather than to pH-dependency in leachability itself. Whether M that was detected is derived from hydrolysis in the saliva of MMA leached from the resins or whether the leaching of M is present in the resins as the hydrolysate of MMA is inconclusive. Anyhow, it is apparent that M concentrations increase remarkably when salivary acidity decreases. M produces adverse effects similar to those of MMA (Mir et al., 1973). Our preliminary cell culture study using HeLa cells or fibroblasts showed that cytotoxicity of M was significant in the concentration ranges obtained from the present leaching experiments. That study also suggested that the toxic potency of M might be corresponding to or greater than that of MMA. M may participate in the occurrence of mucosal damage by acrylic resins, especially under neutral conditions. The present study does not necessarily allow for conclusions to be drawn on the possible association between salivary acidity and leachability itself. However, differences in acidity considerably influence concentrations of both MMA and M in immersion saliva, leading to higher MMA concentrations at acidic pH and higher M concentrations at neutral
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Fig. Z Leachability of MMA, M, and BA from auto-polymerized acrylic resins immersed in artificial saliva of different pH. Each point and bar indicate mean value and SE.
Dental Materials/Januar?/1990 15
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Retention time
(min)
Fig. 3. High-performance liquid chromatograms obtained from a standard solution (A) and heat-polymerized acrylic resins (B-E). The standard solution [containing M (5.0 nmol/mL), BA (10.0 nmol/mL), and MMA (10.0 nmol/mL)] and the MA solution (20.0 nmol/mL) were used. The resins were immersed in the artificial saliva of pH 4.0 for one (B) and six days (C), and pH 6.8 for one (D) and six days (E). Peak numbers as in Fig. 1.
pH. Variations in salivary acidity may modify adverse actions of autopolymerized resins by altering relative concentration ratios between cytotoxic MMA and M in saliva. REFERENCES
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