Journal of Dental Research http://jdr.sagepub.com/
Amine-induced Polymerization of Aqueous HEMA/Aldehyde During Action as a Dentin Bonding Agent E.C. Munksgaard J DENT RES 1990 69: 1236 DOI: 10.1177/00220345900690060201 The online version of this article can be found at: http://jdr.sagepub.com/content/69/6/1236
Published by: http://www.sagepublications.com
On behalf of: International and American Associations for Dental Research
Additional services and information for Journal of Dental Research can be found at: Email Alerts: http://jdr.sagepub.com/cgi/alerts Subscriptions: http://jdr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Citations: http://jdr.sagepub.com/content/69/6/1236.refs.html
>> Version of Record - Jun 1, 1990 What is This?
Downloaded from jdr.sagepub.com at Monash University on October 24, 2014 For personal use only. No other uses without permission.
Amine-induced Polymerization of Aqueous HEMA/Aldehyde During Action as a Dentin Bonding Agent E.C. MUNKSGAARD Department of Dental Materials & Technology, Royal Dental College, Nirre Alle6 20, DK-2200 Copenhagen N. Denmark
Aqueous mixtures of HEMA with glutaraldehyde or propionaldehyde polymerize by addition of catalytic amounts of amines or amino acids. The maximal reaction velocity of the transformation of HEMA/glutaraldehyde with glycine was obtained at pH 0.8. Kinetic data suggested a second-order reaction between glutaraldehyde and glycine, and solubility data suggested formation of a cross-linked polymer. A relatively high bond strength between dentin and resin composite was obtained by pre-treatment of dentin with Gluma (35% HEMA, 5% glutaraldehyde in water) adjusted to pH 1.0 with hydrochloric acid. It is proposed that on application of Gluma, aminogroup-containing substances in dentin react with glutaraldehyde and start the formation of a HEMA polymer. This product may be cross-linked by an ox,3-unsaturated glutaraldehyde aldol condensation product and may bond to dentin by aldehyde fixation to dentin proteins. Resin composite will bond to this product by copolymerization. J Dent Res 69(6):1236-1239, June, 1990
Introduction.
Materials and methods. We obtained 2-hydroxyethyl methacrylate (HEMA) from Fluka AG, Buschs, Switzerland, and 25% glutaraldehyde, propionaldehyde, ammonia, ethylenediamine, and various amino acids from E. Merck, Darmstadt, FRG. Concise was obtained from 3M Co., St. Paul, MN, and 0.5 mol/L EDTA, pH 7.4, was prepared and used as previously described (Munksgaard and Asmussen, 1984). Small amounts of glycine were added to 1.0-mL solutions of 35% HEMA, water, glutaraldehyde or propionaldehyde, with or without pH-adjustment with hydrochloric acid, at 37°C in 1.2-mL polyethylene vials (Eppendorf, Hamburg, FRG) equipped with a lid. The time elapsed-from addition of the amino acid until a white precipitate formed in the center of the tube-was noted and designated the gel time. In some experiments, ammonia, ethylenediamine, lysine, aspartic acid, glutaric acid, or phenylalanine was used instead of glycine. In other experiments, helium or air was bubbled through the solution during the reaction. Six-day-old reaction mixtures of 35% HEMA in 5% glutaraldehyde with 10 Wxmol glycine/mL were minced, extracted with methanol, air-dried, and weighed. All the above-mentioned measurements were performed in duplicate and calculated as the mean SD. Bond strength test. -Human teeth were embedded in a slowly setting epoxy resin (Epofix resin, Struers, Copenhagen, Denmark), and flat dentin was produced by grinding. The grinding was performed wet on carborundum paper No. 220 and on No. 1000 at the final stage. The surfaces were pre-treated with pHadjusted Gluma for 20 s, followed by a blast of compressed air for five s. The pH adjustment was performed with either 1 mol/L or 6 mol/L hydrochloric acid. A split Teflon mold with a cylindrical hole (diameter, 3.6 mm; height, 5 mm) was clamped to a prepared surface. One drop of Concise Enamel Bond was placed inside the mold, and excess was removed by a blast of compressed air for five s, leaving a film of resin on the prepared tooth surface. The mold was then filled with Concise composite by use of a Hawe-Neos syringe (Hawe-Neos Dental, Gentilino, Switzerland). The specimens were stored in water for 24 h at room temperature. The mold was removed, and the specimen mounted in an Instron Testing Machine. A loop made of steel wire (diameter, 0.6 mm) was placed around the composite rod of the test specimen and in contact with the tooth surface. The other end of the loop was attached to the cross-head, and the bond was ruptured parallel to the tooth surface at a speed of 0.5 mm/min. The bond strength was calculated as the mean breaking force per area and expressed in MPa. Six specimens were prepared in each group. SEM studies were performed on ground dentin surfaces treated with 0.5 mol/L EDTA (pH 7.4) for 40 s, rinsed with water for five s, and air-dried for five s. Gluma was then applied, and excess reagent was removed with a blast of air after 40 s. Other specimens for SEM were treated with Gluma adjusted to pH 1.00 with hydrochloric acid but without the EDTA pretreatment. Some specimens were given this treatment for 40 s and were then air-sprayed for five s; others were given treat±
The Gluma dentin bonding agent (35% HEMA and 5% glutaraldehyde in water) has been investigated in a number of studies since its introduction (Munksgaard and Asmussen, 1984). The agent mediates a reasonably high bond strength between dentin and resin composites (Eliades et al., 1985; Komatsu and Finger, 1986; Retief et al., 1988; Munksgaard and Asmussen, 1987; Asmussen and Bowen, 1987), and composite fillings in dentin cavities, made with this agent, exhibit no or relatively small marginal gaps (Munksgaard and Irie, 1988; Hansen and Asmussen, 1989). It has previously been proposed (Asmussen and Munksgaard, 1985) that an N-(hydroxyalkyl) compound produced by a reaction of aldehyde and active nitrogen groups in collagen will, by loss of water, react with the hydroxyl group in HEMA, producing a dentin surface with a polymerizable layer of double bonds. This mechanism has been questioned (Munksgaard and Asmussen, 1984) because it does not seem concordant with the observation that an increase in HEMA concentration beyond 35% in an aqueous HEMA/glutaraldehyde mixture results in a decrease in bond strength. Nor is the mechanism concordant with measurements of the reaction products of glutaraldehyde and amino acids or proteins. The products are composed of various heterocyclic substances, some of which have complicated structures (Hardy et al., 1976; Johnson, 1985a,b; Lubig et al., 1981; Nimni et al., 1987). This study was designed to investigate other possible mechanisms that may explain the action of Gluma as a dentin bonding agent. Received for publication September 13, 1989 Accepted for publication January 5, 1990
1236
Downloaded from jdr.sagepub.com at Monash University on October 24, 2014 For personal use only. No other uses without permission.
AMINES AND POLYMERIZATION OF HEMA/ALDEHYDE
Vol. 69 No. 6
pH 2.9
100
1237
F
60 0
90
pH 1
250 .
80 iod30 z 10 x
70 60
I-
0.15 0.20 0.05 0.10 CONCENTRATION OF GLYCINE, MROMOU1 ML
0.25
111 -J
Fig. 1-Gel times at 37'C in Gluma at pH 1.0 and 2.9, respectively, as a function of the reciprocal initial concentration of glycine. The following numbers indicate coefficient of estimation, slope, and intersection calculated by linear regression: 0.994, 13.1, and 154 for pH 1.0 and 0.996, 34.3, and 145 for pH 2.9.
50
w
40
F
30 _
20 _
,200j
10
I I
I
50
TABLE 10
20
CONCENTRATION
Fig. 2-Gel times at aqueous HEMA with 10
37°C pWmol
OF
30
40
GLUTALEHYDE.
at pH 1.0 and 2.9,
MMO-1-
50 ML
respectively,
in
35%o
glycine/mL as a function of the reciprocal
initial concentration of glutaraldehyde. The two lines intersect at a glu-
taraldehyde concentration of 0.17 mmol/mL. The following numbers indicate coefficient of estimation, slope, and intersection calculated by linear regression:
for pH
I
0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 pH Fig. 3-Gel time at 37°C in Gluma containing 8 pimol glycine/mL as a function of pH in the aqueous phase.
0.999,
18.7, and 7.69 for pH
1.0
and 0.987,
44.4,
and 3.93
2.9.
ment with the same reagent for 40 s, but followed by a rinse of water for 10-20 s. All specimens were air-dried before ap-
plication of a 20-nm film of Pd-Au and inspection under the microscope.
Results. Glycine, when added in minute amounts to 1.00 mL of a solution of 5% glutaraldehyde (0.5 mmol/mL) and 35% HEMA (2.69 mmol/mL) (Gluma), initiated formation of a gel, which in time developed to a rubber-like mass. This mass had a dry weight + SD of 340 + 15 mg after extraction with water and methanol, which corresponds to the amount of HEMA originally present in the mixture (350 mg). The mass was insoluble
in methanol, chloroform, dimethyl-sulfoxide, dimethylformamide, acetone, and hexane. Gluma prepared as above had a pH of 2.9. After addition of glycine in various quantities (4 to 100 Aimol/mL), a linear relationship was observed between gel time and the reciprocal of the concentration of glycine ( Fig. 1). When Gluma adjusted with hydrochloric acid to pH 1.0 was used in a similar experiment, a linear relationship was also obtained. The two lines
1
GEL TIMES OF GLUMA AT 37°C, pH 2.9, AFTER ADDITION OF 16 ,umol/mL OF ONE OF VARIOUS NH2-CONTAINING SUBSTANCES Name Gel time, minutes Ammonia 195.2 + 5.7 Lysine 71.9 1.4 Aspartic acid 68.3 + 1.2 Ethylenediamine 66.1 ± 0.4 Glycine 43.8 0.9 Glutamic acid 39.5 + 0.7 Phenylalanine 38.0 ± 0.8
were
parallel, and the gel times were shorter (by about 20 min)
at pH 1.0 than at pH 2.9.
Ten [Lmol glycine was added to 1.00-mL portions of aqueous HEMA (2.69 mmol/mL) containing glutaraldehyde in various concentrations. The gel time decreased when the glutaraldehyde concentration was increased from 0.02 to 1.00 mmol/ mL. Experiments were performed at pH 2.9 and 1.0, respectively. A linear relationship between gel time and the reciprocal concentration of glutaraldehyde could be demonstrated, as seen in Fig. 2. The two lines in the Fig. representing the two experiments cross each other at a glutaraldehyde concentration of 0.17 mmol/mL. Below this concentration, the gel times were shorter at pH 1.0 than at pH 2.9, and above, the shorter gel times were obtained at pH 2.9. Fig. 3 shows the gel times in vials containing 1.00-mL portions of Gluma at various pH values. Eight pmol of glycine was added to each portion for initiation of gelation. The shortest gel time was obtained when Gluma had a pH of 0.8. Table 1 gives the gel times when ammonia, ethylenediamine, or one of four amino acids was used for initiation of
Downloaded from jdr.sagepub.com at Monash University on October 24, 2014 For personal use only. No other uses without permission.
1238
J Dent Res June 1990
MUNKSGARRD
the gel of Gluma, pH 2.9. No relationship between pKB of these substances and the gel time could be established. The average coefficient of estimation of the results in Figs. 1, 2, and 3 was 1.2%, with a range of 0.4 to 3.0%. When air was bubbled through the reaction mixture of Gluma and glycine at pH 1.0, no precipitate could be observed over a period of six h. The use of helium instead of air gave a gel time of 41 + 2 min. compared with a gel time of 38 ± 3 min in the closed vial that contained some air, cf. Table 2. The closed vials were rotated so that the gel time measurements would be comparable with the measurements performed with the helium-treated vials. Observation of the time of initial precipitation in the helium-treated and rotated vials was difficult because of the constant movement in the mixtures. Table 3 gives the shear bond strengths between composite and dentin pre-treated with one of various Gluma modifications. The modification consisted of the preparation of the solution with dilute hydrochloric acid so that the pH in the aqueous phase varied from 0.2 to 2.9. A distinct maximum in bond strength occurred when Gluma had a pH of 1.0 (Table 3). Propionaldehyde in 10 wt% concentration was used instead of glutaraldehyde in experiments similar to those presented in Fig. 1. Precipitation occurred at pH 2.0 as well as at pH 3.2 (unadjusted). Decreasing gel time occurred as the glycine concentration was raised. The gel time with 40 Amol glycine/mL was 172 + 7.8 min at pH 2.0 and 246 ± 12.8 min at pH 3.2. The SEM pictures in Fig. 4 show dentin surfaces upon treatment with (a) 0.5 mol/L EDTA (pH 7.4) and Gluma, (b) Gluma (pH 1.0) followed by a blast of compressed air, and (c) Gluma (pH 1.0) followed by a 10-20-second water rinse. At higher magnification (x20,000), a faint layer could be distinguished on the surface of specimen (a). The cracks observed in (b) were visible only on a small area of the prepared surface, but gave the impression of a polymerized layer of some thickness. The layer seen in (c) was clearly thinner than the layer in (b), probably due to partial removal by the water rinse.
Discussion.
Fig. 4-SEM pictures of dentin surfaces treated with (a) 0.5 mol/mL EDTA, pH 7.4, followed by Gluma, (b) Gluma at pH 1.0 without water spray, and (c) Gluma at pH 1.0 for 40 s, followed by a 10-20-second water rinse. The pH adjustment was performed with hydrochloric acid. The treatment of the dentin surface in (b) was identical to the pre-treatment performed on the specimens used for bond strength measurements at pH 1.0 (Table 3) (SEM X 2500, bar = 10 Aim).
Aqueous glutaraldehyde solution consists of no more than 15% of free aldehyde in equilibrium with glutaraldehydemonohydrate transformed to a cyclic hemi-acetal and its ohgomers (Korn et al., 1972). A number of reaction products have been postulated for the reaction between glutaraldehyde and amino acids, as well as proteins. Among these are (1) Schiff's bases of mono-aldehyde or its polymeric aldol condensation products; (2) formation of cyclic carbinolamines, dihydropyridinium, as well as pyridinium compounds derived from the cyclic hemiacetal, and polymeric products derived from condensation reactions with linear glutaraldehyde; and (3) Michael adducts of the amine to ox,4-unsaturated glutaraldehyde aldol condensation products (Hardy et al., 1976; Johnson, 1985a,b; Lubig et al., 1981; Nimni et al., 1987). These reaction mechanisms are not readily able to explain the results in the present study. For example, glycine and propionaldehyde/HEMA are unlikely to form pyridinium derivatives. Lubig et al. (1981) and Johnson (1985b) observed that the reaction between glycine and glutaraldehyde was followed by formation of H+ in the initial phase and later on by a consumption of H+. This might explain the pH dependence of the reaction as seen in Figs. 1, 2, and 3, as well as the results with propionaldehyde. The results in Fig. 2 might be explained in much the same way; that is, at low glutaraldehyde concentrations, the formation of H+ is the rate-determining process,
Downloaded from jdr.sagepub.com at Monash University on October 24, 2014 For personal use only. No other uses without permission.
AMINES AND POLYMERIZATION OF HEMA/ALDEHYDE
Vol. 69 No. 6
1239
TABLE 2
TABLE 3
GEL TIMES OF GLUMA, pH 1.0, WITH 8 pumol GLYCINE/mL at 370C IN CLOSED VIALS OR IN VIALS UNDER AIR OR HELIUM Gel time, minutes Condition 38 + 3 Closed vial Air bubbling no gelation within 6 h Helium bubbling 41 + 2
SHEAR BOND STRENGTH BETWEEN RESIN COMPOSITE AND DENTIN PRE-TREATED WITH pH-ADJUSTED GLUMA Bond strength, pH in Gluma MPa (SD) 0.2 1.22 (1.43) 0.6 11.72 (7.25) 1.0 27.79 (8.23) 1.4 15.65 (9.67) 1.8 9.29 (6.63) 2.9 7.31 (3.18)
but at higher concentrations, the process involving consumption of H+ is rate-determining. Johnson (1985a) observed that rapid oxygen consumption takes place during the reaction between amines, as well as proteins and glutaraldehyde. This agrees well with the results presented in Table 2, which can be interpreted in at least two ways for explanation of the polymerization of HEMA, presumably taking place during the reaction with amines and glutaraldehyde/HEMA. One interpretation is that the polymerization was caused by removal of oxygen during the reaction. Another interpretation is that a product, which acted as a polymerization initiator, was formed under anaerobic conditions. The linear relationship between gel time and the reciprocal of the glycine concentration (Fig. 1), as well as the reciprocal of the glutaraldehyde concentration (Fig. 2), suggest second-order reaction kinetics. The results from the experiment in which the reaction product of Gluma and glycine was extracted and dried indicate that, in time, nearly all HEMA in such solutions will be transformed. Because of the variation between duplicate experiments, it was not possible for us to decide to what extent glutaraldehyde took part in the process. The insolubility of the reaction product in a number of solvents indicates a crosslinked polymer. Such cross-linking might occur between polyHEMA chains, either by hemi-acetal formation by reaction with dialdehyde or by polymerization of HEMA together with oligomeric polyaldehyde chains with ox,3-unsaturated bonds. Aldehydes, together with small amounts of amine (Table 1), may represent an alternative way of polymerizing methacrylates, and work is in progress so that knowledge about this possibility may be increased. The coincidence between the pH at which gelation occurred at its maximum velocity (Fig. 3) and the pH at which the maximum shear bond strength occurred (Table 3) supports the assumption that the transformation of HEMA discussed above may play a role when Gluma is used as a dentin bonding agent. It is proposed that on application of Gluma, substances in dentin containing amino groups react with glutaraldehyde and start the formation of a HEMA polymer. Such substances might be collagen, non-collagenous proteins, small peptides produced by grinding, and a number of other substances containing amino groups left over from the cell processes. This product may be cross-linked by ox,P-unsaturated glutaraldehyde aldol condensation product and bond to dentin by aldehyde fixation to dentin proteins. Resin composite will bond to this product by copolymerization. This is supported by the results presented in Fig. 4. Other mechanisms may also be of importance, such as the penetration into dentin of HEMA, which interlocks during polymerization, and a strengthening of the surface collagen layer by aldehyde fixation. The results may also explain the results of Asmussen and Bowen (1987). In these experiments, relatively high bond strengths between composite and Glumatreated dentin were obtained when the dentin was pre-treated with certain acidic glycine solutions.
Acknowledgment. Technical assistant Vivi R0nne is complimented for care and
competence during performance of the experiments. REFERENCES ASMUSSEN, E. and BOWEN, R.L. (1987): Effect of Acidic Pretreatment on Adhesion to Dentin Mediated by Gluma, JDent Res 66:13861388. ASMUSSEN, E. and MUNKSGAARD, E.C. (1985): Bonding of Restorative Resins to Dentin Promoted by Aqueous Mixtures of Aldehydes and Reactive Monomers, Int Dent J 35:160-165. ELIADES, G.C.; CAPUTO, A.A.; and VOUGIOUKLAKIS, G.J. (1985): Composition, Wetting Properties and Bond Strength with Dentin of 6 New Dentin Adhesives, Dent Mater 1:170-176. HANSEN, E.K. and ASMUSSEN, E. (1989): Marginal Adaptation of Posterior Resins: Effect of Dentin-Bonding Agent and Hygroscopic Expansion, Dent Mater 5:122-126. HARDY, P.M.; HUGHES, G.J.; and RYDON, H.N. (1976): Formation of Quaternary Pyridinium Compounds by the Action of Glutaraldehyde on Proteins, J Chem Soc Chem Comm 5:157-158. JOHNSON, T.J.A. (1985a): Glutaraldehyde Fixation Chemistry. Scheme for Rapid Crosslinking and Evidence for Rapid Oxygen Consumption. In: Science of Biological Specimen Preparation, M. Mueller, R.P. Becker, A. Boyde, and J.J. Volosewich, Eds., Chicago: SEM Inc., AMF O'Hare, USA, pp. 51-62. JOHNSON, T.J.A. (1985b): Aldehyde Fixatives: Quantification of Acidproducing Reactions, J Electron Microsc Tech 2:129-138. KOMATSU, M. and FINGER, W. (1986): Dentin Bonding Agents: Correlation of Early Bond Strength with Margin Gaps, Dent Mater 2:257262. KORN, A.H.; FAIRHELLER, S.H.; and FILACHIONE, E.M. (1972): Glutaraldehyde: Nature of the Reagent, J Mol Biol 65:525-529. LUBIG, R.; KUSCH, P.; ROPER, K.; and ZAHN, H. (1981): Zum Reaktionsmechanismus von Glutaraldehyd mit Proteinen, Monatshefte fUr Chemie 112:1313-1323. MUNKSGAARD, E.C. and ASMUSSEN, E. (1984): Bond Strength Between Dentin and Restorative Resins Mediated by Mixtures of HEMA and Glutaraldehyde, J Dent Res 63:1087-1089. MUNKSGAARD, E.C. and ASMUSSEN, E. (1985): Dentin-Polymer Bond Mediated by Glutaraldehyde/HEMA, Scand J Dent Res 93:463-466. MUNKSGAARD, E.C. and ASMUSSEN, E. (1987): Methacrylate-Bonding to Dentin. In: Dentine and Dentine Reactions in the Oral Cavity, A. Thylstrup, S.A. Leach, and V. Qvist, Eds., Oxford: IRL Press Ltd., pp. 209-218. MUNKSGAARD, E.C. and IRIE, M. (1988): Effect of Load-Cycling on Bond Between Composite Fillings and Dentin Established by Gluma and Various Resins, Scand J Dent Res 96:579-583. NIMNI, M.E.; CHEUNG, D.; STRATES, B.; KODAMA, M.; and SHEIKH, K. (1987): Chemically Modified Collagen: A Natural Biomaterial for Tissue Replacement, J Biomed Mater Res 21:741-771. RETIEF, D.H.; O'BRIEN, J.A.; SMITH, L.A.; and MARCHMAN, J.L. (1988): In vitro Investigation and Evaluation of Dentin Bonding Agents, Am J Dent 1:176-183.
Downloaded from jdr.sagepub.com at Monash University on October 24, 2014 For personal use only. No other uses without permission.
Amine-induced Polymerization of Aqueous HEMA/Aldehyde During Action as a Dentin Bonding Agent E.C. Munksgaard J DENT RES 1990 69: 1236 DOI: 10.1177/00220345900690060201 The online version of this article can be found at: http://jdr.sagepub.com/content/69/6/1236
Published by: http://www.sagepublications.com
On behalf of: International and American Associations for Dental Research
Additional services and information for Journal of Dental Research can be found at: Email Alerts: http://jdr.sagepub.com/cgi/alerts Subscriptions: http://jdr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Citations: http://jdr.sagepub.com/content/69/6/1236.refs.html
>> Version of Record - Jun 1, 1990 What is This?
Downloaded from jdr.sagepub.com at Monash University on October 24, 2014 For personal use only. No other uses without permission.
Amine-induced Polymerization of Aqueous HEMA/Aldehyde During Action as a Dentin Bonding Agent E.C. MUNKSGAARD Department of Dental Materials & Technology, Royal Dental College, Nirre Alle6 20, DK-2200 Copenhagen N. Denmark
Aqueous mixtures of HEMA with glutaraldehyde or propionaldehyde polymerize by addition of catalytic amounts of amines or amino acids. The maximal reaction velocity of the transformation of HEMA/glutaraldehyde with glycine was obtained at pH 0.8. Kinetic data suggested a second-order reaction between glutaraldehyde and glycine, and solubility data suggested formation of a cross-linked polymer. A relatively high bond strength between dentin and resin composite was obtained by pre-treatment of dentin with Gluma (35% HEMA, 5% glutaraldehyde in water) adjusted to pH 1.0 with hydrochloric acid. It is proposed that on application of Gluma, aminogroup-containing substances in dentin react with glutaraldehyde and start the formation of a HEMA polymer. This product may be cross-linked by an ox,3-unsaturated glutaraldehyde aldol condensation product and may bond to dentin by aldehyde fixation to dentin proteins. Resin composite will bond to this product by copolymerization. J Dent Res 69(6):1236-1239, June, 1990
Introduction.
Materials and methods. We obtained 2-hydroxyethyl methacrylate (HEMA) from Fluka AG, Buschs, Switzerland, and 25% glutaraldehyde, propionaldehyde, ammonia, ethylenediamine, and various amino acids from E. Merck, Darmstadt, FRG. Concise was obtained from 3M Co., St. Paul, MN, and 0.5 mol/L EDTA, pH 7.4, was prepared and used as previously described (Munksgaard and Asmussen, 1984). Small amounts of glycine were added to 1.0-mL solutions of 35% HEMA, water, glutaraldehyde or propionaldehyde, with or without pH-adjustment with hydrochloric acid, at 37°C in 1.2-mL polyethylene vials (Eppendorf, Hamburg, FRG) equipped with a lid. The time elapsed-from addition of the amino acid until a white precipitate formed in the center of the tube-was noted and designated the gel time. In some experiments, ammonia, ethylenediamine, lysine, aspartic acid, glutaric acid, or phenylalanine was used instead of glycine. In other experiments, helium or air was bubbled through the solution during the reaction. Six-day-old reaction mixtures of 35% HEMA in 5% glutaraldehyde with 10 Wxmol glycine/mL were minced, extracted with methanol, air-dried, and weighed. All the above-mentioned measurements were performed in duplicate and calculated as the mean SD. Bond strength test. -Human teeth were embedded in a slowly setting epoxy resin (Epofix resin, Struers, Copenhagen, Denmark), and flat dentin was produced by grinding. The grinding was performed wet on carborundum paper No. 220 and on No. 1000 at the final stage. The surfaces were pre-treated with pHadjusted Gluma for 20 s, followed by a blast of compressed air for five s. The pH adjustment was performed with either 1 mol/L or 6 mol/L hydrochloric acid. A split Teflon mold with a cylindrical hole (diameter, 3.6 mm; height, 5 mm) was clamped to a prepared surface. One drop of Concise Enamel Bond was placed inside the mold, and excess was removed by a blast of compressed air for five s, leaving a film of resin on the prepared tooth surface. The mold was then filled with Concise composite by use of a Hawe-Neos syringe (Hawe-Neos Dental, Gentilino, Switzerland). The specimens were stored in water for 24 h at room temperature. The mold was removed, and the specimen mounted in an Instron Testing Machine. A loop made of steel wire (diameter, 0.6 mm) was placed around the composite rod of the test specimen and in contact with the tooth surface. The other end of the loop was attached to the cross-head, and the bond was ruptured parallel to the tooth surface at a speed of 0.5 mm/min. The bond strength was calculated as the mean breaking force per area and expressed in MPa. Six specimens were prepared in each group. SEM studies were performed on ground dentin surfaces treated with 0.5 mol/L EDTA (pH 7.4) for 40 s, rinsed with water for five s, and air-dried for five s. Gluma was then applied, and excess reagent was removed with a blast of air after 40 s. Other specimens for SEM were treated with Gluma adjusted to pH 1.00 with hydrochloric acid but without the EDTA pretreatment. Some specimens were given this treatment for 40 s and were then air-sprayed for five s; others were given treat±
The Gluma dentin bonding agent (35% HEMA and 5% glutaraldehyde in water) has been investigated in a number of studies since its introduction (Munksgaard and Asmussen, 1984). The agent mediates a reasonably high bond strength between dentin and resin composites (Eliades et al., 1985; Komatsu and Finger, 1986; Retief et al., 1988; Munksgaard and Asmussen, 1987; Asmussen and Bowen, 1987), and composite fillings in dentin cavities, made with this agent, exhibit no or relatively small marginal gaps (Munksgaard and Irie, 1988; Hansen and Asmussen, 1989). It has previously been proposed (Asmussen and Munksgaard, 1985) that an N-(hydroxyalkyl) compound produced by a reaction of aldehyde and active nitrogen groups in collagen will, by loss of water, react with the hydroxyl group in HEMA, producing a dentin surface with a polymerizable layer of double bonds. This mechanism has been questioned (Munksgaard and Asmussen, 1984) because it does not seem concordant with the observation that an increase in HEMA concentration beyond 35% in an aqueous HEMA/glutaraldehyde mixture results in a decrease in bond strength. Nor is the mechanism concordant with measurements of the reaction products of glutaraldehyde and amino acids or proteins. The products are composed of various heterocyclic substances, some of which have complicated structures (Hardy et al., 1976; Johnson, 1985a,b; Lubig et al., 1981; Nimni et al., 1987). This study was designed to investigate other possible mechanisms that may explain the action of Gluma as a dentin bonding agent. Received for publication September 13, 1989 Accepted for publication January 5, 1990
1236
Downloaded from jdr.sagepub.com at Monash University on October 24, 2014 For personal use only. No other uses without permission.
AMINES AND POLYMERIZATION OF HEMA/ALDEHYDE
Vol. 69 No. 6
pH 2.9
100
1237
F
60 0
90
pH 1
250 .
80 iod30 z 10 x
70 60
I-
0.15 0.20 0.05 0.10 CONCENTRATION OF GLYCINE, MROMOU1 ML
0.25
111 -J
Fig. 1-Gel times at 37'C in Gluma at pH 1.0 and 2.9, respectively, as a function of the reciprocal initial concentration of glycine. The following numbers indicate coefficient of estimation, slope, and intersection calculated by linear regression: 0.994, 13.1, and 154 for pH 1.0 and 0.996, 34.3, and 145 for pH 2.9.
50
w
40
F
30 _
20 _
,200j
10
I I
I
50
TABLE 10
20
CONCENTRATION
Fig. 2-Gel times at aqueous HEMA with 10
37°C pWmol
OF
30
40
GLUTALEHYDE.
at pH 1.0 and 2.9,
MMO-1-
50 ML
respectively,
in
35%o
glycine/mL as a function of the reciprocal
initial concentration of glutaraldehyde. The two lines intersect at a glu-
taraldehyde concentration of 0.17 mmol/mL. The following numbers indicate coefficient of estimation, slope, and intersection calculated by linear regression:
for pH
I
0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 pH Fig. 3-Gel time at 37°C in Gluma containing 8 pimol glycine/mL as a function of pH in the aqueous phase.
0.999,
18.7, and 7.69 for pH
1.0
and 0.987,
44.4,
and 3.93
2.9.
ment with the same reagent for 40 s, but followed by a rinse of water for 10-20 s. All specimens were air-dried before ap-
plication of a 20-nm film of Pd-Au and inspection under the microscope.
Results. Glycine, when added in minute amounts to 1.00 mL of a solution of 5% glutaraldehyde (0.5 mmol/mL) and 35% HEMA (2.69 mmol/mL) (Gluma), initiated formation of a gel, which in time developed to a rubber-like mass. This mass had a dry weight + SD of 340 + 15 mg after extraction with water and methanol, which corresponds to the amount of HEMA originally present in the mixture (350 mg). The mass was insoluble
in methanol, chloroform, dimethyl-sulfoxide, dimethylformamide, acetone, and hexane. Gluma prepared as above had a pH of 2.9. After addition of glycine in various quantities (4 to 100 Aimol/mL), a linear relationship was observed between gel time and the reciprocal of the concentration of glycine ( Fig. 1). When Gluma adjusted with hydrochloric acid to pH 1.0 was used in a similar experiment, a linear relationship was also obtained. The two lines
1
GEL TIMES OF GLUMA AT 37°C, pH 2.9, AFTER ADDITION OF 16 ,umol/mL OF ONE OF VARIOUS NH2-CONTAINING SUBSTANCES Name Gel time, minutes Ammonia 195.2 + 5.7 Lysine 71.9 1.4 Aspartic acid 68.3 + 1.2 Ethylenediamine 66.1 ± 0.4 Glycine 43.8 0.9 Glutamic acid 39.5 + 0.7 Phenylalanine 38.0 ± 0.8
were
parallel, and the gel times were shorter (by about 20 min)
at pH 1.0 than at pH 2.9.
Ten [Lmol glycine was added to 1.00-mL portions of aqueous HEMA (2.69 mmol/mL) containing glutaraldehyde in various concentrations. The gel time decreased when the glutaraldehyde concentration was increased from 0.02 to 1.00 mmol/ mL. Experiments were performed at pH 2.9 and 1.0, respectively. A linear relationship between gel time and the reciprocal concentration of glutaraldehyde could be demonstrated, as seen in Fig. 2. The two lines in the Fig. representing the two experiments cross each other at a glutaraldehyde concentration of 0.17 mmol/mL. Below this concentration, the gel times were shorter at pH 1.0 than at pH 2.9, and above, the shorter gel times were obtained at pH 2.9. Fig. 3 shows the gel times in vials containing 1.00-mL portions of Gluma at various pH values. Eight pmol of glycine was added to each portion for initiation of gelation. The shortest gel time was obtained when Gluma had a pH of 0.8. Table 1 gives the gel times when ammonia, ethylenediamine, or one of four amino acids was used for initiation of
Downloaded from jdr.sagepub.com at Monash University on October 24, 2014 For personal use only. No other uses without permission.
1238
J Dent Res June 1990
MUNKSGARRD
the gel of Gluma, pH 2.9. No relationship between pKB of these substances and the gel time could be established. The average coefficient of estimation of the results in Figs. 1, 2, and 3 was 1.2%, with a range of 0.4 to 3.0%. When air was bubbled through the reaction mixture of Gluma and glycine at pH 1.0, no precipitate could be observed over a period of six h. The use of helium instead of air gave a gel time of 41 + 2 min. compared with a gel time of 38 ± 3 min in the closed vial that contained some air, cf. Table 2. The closed vials were rotated so that the gel time measurements would be comparable with the measurements performed with the helium-treated vials. Observation of the time of initial precipitation in the helium-treated and rotated vials was difficult because of the constant movement in the mixtures. Table 3 gives the shear bond strengths between composite and dentin pre-treated with one of various Gluma modifications. The modification consisted of the preparation of the solution with dilute hydrochloric acid so that the pH in the aqueous phase varied from 0.2 to 2.9. A distinct maximum in bond strength occurred when Gluma had a pH of 1.0 (Table 3). Propionaldehyde in 10 wt% concentration was used instead of glutaraldehyde in experiments similar to those presented in Fig. 1. Precipitation occurred at pH 2.0 as well as at pH 3.2 (unadjusted). Decreasing gel time occurred as the glycine concentration was raised. The gel time with 40 Amol glycine/mL was 172 + 7.8 min at pH 2.0 and 246 ± 12.8 min at pH 3.2. The SEM pictures in Fig. 4 show dentin surfaces upon treatment with (a) 0.5 mol/L EDTA (pH 7.4) and Gluma, (b) Gluma (pH 1.0) followed by a blast of compressed air, and (c) Gluma (pH 1.0) followed by a 10-20-second water rinse. At higher magnification (x20,000), a faint layer could be distinguished on the surface of specimen (a). The cracks observed in (b) were visible only on a small area of the prepared surface, but gave the impression of a polymerized layer of some thickness. The layer seen in (c) was clearly thinner than the layer in (b), probably due to partial removal by the water rinse.
Discussion.
Fig. 4-SEM pictures of dentin surfaces treated with (a) 0.5 mol/mL EDTA, pH 7.4, followed by Gluma, (b) Gluma at pH 1.0 without water spray, and (c) Gluma at pH 1.0 for 40 s, followed by a 10-20-second water rinse. The pH adjustment was performed with hydrochloric acid. The treatment of the dentin surface in (b) was identical to the pre-treatment performed on the specimens used for bond strength measurements at pH 1.0 (Table 3) (SEM X 2500, bar = 10 Aim).
Aqueous glutaraldehyde solution consists of no more than 15% of free aldehyde in equilibrium with glutaraldehydemonohydrate transformed to a cyclic hemi-acetal and its ohgomers (Korn et al., 1972). A number of reaction products have been postulated for the reaction between glutaraldehyde and amino acids, as well as proteins. Among these are (1) Schiff's bases of mono-aldehyde or its polymeric aldol condensation products; (2) formation of cyclic carbinolamines, dihydropyridinium, as well as pyridinium compounds derived from the cyclic hemiacetal, and polymeric products derived from condensation reactions with linear glutaraldehyde; and (3) Michael adducts of the amine to ox,4-unsaturated glutaraldehyde aldol condensation products (Hardy et al., 1976; Johnson, 1985a,b; Lubig et al., 1981; Nimni et al., 1987). These reaction mechanisms are not readily able to explain the results in the present study. For example, glycine and propionaldehyde/HEMA are unlikely to form pyridinium derivatives. Lubig et al. (1981) and Johnson (1985b) observed that the reaction between glycine and glutaraldehyde was followed by formation of H+ in the initial phase and later on by a consumption of H+. This might explain the pH dependence of the reaction as seen in Figs. 1, 2, and 3, as well as the results with propionaldehyde. The results in Fig. 2 might be explained in much the same way; that is, at low glutaraldehyde concentrations, the formation of H+ is the rate-determining process,
Downloaded from jdr.sagepub.com at Monash University on October 24, 2014 For personal use only. No other uses without permission.
AMINES AND POLYMERIZATION OF HEMA/ALDEHYDE
Vol. 69 No. 6
1239
TABLE 2
TABLE 3
GEL TIMES OF GLUMA, pH 1.0, WITH 8 pumol GLYCINE/mL at 370C IN CLOSED VIALS OR IN VIALS UNDER AIR OR HELIUM Gel time, minutes Condition 38 + 3 Closed vial Air bubbling no gelation within 6 h Helium bubbling 41 + 2
SHEAR BOND STRENGTH BETWEEN RESIN COMPOSITE AND DENTIN PRE-TREATED WITH pH-ADJUSTED GLUMA Bond strength, pH in Gluma MPa (SD) 0.2 1.22 (1.43) 0.6 11.72 (7.25) 1.0 27.79 (8.23) 1.4 15.65 (9.67) 1.8 9.29 (6.63) 2.9 7.31 (3.18)
but at higher concentrations, the process involving consumption of H+ is rate-determining. Johnson (1985a) observed that rapid oxygen consumption takes place during the reaction between amines, as well as proteins and glutaraldehyde. This agrees well with the results presented in Table 2, which can be interpreted in at least two ways for explanation of the polymerization of HEMA, presumably taking place during the reaction with amines and glutaraldehyde/HEMA. One interpretation is that the polymerization was caused by removal of oxygen during the reaction. Another interpretation is that a product, which acted as a polymerization initiator, was formed under anaerobic conditions. The linear relationship between gel time and the reciprocal of the glycine concentration (Fig. 1), as well as the reciprocal of the glutaraldehyde concentration (Fig. 2), suggest second-order reaction kinetics. The results from the experiment in which the reaction product of Gluma and glycine was extracted and dried indicate that, in time, nearly all HEMA in such solutions will be transformed. Because of the variation between duplicate experiments, it was not possible for us to decide to what extent glutaraldehyde took part in the process. The insolubility of the reaction product in a number of solvents indicates a crosslinked polymer. Such cross-linking might occur between polyHEMA chains, either by hemi-acetal formation by reaction with dialdehyde or by polymerization of HEMA together with oligomeric polyaldehyde chains with ox,3-unsaturated bonds. Aldehydes, together with small amounts of amine (Table 1), may represent an alternative way of polymerizing methacrylates, and work is in progress so that knowledge about this possibility may be increased. The coincidence between the pH at which gelation occurred at its maximum velocity (Fig. 3) and the pH at which the maximum shear bond strength occurred (Table 3) supports the assumption that the transformation of HEMA discussed above may play a role when Gluma is used as a dentin bonding agent. It is proposed that on application of Gluma, substances in dentin containing amino groups react with glutaraldehyde and start the formation of a HEMA polymer. Such substances might be collagen, non-collagenous proteins, small peptides produced by grinding, and a number of other substances containing amino groups left over from the cell processes. This product may be cross-linked by ox,P-unsaturated glutaraldehyde aldol condensation product and bond to dentin by aldehyde fixation to dentin proteins. Resin composite will bond to this product by copolymerization. This is supported by the results presented in Fig. 4. Other mechanisms may also be of importance, such as the penetration into dentin of HEMA, which interlocks during polymerization, and a strengthening of the surface collagen layer by aldehyde fixation. The results may also explain the results of Asmussen and Bowen (1987). In these experiments, relatively high bond strengths between composite and Glumatreated dentin were obtained when the dentin was pre-treated with certain acidic glycine solutions.
Acknowledgment. Technical assistant Vivi R0nne is complimented for care and
competence during performance of the experiments. REFERENCES ASMUSSEN, E. and BOWEN, R.L. (1987): Effect of Acidic Pretreatment on Adhesion to Dentin Mediated by Gluma, JDent Res 66:13861388. ASMUSSEN, E. and MUNKSGAARD, E.C. (1985): Bonding of Restorative Resins to Dentin Promoted by Aqueous Mixtures of Aldehydes and Reactive Monomers, Int Dent J 35:160-165. ELIADES, G.C.; CAPUTO, A.A.; and VOUGIOUKLAKIS, G.J. (1985): Composition, Wetting Properties and Bond Strength with Dentin of 6 New Dentin Adhesives, Dent Mater 1:170-176. HANSEN, E.K. and ASMUSSEN, E. (1989): Marginal Adaptation of Posterior Resins: Effect of Dentin-Bonding Agent and Hygroscopic Expansion, Dent Mater 5:122-126. HARDY, P.M.; HUGHES, G.J.; and RYDON, H.N. (1976): Formation of Quaternary Pyridinium Compounds by the Action of Glutaraldehyde on Proteins, J Chem Soc Chem Comm 5:157-158. JOHNSON, T.J.A. (1985a): Glutaraldehyde Fixation Chemistry. Scheme for Rapid Crosslinking and Evidence for Rapid Oxygen Consumption. In: Science of Biological Specimen Preparation, M. Mueller, R.P. Becker, A. Boyde, and J.J. Volosewich, Eds., Chicago: SEM Inc., AMF O'Hare, USA, pp. 51-62. JOHNSON, T.J.A. (1985b): Aldehyde Fixatives: Quantification of Acidproducing Reactions, J Electron Microsc Tech 2:129-138. KOMATSU, M. and FINGER, W. (1986): Dentin Bonding Agents: Correlation of Early Bond Strength with Margin Gaps, Dent Mater 2:257262. KORN, A.H.; FAIRHELLER, S.H.; and FILACHIONE, E.M. (1972): Glutaraldehyde: Nature of the Reagent, J Mol Biol 65:525-529. LUBIG, R.; KUSCH, P.; ROPER, K.; and ZAHN, H. (1981): Zum Reaktionsmechanismus von Glutaraldehyd mit Proteinen, Monatshefte fUr Chemie 112:1313-1323. MUNKSGAARD, E.C. and ASMUSSEN, E. (1984): Bond Strength Between Dentin and Restorative Resins Mediated by Mixtures of HEMA and Glutaraldehyde, J Dent Res 63:1087-1089. MUNKSGAARD, E.C. and ASMUSSEN, E. (1985): Dentin-Polymer Bond Mediated by Glutaraldehyde/HEMA, Scand J Dent Res 93:463-466. MUNKSGAARD, E.C. and ASMUSSEN, E. (1987): Methacrylate-Bonding to Dentin. In: Dentine and Dentine Reactions in the Oral Cavity, A. Thylstrup, S.A. Leach, and V. Qvist, Eds., Oxford: IRL Press Ltd., pp. 209-218. MUNKSGAARD, E.C. and IRIE, M. (1988): Effect of Load-Cycling on Bond Between Composite Fillings and Dentin Established by Gluma and Various Resins, Scand J Dent Res 96:579-583. NIMNI, M.E.; CHEUNG, D.; STRATES, B.; KODAMA, M.; and SHEIKH, K. (1987): Chemically Modified Collagen: A Natural Biomaterial for Tissue Replacement, J Biomed Mater Res 21:741-771. RETIEF, D.H.; O'BRIEN, J.A.; SMITH, L.A.; and MARCHMAN, J.L. (1988): In vitro Investigation and Evaluation of Dentin Bonding Agents, Am J Dent 1:176-183.
Downloaded from jdr.sagepub.com at Monash University on October 24, 2014 For personal use only. No other uses without permission.
