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Effects of acid-etching on the tensile properties of demineralized dentin matrix
Dent Mater 14:222–228, June, 1998
Effects of acid-etching on the tensile properties of demineralized dentin matrix Yi Zhang 1, Kelli Agee 1, Jacques No¨r 2, Ricardo Carvalho 3, Bhupinder Sachar 1, Carl Russell 4, David Pashley 1
a1
Department of Oral Biology, School of Dentistry and 4Office of Biostatistics, Medical College of Georgia, Augusta, Georgia, USA 2 School of Graduate Studies, University of Michigan, Ann Arbor, Michigan, USA 3 Department of Restorative Dentistry, Bauru Dental School, University of Sa˜o Paulo, Bauru, Sa˜o Paulo, BRAZIL
ABSTRACT
INTRODUCTION
Objectives. Little research has been done to evaluate the effects of acids commonly used in adhesive dentistry, on the tensile properties of the demineralized dentin matrix. The purpose of this study was to evaluate the effects of a number of acidic conditioners on the ultimate tensile strength (UTS) and modulus of elasticity (E) of human coronal dentin matrix. Methods. Small hour-glass shaped (for UTS) or I-beam shaped (for determination of E) were prepared from mid-coronal dentin of extracted human third molars. After protecting the ends with varnish, the middle of the specimens was completely demineralized in 0.5 M EDTA (pH 7). UTS was determined by tensile stressing to failure. Modulus of elasticity was calculated from stress strain curves. The results were analyzed by ANOVA and Student–Neuman–Keuls test at the 95% confidence level. Results. Brief (ca. 1–2 min) exposure of demineralized dentin matrix to acids had no measurable effects on its tensile properties. Tenminute exposures to 2.5% and 17.5% nitric acid lowered ( p ⬍ 0.05) the UTS compared to phosphate buffered saline (PBS)-exposed controls. Exposure of the decalcified dentin to 10% citric acid containing 3% ferric chloride, 10% citric acid, 37% phosphoric acid or 17.5% nitric acid containing 3% ferric chloride for 10 min had no effect on UTS. None of these acids consistently lowered stiffness. Significance. The results indicate that relatively long exposures to acids are required to alter the tensile properties of demineralized dentin. It is unlikely that the brief exposures to acids that are used in adhesive dentistry would acutely weaken the physical properties of demineralized dentin. However, long-term studies should be done to determine if such treatment increases the susceptibility of the matrix to hydrolysis. 䉷 1998 Published by Elsevier Science Ltd on behalf of the Academy of Dental Materials
In order to provide micromechanical retention of adhesive resins to dentin, most bonding systems employ an acidic conditioner to remove the smear layer and demineralize the top 5–6 mm of dentin (Van Meerbeek et al., 1992; Perdiga˜o et al., 1996). There is concern, however, that these acids might have an adverse effect on the collagen that makes up 90% of the dentin matrix (Butler, 1995). Veis and Schlueter, 1964 demonstrated that demineralized dentin collagen was dimensionally stable in acidic solutions, unlike dermal collagen that exhibited extensive swelling. Several groups have shown that exposure of collagen to acids or other denaturing agents, makes collagen susceptible to proteolysis by trypsin (Scott and Leaver, 1974; Mizunuma, 1986; Dung et al., 1994). Mizunuma (1986) reported that 37% phosphoric acid made bovine dentin matrix more susceptible to the proteolytic effects of trypsin than did 10% citric acid, and that addition of 3% ferric chloride to 10% citric acid offered protection of the collagen to the action of trypsin. Okamoto et al., 1991 blocked the denaturing effect of phosphoric acid on dentin collagen by pretreatment with tannic acid, which was thought to cause crosslinking of the collagen, thereby increasing its stability to acids. Nimni and Cheung (1994) discovered that they could preserve the fibrillar structure of bone collagen in 0.5 M acetic acid/ pepsin solutions by increasing the concentration of NaCl in the medium from 0.9 to 5%. It was thought that the high ionic strength of the solution prevented the disaggregation of the collagen fibrils in acetic acid.
222 Zhang et al./Effects of acid on collagen strength
PII: S0109-5641(98)00035-9
Fig. 1. Schematic of sample preparation from mid-coronal human dentin disk 0.5 mm thick (A). For measurements of ultimate tensile strength, hour-glass shaped specimens were cut from the disk with a bur (B). For measurements of modulus of elasticity or stiffness, ‘‘I’’ beam shaped specimens were cut from the disks (C). After covering the ends of the specimens with varnish, the middle region was demineralized in EDTA.
Type I collagen is an unusual protein in that it exists in situ as an insoluble, fibrous structural protein without any enzymatic activity (Nimni, 1991). Thus, the usual properties that characterize denatured proteins, such as loss of enzymatic activity or precipitation from solution, can not be used to identify denaturation. However, the tensile properties of this structural protein can be evaluated as an indication of its integrity. Little work has been done on the effects of acids on the tensile properties of decalcified dentin. Akimoto (1991) reported that the ultimate tensile strength of 0.5 M EDTA-demineralized bovine dentin fell from 28 MPa to 15 MPa when it was immersed in 37% phosphoric acid for 1 min. However, No¨r et al. (1996) were unable to confirm that observation. The purpose of this study was to evaluate the effects of acids commonly used in adhesive dentistry, on the tensile properties of human demineralized dentin. The null hypothesis to be tested is that acids commonly used with dental adhesives have no adverse effect on the ultimate tensile strength and the modulus of elasticity of demineralized dentin matrix.
MATERIALS AND METHODS Teeth. All specimens were prepared from mid-coronal dentin of extracted, unerupted human third molars. The teeth were stored at 4⬚C in isotonic saline, saturated with thymol, to inhibit microbial growth. They were used within 1 month of extraction. Using a diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA), the occlusal enamel was removed from the crown, and dentin disks 0.5 mm in thickness were prepared (Fig. 1). Either hour-glass or an ‘‘I’’ beam pattern were drawn on the disk with a pencil. The dentin outside the pattern was cut away with an ultrafine diamond bur in a highspeed handpiece using copious air–water spray, to leave the dentin in one of the two patterns (Fig. 1).
Both ends of the specimens were covered with two layers of nail varnish leaving the middle section uncovered and susceptible to 0.5 M EDTA (pH 7.4) demineralization for 5 days at 25⬚C. The EDTA solution contained 0.5 mmol/L phenylmethylsulfonyl fluoride and 0.5 mmol/L N-ethyl-maleimide to inhibit proteolytic enzymes during demineralization. The volume to mass ratio of the EDTA to dentin was 10 5. The decalcification time was selected from microradiography data and by longitudinal evaluation of decreases in the modulus of elasticity of the specimens as previously described (Maciel et al., 1996). Tensile testing. The ultimate tensile strength (UTS) was measured after scraping the nail varnish from each mineralized end using a scalpel, and then fixing each end into the friction grips of a Vitrodyne V1000 tester (Chatillon, Greensboro NC, USA). Specimens were subjected to tensile load at 0.6 mm/min until failure to obtain the ultimate tensile load, which was converted to the ultimate tensile stress by dividing by the crosssectional area of the narrowest part of the hour-glass shape. This was generally 0.5 × 0.5 mm but was measured to the nearest 0.01 mm with a digital micrometer (Sylvae Ultra-Cal II, Fowler Inc., Newton MN, USA) in each specimen. All tensile testing was done with the specimens immersed in 25⬚C phosphate buffered saline (PBS) unless otherwise stated. There were seven groups: PBS, 10% citric acid containing 3% ferric chloride or 10–3 solution, 10% citric acid, 37% phosphoric acid, 2.5% nitric acid, 17.5% nitric acid, and 17.5% nitric acid containing 3% ferric chloride. All concentrations are wt%. Each group consisted of five to six specimens. The specimens were placed in the acidic solution for 10 min, rinsed in water and returned to PBS for testing unless otherwise stated. Modulus of elasticity (E) was measured on ‘‘I’’ beamshaped specimens (Fig. 1) which had gauge-lengths of Dental Materials/June 1998 223
TABLE 1: COMPOSITION AND pH OF TEST SOLUTIONS Test solutions
Additives
pH
PBS
–
7.40
10% citric acid
–
1.89
10% citric acid
3% FeCl3
1.20
37% phosphoric acid
–
0.90
2.5% nitric acid
–
0.92
17.5% nitric acid
–
0.42
17.5% nitric acid
3% FeCl3
0.34
All compositions are wt%.
3.0 ⫾ 0.1 mm. The nail-varnish was scraped off each mineralized end prior to clamping the ends in the friction grips of the Vitrodyne tester. Two different types of tests were performed to measure the modulus of elasticity of the decalcified dentin: destructive versus nondestructive testing. Destructive testing was done by simply measuring the load–displacement relationship of each sample in PBS until failure. Specimens exposed to acids for 10 min were returned to PBS for 10 min before testing. The load–displacement curves were then converted to stress–strain curves using the cross-sectional area, gauge length and elongation of the specimens. The moduli of elasticity (E) were calculated in the regions between 10–12% strain (moderate strain) and 20–24% strain (high strain). Nondestructive testing was done with another set of specimens to provide repeated measures testing, to permit each specimen to serve as its own control, in an attempt to increase the statistical power. Each specimen was strained to 10–12% at 0.6 mm/min to obtain a pretreatment or control stiffness in PBS. Only specimens with a pretreatment or control stiffness between 20 and 35 MPa at 10% strain were included in this experiment, in an effort to reduce the variance. Each specimen was then unloaded and allowed to elastically recover. Then the specimen was immersed in the test acid for 10 min and the stiffness of the specimen was remeasured at 10–12% strain in the acid. After unloading the specimen, it was returned to PBS for a 10 min recovery before remeasuring stiffness again. Thus, in this experiment the stiffness of each specimen was measured prior to exposure to acid, during exposure to acid and again in PBS after recovery. There were six specimens in each (E) group and six groups (10–3 solution, 10% citric acid, 37% phosphoric acid, 2.5% nitric acid and 17.5% nitric acid, 17.5% nitric acid containing 3% ferric chloride). The pH of the solutions (Table 1) was measured with a standard pH meter (Accumet, Fisher Scientific, 224 Zhang et al./Effects of acid on collagen strength
Pittsburgh, PA, USA) calibrated with appropriate standards. Statistics. Power analysis was done to estimate sample size in each group for a one-way ANOVA assuming a difference between the means of 0.5 and a standard deviation of 0.2, with seven groups, a = 0.05 and a power of 0.8. The analysis indicated a sample size of five was adequate. After calculating descriptive statistics, the results were analyzed by one-way ANOVA to identify differences among the groups. Multiple comparisons were done using the Student–Neuman–Keuls test. Statistical significance was considered as p ⬍ 0.05. Power analysis was re-run on the data obtained from nondestructive testing to establish the power of the oneway ANOVA using six specimens in a group and six groups in a repeated measures design.
RESULTS The UTSs of hour-glass-shaped demineralized dentin specimens exposed to various acidic solutions for 10 min are shown in Fig. 2. There were no statistically significant changes produced by 10% citric acid or 10–3 solution compared to the PBS control group. Treatment with 37% phosphoric acid lowered the UTS, but the decrease was not statistically significant. Treatment with either 2.5% or 17.5% nitric acid caused statistically significant (p ⬍ 0.05) reductions in UTS. Addition of 3% ferric chloride to the 17.5% nitric acid provided significant protection to the effects of nitric acid on UTS in that the UTS of that group was not statistically different from PBS controls (Fig. 2). The power of that ANOVA was 0.92. When the modulus of elasticity was measured destructively (Fig. 3), there were no statistically significant differences among the treatment groups when the modulus was calculated from the linear portion of the stress–strain curve at an elongation of 20–24% (just before failure). When the modulus was calculated at 10–12% strain, (moderate strain), there were still few significant differences among the groups (Fig. 3). The power of these analyses ranged from 0.80 (at 20–24% strain) to 0.84 (at 10–12% strain). The lack of consistent, significant changes in stiffness produced by the acidic conditioners in the destructive measurements of modulus of elasticity was due, in part, to the relatively high variance within the groups. The high variance was due to a relatively wide range in moduli of elasticity of our dentin specimens. The distribution of stiffness of 100 specimens of demineralized dentin in PBS is shown in Fig. 4. All of these specimens had been demineralized for 5 days. The specimens exhibiting stiffness values ⬎30 MPa were returned to 0.5 M EDTA for several more days. However, repeated nondestructive measurements of their stiffness were reproducible indicating that the higher stiffness was not due to lack of complete demineralization. All specimens were strained to 10– 12%. Those specimens ⬍20 MPa or ⬎35 MPa were excluded from the nondestructive, repeated measurement of the effects of acids on the stiffness of
Fig. 2. Effects of various treatment solutions on the ultimate tensile strength of demineralized dentin matrix. Bars represent mean ⫾ SD. Numbers in parentheses indicate the number of specimens tested in each group. Each specimen was exposed to one of the acidic solutions for 10 min and then returned to PBS for tensile testing. 10–3, 10% citric acid containing 3% ferric chloride; CA, citric acid; PA, phosphoric acid; NA, nitric acid; FC, ferric chloride. All concentrations are wt%. Groups connected by the same horizontal line are not significantly different (p ⬎ 0.05). Groups not connected by the same horizontal line are significantly different (p ⬍ 0.05).
Fig. 3. Effects of various treatment solutions on the moduli of elasticity of demineralized dentin matrix at moderate (10–12%) and high (20–24%) strains. The height of bar is the mean, brackets indicate ⫾SD, numbers in parentheses are the number of specimens tested. Light bars, 10–12% strain; dark bars, 20–24% strain. Multiple comparison testing indicated that there were no statistically significant differences in modulus of elasticity (20–24% strain) among the treatment groups. At the moderate strain (10–12%), the only significant reduction in modulus was in the 17.5% nitric acid group (indicated by an asterisk). Symbols are the same as in Fig. 2.
Fig. 4. Histogram of the distribution of moduli of elasticity among 100 specimens of demineralized dentin ‘‘I’’ beams nondestructively strained to 10%. For repeated measurements, only specimens with moduli between 20–30 MPa were used.
demineralized dentin, and the groups were balanced so that they all had similar mean values. These specimens were then subjected to repeated strains of 10–12% in PBS before, during and after 10 min treatments in the acidic solutions, to obtain
repeated percent changes in stiffness in the same specimens. The use of those moderate strains avoided irreversible damage to the specimens. This was sufficient to develop reproducible stresses and yet the specimens showed excellent elastic behavior. The upper Dental Materials/June 1998 225
Fig. 5. Repeatability of stress–strain curves of an individual specimen strained to about 10% in PBS three times in succession (upper group of curves). After immersion in 17.5% nitric acid for 10 min, the specimen was returned to PBS and three more stress–strain curves were made (lower group of curves).
Fig. 6. Effects of various acidic solutions on changes in the stiffness of demineralized dentin matrix when strained to 10% in the control PBS solution (c), when strained in the test acid after 10 min (the middle bar graph of each triplet), and again after recovery (R) in PBS for 10 min. The horizontal line at 100% indicates control value. The height of the bar is the mean and the brackets indicate ⫾1 SD. Each specimen served as its own control. There were six specimens in each group. The absolute modulus of elasticity of each group was 28.4 ⫾ 4.1 (mean ⫾ S.D., N = 36). Symbols are the same as in Fig. 2.
curve in Fig. 5 shows a representative example of a specimen that underwent a 10% elongation three times while immersed in PBS. Since there was no evidence of any consistent difference between the stress–strain curves, they were averaged and assigned a value of 100%. The lower three curves in Fig. 5 represent the stress–strain curves of the same sample exposed to 17.5% HN0 3 for 10 min and then tested in PBS. This acidic conditioner produced a significant reduction (p ⬍ 0.05) in the modulus of elasticity of this specimen. Fig. 6 summarizes the effects of the acidic conditioners on the stiffness of decalcified dentin that was tested nondestructively in a repeated manner. The data have been expressed as percent changes from PBS controls to reduce the variance. By limiting the test specimens to those that had stiffness values between 20 and 35 MPa, and by using the pretreatment values as 100%, the standard deviations were reduced to 10– 20%. None of the commonly used acidic conditioners (10% citric acid, 10–3 solution, 37% phosphoric acid) produced any significant change in the stiffness of the decalcified matrix whether tested in the acid or in PBS, 10 min later. However, multiple comparisons revealed 226 Zhang et al./Effects of acid on collagen strength
that all nitric acid formulations lowered the stiffness (p ⬍ 0.001) when tested in acid. The statistical power of this test was 0.99. There were no significant (p = 0.22) changes in stiffness when the specimens were retested in PBS.
DISCUSSION The lack of a significant consistent effect of most of the acidic conditioners on the tensile properties of demineralized dentin indicates that concerns about the denaturing effects of acids on the mechanical properties of decalcified dentin appear largely unwarranted. In the present study, exposure of EDTA-decalcified dentin to 10% citric acid, 10–3 solution, or 37% phosphoric acid, or 17.5% nitric acid containing 3% ferric chloride did not lower the UTS of decalcified dentin (Fig. 2), although 2.5 and 17.5% nitric acid did reduce it (p ⬍ 0.05). These prolonged exposures to acids are far removed from clinical practice, but were used because shorter exposures produced no measurable changes (No¨r et al., 1996). It should be emphasized that what was measured in this study was not the stiffness of collagen but of a
collagenous meshwork that includes noncollagenous proteins and proteoglycans. The modulus of elasticity of collagen molecules was reported to be 2.9–9.0 GPa for Achilles tendon (Sasaki and Odajima, 1996). Unlike tendon, the collagen fibrils in dentin are arranged more randomly. When demineralized dentin matrix is stressed, the load is not evenly distributed but is preferrentially borne by the fibrils oriented in the direction of straining. Thus, it is not surprising that the stress–strain properties of demineralized dentin are different from other connective tissues. Because the organic matrix of dentin only occupies 30–48% of the total volume (Kinney et al., 1994; Sano et al., 1995), the tensile values of the collagen fibrils themselves obtained in this study could be two to three times higher if the stresses were divided by the actual crosssectional areas of the collagen fibrils. When measuring the stress–strain relationships of demineralized dentin matrix, it was not necessary to use a ‘‘preconditioning’’ strain cycle as is often done in collagenous tissues that contain connective tissue sheaths within the tissue (Van Brocklin and Ellis, 1965; Kastelic et al., 1978; Bowman et al., 1996). This was demonstrated by the high reproducibility of the stress–strain curves that were nearly identical (Fig. 5) and showed no hysteresis that has been reported in tendon (Rigby et al., 1959). The effects of acidic conditioners on the tensile properties of demineralized dentin are complex. The low pH of the solutions presumably alters the tertiary configuration of the collagen peptides (Eliades et al., 1997), but the correlation between mild denaturation of collagen and changes in UTS or stiffness is unknown. With some acids, such as 17.5% nitric acid, with or without ferric chloride, the reduction in stiffness was reversible when the specimen was returned to PBS (Fig. 6). Ferric chloride (3%) prevented the decrease in UTS that was obtained following treatment of demineralized dentin with 17.5% nitric acid (Fig. 2). The inability of 3% ferric chloride to prevent the fall in modulus of elasticity produced by 17.5% nitric acid (Figs. 3 and 6) may be explained as follows: Solutions with high hydrogen ion concentrations can change the teritary structure of proteins by protonation of amino groups and by suppression of ionization of carboxyl groups on bifunctional amino acids that have free amino or carboxyl groups. This transient gain of positive charge by basic amino acids and loss of negative charge by glutamic and aspartic acid may induce changes in the configuration of nonhelical portions of collagen peptides. Indeed, the helical portions of the peptides may provide enough structural stability to prevent the disaggregation of collagen peptides during relatively brief exposures to acidic solutions at room temperature. When the acidified demineralized dentin is rinsed with PBS, the amino groups would become deprotonized and carboxyl groups would dissociate and the tertiary structure may return. If very high concentrations of hydrogen ions are used (e.g. 17.5%, HNO 3), there may be disaggregation of collagen fibrils leading to a decrease in the UTS of the demineralized dentin matrix (Figs. 2 and 3).
CONCLUSIONS The results of this study would predict minimal changes in tensile properties of the dentin matrix following acid etching under clinically revelant conditions. ACKNOWLEDGEMENTS This work was supported, in part, by grant DE06427 from the National Institute of Dental Research, by the Medical College of Georgia Biocompatibility Group and by FAPESP grant 95/3895-9 (to RMC). Received April 3, 1998 / Accepted April 20, 1998 Address correspondence and reprint requests to: Dr D.H. Pashley Department of Oral Biology School of Dentistry Medical College of Georgia Augusta GA 30909-1129 USA Tel.: 001-706-721-2033 Fax: 001-706-721-6252 E-mail: [email protected]
REFERENCES Akimoto T (1991). Study of adhesion of MMA-TBBO resin to dentin. J Jpn Soc Dent Mater and Device 10:42–54. Bowman SM, Zeind J, Gibson LJ, Hayes WC, McMahons TA (1996). The tensile behavior of demineralized bovine cortical bone. J Biomechanics 29:1497–1501. Butler WT (1995). The nature and functional significance of dentin extracellular matrix proteins. Int J Dev Biol 39:169–179. Dung S-Z, Li Y, Dumpace AJ, Stookey GK (1994). Degradation of insoluble bovine collagen and human dentine collagen pretreated in vitro with lactic acid, pH 40 and 55. Arch Oral Biol 39:901–905. Eliades G, Palaghias G, Vougiouklakis G (1997). Effect of acidic conditioners on dentin morphology, molecular composition and collagen configuration in situ. Dent Mater 13:24–33. Kastelic J, Galeski A, Baer E (1978). Composite structure of tendon. Connec Tiss Res 6:11–23. Kinney JH, Marshall GW, Marshall SJ (1994). Three dimensional mapping of mineral densities in carious dentin: theory and method. Scanning Microscopy 8:197–205. Maciel KT, Carvallo RM, Ringle PD, Preston CD, Russell CM, Pashley DH (1996). The effects of acetone, ethanol, HEMA and air on the stiffness of human demineralized dentin. J Dent Res 75:1851– 1858. Mizunuma T (1986). Relationship between bond strength of resin to dentin and structural change in dentin collagen during etching—Influence of ferric chloride to structure of the collagen. J Jpn Dent Mater 5:54–64. Dental Materials/June 1998 227
Nimni ME (1991). Collagen, structure and function. In: Dulbecco R, editor, Encyclopedia of Human Biology, vol. 2, 2nd ed. New York: Academic Press, 1991, 559– 575. Nimni ME, Cheung DT (1994). Process for purifying collagen and generating bioprostheses. US Patent 5374539. United States Patent and Trademark Office, Arlington, VA/Washington, DC. No¨r J, Carvalho RM, Pashley DH (1996). Effect of potentially denaturing agents on tensile properties of collagen. J Dent Res Special Issue 75:347. Abst #2635. Okamoto Y, Heeley JD, Dogon IL, Shintani H (1991). Effects of phosphoric acid and tannic acid on dentine collagen. J Oral Rehabilitation 18:507–512. Perdiga˜o J, Lambrechts P, Van Meerbeek B, Tome´ AR, Vanherle G, Lopes AB (1996). Morphological field emission-SEM study of the effect of six phosphoric acid etching agents on human dentin. Dent Mater 12:262–271. Rigby BJ, Hirai N, Spikes JD, Eyring H (1959). The mechanical properties of rat tail tendon. J Gen Physiol 43:265–283.
228 Zhang et al./Effects of acid on collagen strength
Sano H, Takatsu T, Ciucchi B, Russell CM, Pashley DH (1995). Tensile properties of resin-infiltrated demineralized human dentin. J Dent Res 74:1093– 1102. Sasaki N, Odajima S (1996). Stress–strain curve and Young’s modulus of a collagen molecule as determined by the X-ray diffraction technique. J Biomechanics 29:655–658. Scott PG, Leaver AG (1974). The degradation of human collagen by trypsin. Connect Tiss Res 2:299–307. Van Brocklin JD, Ellis DG (1965). A study of the mechanical behavior of toe extensor tendons under applied stress. Arch Phys Med Rehab 46:369–373. Veis A, Schlueter RJ (1964). The macromolecular organization of dentine matrix collagen I Characterization of dentine collagen. Biochem J 3:1650–1657. Van Meerbeek B, Inokoshi S, Braem M, Lambrechts P, Vanderle G (1992). Morphological aspects of the resin–dentin interdiffusion zone with different adhesive systems. J Dent Res 71:1530–1540
Effects of acid-etching on the tensile properties of demineralized dentin matrix Yi Zhang 1, Kelli Agee 1, Jacques No¨r 2, Ricardo Carvalho 3, Bhupinder Sachar 1, Carl Russell 4, David Pashley 1
a1
Department of Oral Biology, School of Dentistry and 4Office of Biostatistics, Medical College of Georgia, Augusta, Georgia, USA 2 School of Graduate Studies, University of Michigan, Ann Arbor, Michigan, USA 3 Department of Restorative Dentistry, Bauru Dental School, University of Sa˜o Paulo, Bauru, Sa˜o Paulo, BRAZIL
ABSTRACT
INTRODUCTION
Objectives. Little research has been done to evaluate the effects of acids commonly used in adhesive dentistry, on the tensile properties of the demineralized dentin matrix. The purpose of this study was to evaluate the effects of a number of acidic conditioners on the ultimate tensile strength (UTS) and modulus of elasticity (E) of human coronal dentin matrix. Methods. Small hour-glass shaped (for UTS) or I-beam shaped (for determination of E) were prepared from mid-coronal dentin of extracted human third molars. After protecting the ends with varnish, the middle of the specimens was completely demineralized in 0.5 M EDTA (pH 7). UTS was determined by tensile stressing to failure. Modulus of elasticity was calculated from stress strain curves. The results were analyzed by ANOVA and Student–Neuman–Keuls test at the 95% confidence level. Results. Brief (ca. 1–2 min) exposure of demineralized dentin matrix to acids had no measurable effects on its tensile properties. Tenminute exposures to 2.5% and 17.5% nitric acid lowered ( p ⬍ 0.05) the UTS compared to phosphate buffered saline (PBS)-exposed controls. Exposure of the decalcified dentin to 10% citric acid containing 3% ferric chloride, 10% citric acid, 37% phosphoric acid or 17.5% nitric acid containing 3% ferric chloride for 10 min had no effect on UTS. None of these acids consistently lowered stiffness. Significance. The results indicate that relatively long exposures to acids are required to alter the tensile properties of demineralized dentin. It is unlikely that the brief exposures to acids that are used in adhesive dentistry would acutely weaken the physical properties of demineralized dentin. However, long-term studies should be done to determine if such treatment increases the susceptibility of the matrix to hydrolysis. 䉷 1998 Published by Elsevier Science Ltd on behalf of the Academy of Dental Materials
In order to provide micromechanical retention of adhesive resins to dentin, most bonding systems employ an acidic conditioner to remove the smear layer and demineralize the top 5–6 mm of dentin (Van Meerbeek et al., 1992; Perdiga˜o et al., 1996). There is concern, however, that these acids might have an adverse effect on the collagen that makes up 90% of the dentin matrix (Butler, 1995). Veis and Schlueter, 1964 demonstrated that demineralized dentin collagen was dimensionally stable in acidic solutions, unlike dermal collagen that exhibited extensive swelling. Several groups have shown that exposure of collagen to acids or other denaturing agents, makes collagen susceptible to proteolysis by trypsin (Scott and Leaver, 1974; Mizunuma, 1986; Dung et al., 1994). Mizunuma (1986) reported that 37% phosphoric acid made bovine dentin matrix more susceptible to the proteolytic effects of trypsin than did 10% citric acid, and that addition of 3% ferric chloride to 10% citric acid offered protection of the collagen to the action of trypsin. Okamoto et al., 1991 blocked the denaturing effect of phosphoric acid on dentin collagen by pretreatment with tannic acid, which was thought to cause crosslinking of the collagen, thereby increasing its stability to acids. Nimni and Cheung (1994) discovered that they could preserve the fibrillar structure of bone collagen in 0.5 M acetic acid/ pepsin solutions by increasing the concentration of NaCl in the medium from 0.9 to 5%. It was thought that the high ionic strength of the solution prevented the disaggregation of the collagen fibrils in acetic acid.
222 Zhang et al./Effects of acid on collagen strength
PII: S0109-5641(98)00035-9
Fig. 1. Schematic of sample preparation from mid-coronal human dentin disk 0.5 mm thick (A). For measurements of ultimate tensile strength, hour-glass shaped specimens were cut from the disk with a bur (B). For measurements of modulus of elasticity or stiffness, ‘‘I’’ beam shaped specimens were cut from the disks (C). After covering the ends of the specimens with varnish, the middle region was demineralized in EDTA.
Type I collagen is an unusual protein in that it exists in situ as an insoluble, fibrous structural protein without any enzymatic activity (Nimni, 1991). Thus, the usual properties that characterize denatured proteins, such as loss of enzymatic activity or precipitation from solution, can not be used to identify denaturation. However, the tensile properties of this structural protein can be evaluated as an indication of its integrity. Little work has been done on the effects of acids on the tensile properties of decalcified dentin. Akimoto (1991) reported that the ultimate tensile strength of 0.5 M EDTA-demineralized bovine dentin fell from 28 MPa to 15 MPa when it was immersed in 37% phosphoric acid for 1 min. However, No¨r et al. (1996) were unable to confirm that observation. The purpose of this study was to evaluate the effects of acids commonly used in adhesive dentistry, on the tensile properties of human demineralized dentin. The null hypothesis to be tested is that acids commonly used with dental adhesives have no adverse effect on the ultimate tensile strength and the modulus of elasticity of demineralized dentin matrix.
MATERIALS AND METHODS Teeth. All specimens were prepared from mid-coronal dentin of extracted, unerupted human third molars. The teeth were stored at 4⬚C in isotonic saline, saturated with thymol, to inhibit microbial growth. They were used within 1 month of extraction. Using a diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA), the occlusal enamel was removed from the crown, and dentin disks 0.5 mm in thickness were prepared (Fig. 1). Either hour-glass or an ‘‘I’’ beam pattern were drawn on the disk with a pencil. The dentin outside the pattern was cut away with an ultrafine diamond bur in a highspeed handpiece using copious air–water spray, to leave the dentin in one of the two patterns (Fig. 1).
Both ends of the specimens were covered with two layers of nail varnish leaving the middle section uncovered and susceptible to 0.5 M EDTA (pH 7.4) demineralization for 5 days at 25⬚C. The EDTA solution contained 0.5 mmol/L phenylmethylsulfonyl fluoride and 0.5 mmol/L N-ethyl-maleimide to inhibit proteolytic enzymes during demineralization. The volume to mass ratio of the EDTA to dentin was 10 5. The decalcification time was selected from microradiography data and by longitudinal evaluation of decreases in the modulus of elasticity of the specimens as previously described (Maciel et al., 1996). Tensile testing. The ultimate tensile strength (UTS) was measured after scraping the nail varnish from each mineralized end using a scalpel, and then fixing each end into the friction grips of a Vitrodyne V1000 tester (Chatillon, Greensboro NC, USA). Specimens were subjected to tensile load at 0.6 mm/min until failure to obtain the ultimate tensile load, which was converted to the ultimate tensile stress by dividing by the crosssectional area of the narrowest part of the hour-glass shape. This was generally 0.5 × 0.5 mm but was measured to the nearest 0.01 mm with a digital micrometer (Sylvae Ultra-Cal II, Fowler Inc., Newton MN, USA) in each specimen. All tensile testing was done with the specimens immersed in 25⬚C phosphate buffered saline (PBS) unless otherwise stated. There were seven groups: PBS, 10% citric acid containing 3% ferric chloride or 10–3 solution, 10% citric acid, 37% phosphoric acid, 2.5% nitric acid, 17.5% nitric acid, and 17.5% nitric acid containing 3% ferric chloride. All concentrations are wt%. Each group consisted of five to six specimens. The specimens were placed in the acidic solution for 10 min, rinsed in water and returned to PBS for testing unless otherwise stated. Modulus of elasticity (E) was measured on ‘‘I’’ beamshaped specimens (Fig. 1) which had gauge-lengths of Dental Materials/June 1998 223
TABLE 1: COMPOSITION AND pH OF TEST SOLUTIONS Test solutions
Additives
pH
PBS
–
7.40
10% citric acid
–
1.89
10% citric acid
3% FeCl3
1.20
37% phosphoric acid
–
0.90
2.5% nitric acid
–
0.92
17.5% nitric acid
–
0.42
17.5% nitric acid
3% FeCl3
0.34
All compositions are wt%.
3.0 ⫾ 0.1 mm. The nail-varnish was scraped off each mineralized end prior to clamping the ends in the friction grips of the Vitrodyne tester. Two different types of tests were performed to measure the modulus of elasticity of the decalcified dentin: destructive versus nondestructive testing. Destructive testing was done by simply measuring the load–displacement relationship of each sample in PBS until failure. Specimens exposed to acids for 10 min were returned to PBS for 10 min before testing. The load–displacement curves were then converted to stress–strain curves using the cross-sectional area, gauge length and elongation of the specimens. The moduli of elasticity (E) were calculated in the regions between 10–12% strain (moderate strain) and 20–24% strain (high strain). Nondestructive testing was done with another set of specimens to provide repeated measures testing, to permit each specimen to serve as its own control, in an attempt to increase the statistical power. Each specimen was strained to 10–12% at 0.6 mm/min to obtain a pretreatment or control stiffness in PBS. Only specimens with a pretreatment or control stiffness between 20 and 35 MPa at 10% strain were included in this experiment, in an effort to reduce the variance. Each specimen was then unloaded and allowed to elastically recover. Then the specimen was immersed in the test acid for 10 min and the stiffness of the specimen was remeasured at 10–12% strain in the acid. After unloading the specimen, it was returned to PBS for a 10 min recovery before remeasuring stiffness again. Thus, in this experiment the stiffness of each specimen was measured prior to exposure to acid, during exposure to acid and again in PBS after recovery. There were six specimens in each (E) group and six groups (10–3 solution, 10% citric acid, 37% phosphoric acid, 2.5% nitric acid and 17.5% nitric acid, 17.5% nitric acid containing 3% ferric chloride). The pH of the solutions (Table 1) was measured with a standard pH meter (Accumet, Fisher Scientific, 224 Zhang et al./Effects of acid on collagen strength
Pittsburgh, PA, USA) calibrated with appropriate standards. Statistics. Power analysis was done to estimate sample size in each group for a one-way ANOVA assuming a difference between the means of 0.5 and a standard deviation of 0.2, with seven groups, a = 0.05 and a power of 0.8. The analysis indicated a sample size of five was adequate. After calculating descriptive statistics, the results were analyzed by one-way ANOVA to identify differences among the groups. Multiple comparisons were done using the Student–Neuman–Keuls test. Statistical significance was considered as p ⬍ 0.05. Power analysis was re-run on the data obtained from nondestructive testing to establish the power of the oneway ANOVA using six specimens in a group and six groups in a repeated measures design.
RESULTS The UTSs of hour-glass-shaped demineralized dentin specimens exposed to various acidic solutions for 10 min are shown in Fig. 2. There were no statistically significant changes produced by 10% citric acid or 10–3 solution compared to the PBS control group. Treatment with 37% phosphoric acid lowered the UTS, but the decrease was not statistically significant. Treatment with either 2.5% or 17.5% nitric acid caused statistically significant (p ⬍ 0.05) reductions in UTS. Addition of 3% ferric chloride to the 17.5% nitric acid provided significant protection to the effects of nitric acid on UTS in that the UTS of that group was not statistically different from PBS controls (Fig. 2). The power of that ANOVA was 0.92. When the modulus of elasticity was measured destructively (Fig. 3), there were no statistically significant differences among the treatment groups when the modulus was calculated from the linear portion of the stress–strain curve at an elongation of 20–24% (just before failure). When the modulus was calculated at 10–12% strain, (moderate strain), there were still few significant differences among the groups (Fig. 3). The power of these analyses ranged from 0.80 (at 20–24% strain) to 0.84 (at 10–12% strain). The lack of consistent, significant changes in stiffness produced by the acidic conditioners in the destructive measurements of modulus of elasticity was due, in part, to the relatively high variance within the groups. The high variance was due to a relatively wide range in moduli of elasticity of our dentin specimens. The distribution of stiffness of 100 specimens of demineralized dentin in PBS is shown in Fig. 4. All of these specimens had been demineralized for 5 days. The specimens exhibiting stiffness values ⬎30 MPa were returned to 0.5 M EDTA for several more days. However, repeated nondestructive measurements of their stiffness were reproducible indicating that the higher stiffness was not due to lack of complete demineralization. All specimens were strained to 10– 12%. Those specimens ⬍20 MPa or ⬎35 MPa were excluded from the nondestructive, repeated measurement of the effects of acids on the stiffness of
Fig. 2. Effects of various treatment solutions on the ultimate tensile strength of demineralized dentin matrix. Bars represent mean ⫾ SD. Numbers in parentheses indicate the number of specimens tested in each group. Each specimen was exposed to one of the acidic solutions for 10 min and then returned to PBS for tensile testing. 10–3, 10% citric acid containing 3% ferric chloride; CA, citric acid; PA, phosphoric acid; NA, nitric acid; FC, ferric chloride. All concentrations are wt%. Groups connected by the same horizontal line are not significantly different (p ⬎ 0.05). Groups not connected by the same horizontal line are significantly different (p ⬍ 0.05).
Fig. 3. Effects of various treatment solutions on the moduli of elasticity of demineralized dentin matrix at moderate (10–12%) and high (20–24%) strains. The height of bar is the mean, brackets indicate ⫾SD, numbers in parentheses are the number of specimens tested. Light bars, 10–12% strain; dark bars, 20–24% strain. Multiple comparison testing indicated that there were no statistically significant differences in modulus of elasticity (20–24% strain) among the treatment groups. At the moderate strain (10–12%), the only significant reduction in modulus was in the 17.5% nitric acid group (indicated by an asterisk). Symbols are the same as in Fig. 2.
Fig. 4. Histogram of the distribution of moduli of elasticity among 100 specimens of demineralized dentin ‘‘I’’ beams nondestructively strained to 10%. For repeated measurements, only specimens with moduli between 20–30 MPa were used.
demineralized dentin, and the groups were balanced so that they all had similar mean values. These specimens were then subjected to repeated strains of 10–12% in PBS before, during and after 10 min treatments in the acidic solutions, to obtain
repeated percent changes in stiffness in the same specimens. The use of those moderate strains avoided irreversible damage to the specimens. This was sufficient to develop reproducible stresses and yet the specimens showed excellent elastic behavior. The upper Dental Materials/June 1998 225
Fig. 5. Repeatability of stress–strain curves of an individual specimen strained to about 10% in PBS three times in succession (upper group of curves). After immersion in 17.5% nitric acid for 10 min, the specimen was returned to PBS and three more stress–strain curves were made (lower group of curves).
Fig. 6. Effects of various acidic solutions on changes in the stiffness of demineralized dentin matrix when strained to 10% in the control PBS solution (c), when strained in the test acid after 10 min (the middle bar graph of each triplet), and again after recovery (R) in PBS for 10 min. The horizontal line at 100% indicates control value. The height of the bar is the mean and the brackets indicate ⫾1 SD. Each specimen served as its own control. There were six specimens in each group. The absolute modulus of elasticity of each group was 28.4 ⫾ 4.1 (mean ⫾ S.D., N = 36). Symbols are the same as in Fig. 2.
curve in Fig. 5 shows a representative example of a specimen that underwent a 10% elongation three times while immersed in PBS. Since there was no evidence of any consistent difference between the stress–strain curves, they were averaged and assigned a value of 100%. The lower three curves in Fig. 5 represent the stress–strain curves of the same sample exposed to 17.5% HN0 3 for 10 min and then tested in PBS. This acidic conditioner produced a significant reduction (p ⬍ 0.05) in the modulus of elasticity of this specimen. Fig. 6 summarizes the effects of the acidic conditioners on the stiffness of decalcified dentin that was tested nondestructively in a repeated manner. The data have been expressed as percent changes from PBS controls to reduce the variance. By limiting the test specimens to those that had stiffness values between 20 and 35 MPa, and by using the pretreatment values as 100%, the standard deviations were reduced to 10– 20%. None of the commonly used acidic conditioners (10% citric acid, 10–3 solution, 37% phosphoric acid) produced any significant change in the stiffness of the decalcified matrix whether tested in the acid or in PBS, 10 min later. However, multiple comparisons revealed 226 Zhang et al./Effects of acid on collagen strength
that all nitric acid formulations lowered the stiffness (p ⬍ 0.001) when tested in acid. The statistical power of this test was 0.99. There were no significant (p = 0.22) changes in stiffness when the specimens were retested in PBS.
DISCUSSION The lack of a significant consistent effect of most of the acidic conditioners on the tensile properties of demineralized dentin indicates that concerns about the denaturing effects of acids on the mechanical properties of decalcified dentin appear largely unwarranted. In the present study, exposure of EDTA-decalcified dentin to 10% citric acid, 10–3 solution, or 37% phosphoric acid, or 17.5% nitric acid containing 3% ferric chloride did not lower the UTS of decalcified dentin (Fig. 2), although 2.5 and 17.5% nitric acid did reduce it (p ⬍ 0.05). These prolonged exposures to acids are far removed from clinical practice, but were used because shorter exposures produced no measurable changes (No¨r et al., 1996). It should be emphasized that what was measured in this study was not the stiffness of collagen but of a
collagenous meshwork that includes noncollagenous proteins and proteoglycans. The modulus of elasticity of collagen molecules was reported to be 2.9–9.0 GPa for Achilles tendon (Sasaki and Odajima, 1996). Unlike tendon, the collagen fibrils in dentin are arranged more randomly. When demineralized dentin matrix is stressed, the load is not evenly distributed but is preferrentially borne by the fibrils oriented in the direction of straining. Thus, it is not surprising that the stress–strain properties of demineralized dentin are different from other connective tissues. Because the organic matrix of dentin only occupies 30–48% of the total volume (Kinney et al., 1994; Sano et al., 1995), the tensile values of the collagen fibrils themselves obtained in this study could be two to three times higher if the stresses were divided by the actual crosssectional areas of the collagen fibrils. When measuring the stress–strain relationships of demineralized dentin matrix, it was not necessary to use a ‘‘preconditioning’’ strain cycle as is often done in collagenous tissues that contain connective tissue sheaths within the tissue (Van Brocklin and Ellis, 1965; Kastelic et al., 1978; Bowman et al., 1996). This was demonstrated by the high reproducibility of the stress–strain curves that were nearly identical (Fig. 5) and showed no hysteresis that has been reported in tendon (Rigby et al., 1959). The effects of acidic conditioners on the tensile properties of demineralized dentin are complex. The low pH of the solutions presumably alters the tertiary configuration of the collagen peptides (Eliades et al., 1997), but the correlation between mild denaturation of collagen and changes in UTS or stiffness is unknown. With some acids, such as 17.5% nitric acid, with or without ferric chloride, the reduction in stiffness was reversible when the specimen was returned to PBS (Fig. 6). Ferric chloride (3%) prevented the decrease in UTS that was obtained following treatment of demineralized dentin with 17.5% nitric acid (Fig. 2). The inability of 3% ferric chloride to prevent the fall in modulus of elasticity produced by 17.5% nitric acid (Figs. 3 and 6) may be explained as follows: Solutions with high hydrogen ion concentrations can change the teritary structure of proteins by protonation of amino groups and by suppression of ionization of carboxyl groups on bifunctional amino acids that have free amino or carboxyl groups. This transient gain of positive charge by basic amino acids and loss of negative charge by glutamic and aspartic acid may induce changes in the configuration of nonhelical portions of collagen peptides. Indeed, the helical portions of the peptides may provide enough structural stability to prevent the disaggregation of collagen peptides during relatively brief exposures to acidic solutions at room temperature. When the acidified demineralized dentin is rinsed with PBS, the amino groups would become deprotonized and carboxyl groups would dissociate and the tertiary structure may return. If very high concentrations of hydrogen ions are used (e.g. 17.5%, HNO 3), there may be disaggregation of collagen fibrils leading to a decrease in the UTS of the demineralized dentin matrix (Figs. 2 and 3).
CONCLUSIONS The results of this study would predict minimal changes in tensile properties of the dentin matrix following acid etching under clinically revelant conditions. ACKNOWLEDGEMENTS This work was supported, in part, by grant DE06427 from the National Institute of Dental Research, by the Medical College of Georgia Biocompatibility Group and by FAPESP grant 95/3895-9 (to RMC). Received April 3, 1998 / Accepted April 20, 1998 Address correspondence and reprint requests to: Dr D.H. Pashley Department of Oral Biology School of Dentistry Medical College of Georgia Augusta GA 30909-1129 USA Tel.: 001-706-721-2033 Fax: 001-706-721-6252 E-mail: [email protected]
REFERENCES Akimoto T (1991). Study of adhesion of MMA-TBBO resin to dentin. J Jpn Soc Dent Mater and Device 10:42–54. Bowman SM, Zeind J, Gibson LJ, Hayes WC, McMahons TA (1996). The tensile behavior of demineralized bovine cortical bone. J Biomechanics 29:1497–1501. Butler WT (1995). The nature and functional significance of dentin extracellular matrix proteins. Int J Dev Biol 39:169–179. Dung S-Z, Li Y, Dumpace AJ, Stookey GK (1994). Degradation of insoluble bovine collagen and human dentine collagen pretreated in vitro with lactic acid, pH 40 and 55. Arch Oral Biol 39:901–905. Eliades G, Palaghias G, Vougiouklakis G (1997). Effect of acidic conditioners on dentin morphology, molecular composition and collagen configuration in situ. Dent Mater 13:24–33. Kastelic J, Galeski A, Baer E (1978). Composite structure of tendon. Connec Tiss Res 6:11–23. Kinney JH, Marshall GW, Marshall SJ (1994). Three dimensional mapping of mineral densities in carious dentin: theory and method. Scanning Microscopy 8:197–205. Maciel KT, Carvallo RM, Ringle PD, Preston CD, Russell CM, Pashley DH (1996). The effects of acetone, ethanol, HEMA and air on the stiffness of human demineralized dentin. J Dent Res 75:1851– 1858. Mizunuma T (1986). Relationship between bond strength of resin to dentin and structural change in dentin collagen during etching—Influence of ferric chloride to structure of the collagen. J Jpn Dent Mater 5:54–64. Dental Materials/June 1998 227
Nimni ME (1991). Collagen, structure and function. In: Dulbecco R, editor, Encyclopedia of Human Biology, vol. 2, 2nd ed. New York: Academic Press, 1991, 559– 575. Nimni ME, Cheung DT (1994). Process for purifying collagen and generating bioprostheses. US Patent 5374539. United States Patent and Trademark Office, Arlington, VA/Washington, DC. No¨r J, Carvalho RM, Pashley DH (1996). Effect of potentially denaturing agents on tensile properties of collagen. J Dent Res Special Issue 75:347. Abst #2635. Okamoto Y, Heeley JD, Dogon IL, Shintani H (1991). Effects of phosphoric acid and tannic acid on dentine collagen. J Oral Rehabilitation 18:507–512. Perdiga˜o J, Lambrechts P, Van Meerbeek B, Tome´ AR, Vanherle G, Lopes AB (1996). Morphological field emission-SEM study of the effect of six phosphoric acid etching agents on human dentin. Dent Mater 12:262–271. Rigby BJ, Hirai N, Spikes JD, Eyring H (1959). The mechanical properties of rat tail tendon. J Gen Physiol 43:265–283.
228 Zhang et al./Effects of acid on collagen strength
Sano H, Takatsu T, Ciucchi B, Russell CM, Pashley DH (1995). Tensile properties of resin-infiltrated demineralized human dentin. J Dent Res 74:1093– 1102. Sasaki N, Odajima S (1996). Stress–strain curve and Young’s modulus of a collagen molecule as determined by the X-ray diffraction technique. J Biomechanics 29:655–658. Scott PG, Leaver AG (1974). The degradation of human collagen by trypsin. Connect Tiss Res 2:299–307. Van Brocklin JD, Ellis DG (1965). A study of the mechanical behavior of toe extensor tendons under applied stress. Arch Phys Med Rehab 46:369–373. Veis A, Schlueter RJ (1964). The macromolecular organization of dentine matrix collagen I Characterization of dentine collagen. Biochem J 3:1650–1657. Van Meerbeek B, Inokoshi S, Braem M, Lambrechts P, Vanderle G (1992). Morphological aspects of the resin–dentin interdiffusion zone with different adhesive systems. J Dent Res 71:1530–1540