Effects of phosphoric acid concentration and etch duration on enamel depth of etch: An in vitro study L. R. Legler, BS,* D. H. Retief, BDS, MSc, PhD, DSc,** and E. L. Bradley, PhD*** Birmingham, A/a. In a previous study we reported no significant differences among the shear bond strengths resulting from the application of an orthodontic bonding resin to enamel surfaces etched with three phosphoric acid (HaPO,) concentrations, each for three etch durations. The objective of the current study was to determine the depths of etch on ground enamel surfaces exposed to the nine etching procedures. The facial surfaces of 45 extracted human maxillary permanent central incisors Were ground wet on 600-grit silicon carbide paper. Annular adhesive disks of 6 mm outer diameter and 3 mm inner diameter were positioned on the ground enamel surfaces and etched with 10 mm3 of 37%, 15%, and 5% HaPO, for 60, 30, and 15 seconds, respectively. The calcium concentrations of the etching solutions were determined and the depths of etch calculated. The depths of etch were then measured with a surface profilometer. A stepwise decrease in the calculated depths of etch with decreasing acid concentration and duration of etching was obtained. The calculated etch depths ranged from 27.1 ism by etching with 37% HaPO, for 60 seconds to 3.5 I-tin by etching with 5% HaPO4 for 15 seconds. The measured depths of etch followed a similar pattern. A highly significant correlation between calculated and measured depths of etch was obtained. (AM J ORTHODDENTOFAC ORTHOP 1990;98:154-60.)
S i n c e the introduction of the acid etch technique by Buonocore, ~ the bonding of orthodontic attachments to acid-etched enamel has become an accepted clinical procedure. 2 However, it is impossible to confine the phosphoric acid (HaPO4) etching solution solely to the area that subsequently will be covered by the bonded attachment. Therefore concern has been expressed about the etching of enamel adjacent to the bonded attachments, since removal of the fluoride-rich surface enamel may predispose these surfaces to enamel decalcification during orthodontic treatment. 3 In a recent in vitro study, the effects of phosphoric acid concentration and duration of etching on the shear bond strengths of an orthodontic bonding resin to enamel were determined. 4 It was reported that the shear bond strengths to enamel surfaces etched with 37%, 15%, and 5% wt/wt HaPO4 for 60, 30, and 15 seconds, respectively, were not significantly different. The purpose of this study was to determine the calculated and measured depths of etch and the surface From the University of Alabama School of Dentistry, University of Alabama at Birmingham. Supported by Biomedical Research Support Grant 2 S07 RR 05300-27 from the National Institutes of ttealth, Bethesda, Md. *Undergraduate student. **Professor, Department of Biomaterials. ***Professbr, Department of Biostatisties and Biomathematics. 811115606
154
roughness of ground enamel surfaces etched with various H3PO4 concentrations and for different etch times. MATERIALS AND METHODS Forty-five extracted noncarious human maxillary permanent central incisors were used in the study. After extraction the teeth were cleaned with flour of pumice on a dental lathe and stored in 70% ethanol. The facial surfaces of the teeth were ground wet with 600-grit silicon carbide paper on a polishing machine (Buehler Ltd., Lake Bluff, III.) to produce a flat enamel surface. Calculated depth of etch The ground enamel surfaces were cleaned with a cotton pellet soaked in acetone and dried with oil-free compressed air. Annular adhesive disks with an outer diameter of 6 mm and an inner diameter of 3 mm were punched in No. 471 adhesive tape (3M CompanY, St. Paul, Minn.) with microtrephines (Roboz Surgical, Washington, D.C.). The adhesive rings were burnished to ensure good marginal adaptation of the adhesive tape to the enamel surfaces. Five teeth were etched with each of the following nine procedures: Procedure A - - W i t h 37% HaPO4 solution for 60 seconds. Procedure B R W i t h 37% H3PO4 solution for 30 seconds.
Volume 9 8 Number 2
Effects of tt3PO~ concentration and etch duration 155
Procedure C - - W i t h 37% HaPOa solution for 15 seconds. Procedure D - - W i t h 15% H3PO4 solution for 60 seconds. Procedure E - - W i t h 15% HaPO4 solution for 30 seconds. Procedure F - - W i t h 15% H3PO4 solution for 15 seconds. Procedure G - - W i t h 5% H3PO4 solution for 60 seconds. Procedure H - - W i t h 5% H3PO4 solution for 30 seconds. Procedure I - - W i t h 5% H3PO4 solution for 15 seconds. An adhesive tape was placed over millipore filter paper (Millipore Corporation, Bedford, Mass.) and disks with a 3 mm diameter were punched with the microtrephine. The disks were handled with finepointed tweezers, and 10 mm 3 of the phosphoric etching agent was pipetted onto the disc. The soaked disc was placed on the demarcated enamel surface and etched for the required duration with a light dabbing action. After etching, the demarcated etched enamel surface was washed with 10 mm 3 of distilled water, which was absorbed onto a 3 mm-diameter filter paper disk. The disks with the etching and washing solutions were placed in plastic test tubes containing 1000 mm 3 of distilled water for the 37% and 15% H3PO 4 solutions or 500 mm 3 of distilled water for the 5% H3PO4 solutions. The calcium content in the etching solutions was determined with a No. 940 Coming Calcium Analyzer (Coming Medical, Medfield, Mass.). The dye calcein, a fluorescein derivative, forms an intensely fluorescent, nondissociated complex with calcium in an alkaline medium. The analytical procedure is based on the quenching of this fluorescence by chelation of the calcium ions with the titrant ethyleneglycoltetraacetic acid (EGTA). The mass calcium in the etching solutions was converted to mass enamel with the assumption that human enamel contains 37% calcium. 5 The depth of etch was calculated from the following equation: Depth of etch (p.m) = Mass enamel (isg) Density of enamel × biopsy area (mm2) where density of enamel = 2.95 gm/ml. 6
Measured depth of etch and surface roughness The annular adhesive disks were then removed from the ground enamel surfaces. To measure the depth of etch, the stylus of a surface profilometer (Surfanalyzer System 4000, Federal Products Corp., Providence, R.I.) with a diameter of 2.5 txm was traversed over the
demarcated etched area. A stylus pressure of 100 mg was applied. The depth of etch was calculated from the printout. In addition, the "stylus surface roughness" (Ra) was calculated from the following equationT:
,yfyl.~ lm
Ra = ~
o
where lm = measuring length and Y = the distance of the stylus roughness profile to the center line. The data were analyzed by an analysis of variance s and Duncan's multiple range tests, 9 and the Pearson correlation coefficient I° between calculated and measured depths of etch was determined. All computations were made at the 5% level of significance by the Statistical Analysis System."
RESULTS The calculated depths of etch, presented in Table I, are listed in descending oi'der of magnitude. A oneway analysis of variance indicated that the calculated depths of etch were significantly different (p = 0.0001). Means linked by vertical lines were not significantly different. The mean calculated depth of etch (plus standard deviation) for each method is diagrammatically presented in Fig. 1. Note the stepwise decrease in the calculated depth of etch with decreasing acid concentration and duration of etching. A maximum mean depth of etch of 27.1 FLmwas obtained by etching with 37% H3PO4 for 60 seconds, while a minimum mean etch of 3.5 p.m was obtained by etching with 5% H3PO4 for 15 seconds. The measured depths of etch are presented in Table II in descending order of magnitude. A one-way analysis of variance showed that the etch depths were significantly different (p = 0.0001). Means linked by vertical lines were not significantly different. The mean depth of etch (plus standard deviation) for each method is diagrammatically presented in Fig. 2. The results follow a pattern similar to those obtained for the calculated depths of etch. The mean etch depths ranged from 28.0 I.tm with the 37% H3PO4 solution applied for 60 seconds to 4.0 I.tm when the enamel was etched with 5% H3PO4 solution for 15 seconds. The means - SD of the calculated and measured depths of etch are given in Table III. The correlation between calculated and measured" depths of etch was significant (r = 0.959, p = 0.13001). The surface roughness (Ra) values of the etched enamel surfaces are listed in descending order of magnitude in Table IV and depicted diagrammatically in Fig. 3. A one-way analysis of variance showed that the
156
Am. J. Orthod. Dentofac. Orthop. August 1990
Legler, Retief, and Bradley
Table I. Calculated depths of etch (I-tm) listed in descending order of magnitude Calculated depth of etch (pro) NO. of
Procedure
specbneas
Mean*
A: 37% H3POa 60-second etch
5
D:15% H~PO4 60-second etch
]
SD
Range
27. I
4.3
20.6-32.6
5
21.0
3.2
16.2-25.1
B: 37% H~PO~ 30-second etch
5
16.7
1.0
16.0-18.5
E: 15% H~PO~ 30-second etch
5
16.1
1.7
14.5-18.6
G:
5% H~PO~ 60-second etch
5
9.1
1.0
8.0-10.3
C: 37% H~PO~ 15-second etch
5
8.9
0.6
8.2-9.7
F: 15% tt~PO4 15-second etch
5
7.6
1.2
5.7-8.4
H:
5% tt~PO4 30-second etch
5
6.2
0.5
5.7-6.9
l:
5% H~PO4 15-second etch
5
3.5
0.3
3.2-4.0
I
*Means linked by vertical lines were not significantly different.
"l'abla II. Measured depths of etch (IJ.m) listed in descending order of magnitude Measured depth of etch (ttm) of specimens
Mean*
/~: 37% H~PO~ 60-second etch
5
28.0
5.1
20.0-32.0
D: 15% H3PO4 60-second etch
5
22.8
4.8
16.0-28.0
B: 37% tt~PO4 30-second etch
5
16.4
2.2
14.0-20.0
E: 15% H~PO~ 30-second etch
5
13.8
2.5
10.0-17.0
G:
5% H~PO4 60-second etch
5
9.8
1.8
8.0-12.0
F: 15% H~PO4 15-second etch
5
9.4
0.9
8.0-10.0
C: 37% H~PO4 15-second etch
5
8.8
1.1
8.0-10.0
H:
5% H~PO4 30-second etch
5
6.0
1.4
4.0-8.0
I:
5% H3PO4 15-second etch
5
4.0
0.7
3.0-5.0
No.
Procedure
*Means linked by vertical lines were not significantly different.
I
SD
[
Range
Volume 98 Number 2
Effects of HjPO, concentration and etch duration 157
40 E |
,- 30 ,11 '5
7-
¢,-
~. 20 O '10
,~ -5
10
t~ O
60
30
15
60 30 15 60 Etch Duration - Seconds I
I
37 % H3PO4
I
30
15
I
15 % H3PO4
5 % H3PO4
Fig. 1. Bar graph of calculated depth of etch.
40
E i 1: O
tO. {D
30
T II
T
20
D "10
T
10 Q}
60
30
15
60 30 15 60 Etch Duration - Seconds J
I
37 % H3PO4
I
15 % H3PO4
30
15
I
5 % H3PO4
Fig. 2. Bar graph of measured depth of etch.
Ra values were significantly different (p = 0.0001). There was a stepwise reduction in Ra with a decrease in acid concentration and duration of etching. DISCUSSION
Enamel decalcification frequently occurs during orthodontic treatment. ~2~4 During the clinical application of the enamel etchant it is practically impossible to confine the acid solely to the site on which the bracket will be seated, so surface enamel adjacent to the bonded attachment also is removed. Fluoride is not evenly distributed in enamel; it follows a negative exponential distribution, with the highest fluoride concentration being in the surface enamel, ts Etching of enamel ad-
jacent to the bonded brackets will result in the loss of fluoride-rich surface enamel and may predispose the enamel to decalcification during orthodontic treatment. Etching enamel with H3PO4 results in a superficial etched zone and subsurface qualitative and quantitative porous zones. 16 The subsurface zones remineralize in the oral environment, but the surface-etched zone is permanently lost from the tooth surface. ,7 The effects of phosphoric acid concentration and duration of etching on the enamel etch depth have been studied by several investigators. Silverstone ~8 etched unground enamel surfaces with H~PO4 concentrations ranging from 20% to 70% HaPO 4 for 60 seconds and reported that the loss in depth of surface contour ranged
158
Am. J. Orthod. Dentofac. Orthop. August 1990
Legler, Retief, and Bradley 2.5
T
2.0 ¢/)
1.5
> n-" 1.0
0.5 0.0
60
30
15
I
60 30 15 60 Etch Duration - Seconds I
I
370/o H3PO4
I
15 % H3PO4
30
15
I
50/0 H3PO4
Fig. 3. Bar graph of surface roughness (Ra).
Table III. Calculated and measured depths of etch Calculated depth of etch (tun)
Measured depth of etch (tun)
Procedure
Mean
SD
Mean
[
SD
A: 37% H3PO4 60-second etch
27.1
4.3
28.0
5.1
B: 37% H3PO4 30-second etch
16.7
1.0
16.4
2.2
C: 37% H~PO4 15-second etch
8.9
0.6
8.8
1.1
D" 15% H~PO4 60-second etch
21.0
3.2
22.8
4.8
E: 15% H3PO4 30-second etch
16.1
1.7
13.8
2.5
F: 15% H~PO4 15-second etch
7.6
1.2
9.4
0.9
G:
5% H3PO4 60-second etch
9.1
1.0
9.8
1.8
H:
5% H~POa 30-second etch
6.2
0.5
6.0
1.4
I:
5% H~PO4 15-second etch
3.5
0.3
4.7
0.7
r = 0.959, p = 0.0001.
from 14 I.tm with 20% HaPO4 to 2 I.tm with 70% H3PO4. RetieP 9 etched enamel surfaces ground on 600-grit silicon carbide paper with from 10% to 85% HaPO4 solutions for 60 seconds and recorded depths of etch ranging from 15 p.m to 0.8 i.tm. Etching unground enamel surfaces with 37% H3PO4 for 90 seconds resulted in a mean loss of 6.9 p.m of surface enamel.2° The unground facial surfaces of premolar teeth were etched with three
commercially available etching solutions for 60 seconds and a mean depth of etch of 22.5 I.tm was found. 2~ The depths of etch ranged from 16.6 ism to 29.5 t.tm, depending on the etchant used. These findings can readily be explained by the phase diagram developed by Chow and Brown 22 for the ternary system of calcium hydroxide, phosphoric acid, and water. Etching enamel surfaces with solutons containing more than 27% H3PO4
Volume98 Number2
Effects of H3P04 concentration and etch duration 159
Table IV. Ra values of etched enamel surfaces (~m) listed in descending order of magnitude
Ra values (fun)
of specimens
Mean*
A: 37% tt3PO4 60-second etch
5
1.7
0.5
0.9-2.2
D: 15% H~PO~ 60-second etch
5
1.2
0.2
0.9-1.5
B: 37% H~PO~ 30-second etch
5
1.0
0.3
0.6-1.4
E" 15% H3PO~ 30-second etch
5
0.8
0. I
0.6-0.9
C: 37% H3PO~ 15-second etch
5
0.5
0.1
0.4-0.6
F: 15% H~PO4 i 5-second etch
5
0.5
0.1
0.4-0.6
G:
5% H3PO4 60-second etch
5
0.4
0.1
0.4-0.6
H:
5% H~PO4 30-second etch
5
0.4
0.1
0.4-0.5
5
0.2
0.0
0.2-0.2
Procedure
I:
5% H~PO~ 15-second etch
No.
I
SD
[
Range
*Means linked by vertical lines were not significantly different.
results in the formation of monocalcium phosphate monohydrate (MCPM), while etching with more dilute H3PO~ solutions results in the formation of dicalcium phosphate dihydrate (DCPD). Because MCPM is more soluble than DCPD, more concentrated H a P O 4 solutions are used clinically. The phase diagram clearly indicates that the amount of enamel dissolved will increase with acid concentrations up to 27% H3PO4. With acid concentrations greater than 27% H3PO4, the amount of enamel dissolved will decrease with an increase in acid concentration. The present study determined calculated and measured depths of etch on enamel surfaces ground on 600grit silicon carbide paper. It was not possible to record the surface profiles of the demarcated biopsy sites on unground enamel surfaces because of the curvature of unground enamel surfaces. Orthodontic attachments are bonded to unground enamel surfaces, and the depths of etch on such surfaces are less than the depths recorded in this study. The depth of etch or the amount of surface enamel removed during the etching procedure is dependent on the type of acid used, the concentration of the acid, the duration of etching, and the chemical composition of the enamel. 4 It was shown that etching high-fluoride unground enamel surfaces with perchloric acid resulted in a significantly decreased depth of etch compared with that obtained by grinding. It is interesting to note that, given the same acid
concentrations and durations of etching, significant differences in surface roughness of tooth enamel did not result in significant differences in the shear bond strengths of an orthodontic bonding resin .4 In the study that produced these findings a scanning electron microscope was used to observe great variations in the surface morphology of enamel surfaces etched with the nine etching procedures. These observations suggest that the depth of resin penetration into the etched enamel surfaces is not of prime importance. Etching enamel surfaces with orthophosphoric acid results in a significant increase in the surface free energy of the enamel surfaces, 24 producing greater wettability of the etched enamel surfaces and subsequent penetration of the curing resin into the subsurface porous zones. It is interesting to note that the current study found a highly significant correlation between the calculated and measured depths of etch (Table III). Silverstone ~6 has demonstrated that etching of enamel surfaces with H3PO4 results in a superficial etched zone and subsurface qualitative and quantitative porous zones. Only the superficial etched zone is measured from the surface profilometer tracings, while the calculated depths of etch are obtained from the total calcium (enamel) dissolved during the etching procedures. The results of our study suggest that the amount of calcium (enamel) dissolved from the subsurface zones is minimal. Enamel decalcification occurring during orthodontic
160 Legler, Retief, and Bradley
treatment is reduced b y a p p r o x i m a t e l y 25% b y topical fluoride treatment, z5 H o w e v e r , it has b e e n d e m o n s t r a t e d that m o r e than 5 0 % o f subjects c o m p l y p o o r l y with a p r e v e n t i v e fluoride m o u t h rinse program w h i l e underg o i n g orthodontic treatment. 2s A n o t h e r w a y e n a m e l decalcification during orthodontic treatment m a y be red u c e d is b y reduction o f the phosphoric acid c o n c e n tration and the duration o f etching. A m a j o r c o n c e r n is the effect o f these parameters on the retention o f b o n d e d attachments in the clinical situation. P r e l i m i n a r y studies, however, h a v e s h o w n that reducing the acid c o n centration and duration o f etching had no adverse effect on the clinical retention o f b o n d e d attachments. 27"2s We are indebted to Ms. S. Roper for typing the manuscript. REFERENCES 1. Buonocore MG. A simple method of increasing the adhesion of acrylic filling materials to enamel surfaces. J Dent Res 1955;38:849-53. i 2. Retief DH, Dreyer CJ, Gavron G. The direct bonding of orthodontic attachments to teeth by means of an epoxy adhesive. AM J ORTHOD1970;58:21-40. 3. Ceen RF, Gwinnett AJ. White spot formation associated with sealants in orthodontics. Pediatr Dent 1981;3:174-8. 4. Legler LR, Retief DH, Bradley EL, Denys FR, Sadowsky PL. Effects of phosphoric acid concentration and etch duration on the shear bond strength of an orthodontic bonding resin to enamel: an in vitro study. AM J ORTHODDENTOFACORTHOP1989; 96:485-92. 5. Srremark R, Samsahl K. Gamma-ray spectrophotometric analysis of elements in normal human enamel. Arch Oral Biol 1961 ;6:275-83. 6. Mazily RS, Hodge HS, Ange LE. Density and refractive index studies of dental hard tissues. II. Density distribution curves. J Deni Res 1939;19:203-11. 7. Retief DH, Busscher HJ, de Boer P, Jongebloed WL, Arends J. A laboratory evaluation of three etching solutions. Dent Mater 1986;2:202-6. 8. Ostle B, Mensing R. Statistics in research. 3rd ed. Iowa City, Iowa: Iowa State University Press, 1975:452-61. 9. Steele R , Torrie J. Principles and procedures in statistics. New York: McGraw-Hill, 1960:99-160. 10. Snedecor GW, Cochran WG. Statistical methods. 7th ed. Ames, Iowa: Iowa State University Press, 1980:175-91. 11. SAS/STAT guide for personal computers, version 6 ed. Gary, North Carolina: SAS Institute, 1985:235-6. 12. Gorelick L, Geigei" AM, Gwinnett AJ. Incidence of white spot formation after bonding and banding. AM J OR~IOD 1982;81: 93-8.
Am. J.
Orthod. Dentofac. Orthop. August 1990
13. Mizrahi E. Surface distribution of enamel opacities following orthodontic treatment. AM J ORrHOD 1983;84:323-31. 14. O'Reilly MM, Featherstone JDB. Demineralization and remineralization around orthodontic appliances: an in vivo study. AM J ORTtIODDENTOFACORTHOP 1987;92:33-40. 15. Brudevold F, Gardner DE, Smith FA. The distribution of fluoride in human enamel. J Dent Res 1956;35:420-9. 16. Silverstone LM. The acid etch technique: in vitro studies with special reference to the enamel surface and enamel-resin interface. In: Silverstone LM, Dogon IL. Proceedings of an international symposium on the acid etch technique. St. Paul: North Central, 1975:13-39. 17. Lee H, Ocumpaugh DE, Shaffer J, Sheble AM. Sealing of developmental pits and fissures. IV. Measurements of in vivo fluoride pickup by electron microprobe x-ray spectrophotometry. J Dent Res 1972;51:634-9. 18. Silverstone LM. Fissure sealants: Laboratory studies. Caries Res 1974;8:2-26. 19. Retief DH. The use of 50 per cent phosphoric acid as an etching agent in orthodontics: a rational approach. AM J ORTHOD 1875;68:165-78. 20. Pus MD, Way 1)(2. Enamel loss due to orthodontic bonding with filled and unfilled resins using various clean-up techniques. AM J ORa'HOD1980;77:269-83. 21. SheyZ, BrandtS. Enamel loss due to acid treatment for bonding. J Clin Orthod 1982;16:338-40. 22. Chow LC, Brown WE. Phosphoric acid conditioning of teeth for pit and fissure sealants. J Dent Res 1973;52:1158. 23. Thornton JB, Retief DH, Bradley EL, Denys FR. The effect of fluoride in phosphoric acid on enamel fluoride uptake and the tensile bond strength of an orthodontic bonding resin. AM J ORTHODDErZrOFACORT~OP 1986;90:91-101. 24. BusscherHJ, RetiefDH, Arends J. Relationship between surface free energies of dental resins and bond strengths. Dent Mater 1987;3:60-3. 25. Saloum IS, Sondhi A. Preventing enamel decalcification after orthodontic treatment. J Am Dent Assoe 1985;115:257-61. 26. Gejger AM, Gorelick L, Gwinnett AJ, Griswold PG. The effect of a fluoride progi'am on white spot formation during orthodontic treatment. AM J ORTHODDENTOFACORTHOP1988;93:29-37. 27. Sadowsky PL, Retief DH, Cox PR, Hemandez R, Rape G. Effect of etchant concentration and duration of etching on retention of orthodontic attachments [Abstract]. J Dent Res 1988;67:361. 28. ViljoenWP, Swb.nepoel F, Pretorius LM, DuPlessis LS, Smit HJ, De Mflelenaere JJGG. Shorter etching times in orthodontic bonding: an in vitro study [Abstract]. J Dent Res 1988;67:776. Reprint requests to: Dr. D.H. Retief Department of Biomaterials University of Alabama School of Dentistry University Station Birmingham, AL 35294
S i n c e the introduction of the acid etch technique by Buonocore, ~ the bonding of orthodontic attachments to acid-etched enamel has become an accepted clinical procedure. 2 However, it is impossible to confine the phosphoric acid (HaPO4) etching solution solely to the area that subsequently will be covered by the bonded attachment. Therefore concern has been expressed about the etching of enamel adjacent to the bonded attachments, since removal of the fluoride-rich surface enamel may predispose these surfaces to enamel decalcification during orthodontic treatment. 3 In a recent in vitro study, the effects of phosphoric acid concentration and duration of etching on the shear bond strengths of an orthodontic bonding resin to enamel were determined. 4 It was reported that the shear bond strengths to enamel surfaces etched with 37%, 15%, and 5% wt/wt HaPO4 for 60, 30, and 15 seconds, respectively, were not significantly different. The purpose of this study was to determine the calculated and measured depths of etch and the surface From the University of Alabama School of Dentistry, University of Alabama at Birmingham. Supported by Biomedical Research Support Grant 2 S07 RR 05300-27 from the National Institutes of ttealth, Bethesda, Md. *Undergraduate student. **Professor, Department of Biomaterials. ***Professbr, Department of Biostatisties and Biomathematics. 811115606
154
roughness of ground enamel surfaces etched with various H3PO4 concentrations and for different etch times. MATERIALS AND METHODS Forty-five extracted noncarious human maxillary permanent central incisors were used in the study. After extraction the teeth were cleaned with flour of pumice on a dental lathe and stored in 70% ethanol. The facial surfaces of the teeth were ground wet with 600-grit silicon carbide paper on a polishing machine (Buehler Ltd., Lake Bluff, III.) to produce a flat enamel surface. Calculated depth of etch The ground enamel surfaces were cleaned with a cotton pellet soaked in acetone and dried with oil-free compressed air. Annular adhesive disks with an outer diameter of 6 mm and an inner diameter of 3 mm were punched in No. 471 adhesive tape (3M CompanY, St. Paul, Minn.) with microtrephines (Roboz Surgical, Washington, D.C.). The adhesive rings were burnished to ensure good marginal adaptation of the adhesive tape to the enamel surfaces. Five teeth were etched with each of the following nine procedures: Procedure A - - W i t h 37% HaPO4 solution for 60 seconds. Procedure B R W i t h 37% H3PO4 solution for 30 seconds.
Volume 9 8 Number 2
Effects of tt3PO~ concentration and etch duration 155
Procedure C - - W i t h 37% HaPOa solution for 15 seconds. Procedure D - - W i t h 15% H3PO4 solution for 60 seconds. Procedure E - - W i t h 15% HaPO4 solution for 30 seconds. Procedure F - - W i t h 15% H3PO4 solution for 15 seconds. Procedure G - - W i t h 5% H3PO4 solution for 60 seconds. Procedure H - - W i t h 5% H3PO4 solution for 30 seconds. Procedure I - - W i t h 5% H3PO4 solution for 15 seconds. An adhesive tape was placed over millipore filter paper (Millipore Corporation, Bedford, Mass.) and disks with a 3 mm diameter were punched with the microtrephine. The disks were handled with finepointed tweezers, and 10 mm 3 of the phosphoric etching agent was pipetted onto the disc. The soaked disc was placed on the demarcated enamel surface and etched for the required duration with a light dabbing action. After etching, the demarcated etched enamel surface was washed with 10 mm 3 of distilled water, which was absorbed onto a 3 mm-diameter filter paper disk. The disks with the etching and washing solutions were placed in plastic test tubes containing 1000 mm 3 of distilled water for the 37% and 15% H3PO 4 solutions or 500 mm 3 of distilled water for the 5% H3PO4 solutions. The calcium content in the etching solutions was determined with a No. 940 Coming Calcium Analyzer (Coming Medical, Medfield, Mass.). The dye calcein, a fluorescein derivative, forms an intensely fluorescent, nondissociated complex with calcium in an alkaline medium. The analytical procedure is based on the quenching of this fluorescence by chelation of the calcium ions with the titrant ethyleneglycoltetraacetic acid (EGTA). The mass calcium in the etching solutions was converted to mass enamel with the assumption that human enamel contains 37% calcium. 5 The depth of etch was calculated from the following equation: Depth of etch (p.m) = Mass enamel (isg) Density of enamel × biopsy area (mm2) where density of enamel = 2.95 gm/ml. 6
Measured depth of etch and surface roughness The annular adhesive disks were then removed from the ground enamel surfaces. To measure the depth of etch, the stylus of a surface profilometer (Surfanalyzer System 4000, Federal Products Corp., Providence, R.I.) with a diameter of 2.5 txm was traversed over the
demarcated etched area. A stylus pressure of 100 mg was applied. The depth of etch was calculated from the printout. In addition, the "stylus surface roughness" (Ra) was calculated from the following equationT:
,yfyl.~ lm
Ra = ~
o
where lm = measuring length and Y = the distance of the stylus roughness profile to the center line. The data were analyzed by an analysis of variance s and Duncan's multiple range tests, 9 and the Pearson correlation coefficient I° between calculated and measured depths of etch was determined. All computations were made at the 5% level of significance by the Statistical Analysis System."
RESULTS The calculated depths of etch, presented in Table I, are listed in descending oi'der of magnitude. A oneway analysis of variance indicated that the calculated depths of etch were significantly different (p = 0.0001). Means linked by vertical lines were not significantly different. The mean calculated depth of etch (plus standard deviation) for each method is diagrammatically presented in Fig. 1. Note the stepwise decrease in the calculated depth of etch with decreasing acid concentration and duration of etching. A maximum mean depth of etch of 27.1 FLmwas obtained by etching with 37% H3PO4 for 60 seconds, while a minimum mean etch of 3.5 p.m was obtained by etching with 5% H3PO4 for 15 seconds. The measured depths of etch are presented in Table II in descending order of magnitude. A one-way analysis of variance showed that the etch depths were significantly different (p = 0.0001). Means linked by vertical lines were not significantly different. The mean depth of etch (plus standard deviation) for each method is diagrammatically presented in Fig. 2. The results follow a pattern similar to those obtained for the calculated depths of etch. The mean etch depths ranged from 28.0 I.tm with the 37% H3PO4 solution applied for 60 seconds to 4.0 I.tm when the enamel was etched with 5% H3PO4 solution for 15 seconds. The means - SD of the calculated and measured depths of etch are given in Table III. The correlation between calculated and measured" depths of etch was significant (r = 0.959, p = 0.13001). The surface roughness (Ra) values of the etched enamel surfaces are listed in descending order of magnitude in Table IV and depicted diagrammatically in Fig. 3. A one-way analysis of variance showed that the
156
Am. J. Orthod. Dentofac. Orthop. August 1990
Legler, Retief, and Bradley
Table I. Calculated depths of etch (I-tm) listed in descending order of magnitude Calculated depth of etch (pro) NO. of
Procedure
specbneas
Mean*
A: 37% H3POa 60-second etch
5
D:15% H~PO4 60-second etch
]
SD
Range
27. I
4.3
20.6-32.6
5
21.0
3.2
16.2-25.1
B: 37% H~PO~ 30-second etch
5
16.7
1.0
16.0-18.5
E: 15% H~PO~ 30-second etch
5
16.1
1.7
14.5-18.6
G:
5% H~PO~ 60-second etch
5
9.1
1.0
8.0-10.3
C: 37% H~PO~ 15-second etch
5
8.9
0.6
8.2-9.7
F: 15% tt~PO4 15-second etch
5
7.6
1.2
5.7-8.4
H:
5% tt~PO4 30-second etch
5
6.2
0.5
5.7-6.9
l:
5% H~PO4 15-second etch
5
3.5
0.3
3.2-4.0
I
*Means linked by vertical lines were not significantly different.
"l'abla II. Measured depths of etch (IJ.m) listed in descending order of magnitude Measured depth of etch (ttm) of specimens
Mean*
/~: 37% H~PO~ 60-second etch
5
28.0
5.1
20.0-32.0
D: 15% H3PO4 60-second etch
5
22.8
4.8
16.0-28.0
B: 37% tt~PO4 30-second etch
5
16.4
2.2
14.0-20.0
E: 15% H~PO~ 30-second etch
5
13.8
2.5
10.0-17.0
G:
5% H~PO4 60-second etch
5
9.8
1.8
8.0-12.0
F: 15% H~PO4 15-second etch
5
9.4
0.9
8.0-10.0
C: 37% H~PO4 15-second etch
5
8.8
1.1
8.0-10.0
H:
5% H~PO4 30-second etch
5
6.0
1.4
4.0-8.0
I:
5% H3PO4 15-second etch
5
4.0
0.7
3.0-5.0
No.
Procedure
*Means linked by vertical lines were not significantly different.
I
SD
[
Range
Volume 98 Number 2
Effects of HjPO, concentration and etch duration 157
40 E |
,- 30 ,11 '5
7-
¢,-
~. 20 O '10
,~ -5
10
t~ O
60
30
15
60 30 15 60 Etch Duration - Seconds I
I
37 % H3PO4
I
30
15
I
15 % H3PO4
5 % H3PO4
Fig. 1. Bar graph of calculated depth of etch.
40
E i 1: O
tO. {D
30
T II
T
20
D "10
T
10 Q}
60
30
15
60 30 15 60 Etch Duration - Seconds J
I
37 % H3PO4
I
15 % H3PO4
30
15
I
5 % H3PO4
Fig. 2. Bar graph of measured depth of etch.
Ra values were significantly different (p = 0.0001). There was a stepwise reduction in Ra with a decrease in acid concentration and duration of etching. DISCUSSION
Enamel decalcification frequently occurs during orthodontic treatment. ~2~4 During the clinical application of the enamel etchant it is practically impossible to confine the acid solely to the site on which the bracket will be seated, so surface enamel adjacent to the bonded attachment also is removed. Fluoride is not evenly distributed in enamel; it follows a negative exponential distribution, with the highest fluoride concentration being in the surface enamel, ts Etching of enamel ad-
jacent to the bonded brackets will result in the loss of fluoride-rich surface enamel and may predispose the enamel to decalcification during orthodontic treatment. Etching enamel with H3PO4 results in a superficial etched zone and subsurface qualitative and quantitative porous zones. 16 The subsurface zones remineralize in the oral environment, but the surface-etched zone is permanently lost from the tooth surface. ,7 The effects of phosphoric acid concentration and duration of etching on the enamel etch depth have been studied by several investigators. Silverstone ~8 etched unground enamel surfaces with H~PO4 concentrations ranging from 20% to 70% HaPO 4 for 60 seconds and reported that the loss in depth of surface contour ranged
158
Am. J. Orthod. Dentofac. Orthop. August 1990
Legler, Retief, and Bradley 2.5
T
2.0 ¢/)
1.5
> n-" 1.0
0.5 0.0
60
30
15
I
60 30 15 60 Etch Duration - Seconds I
I
370/o H3PO4
I
15 % H3PO4
30
15
I
50/0 H3PO4
Fig. 3. Bar graph of surface roughness (Ra).
Table III. Calculated and measured depths of etch Calculated depth of etch (tun)
Measured depth of etch (tun)
Procedure
Mean
SD
Mean
[
SD
A: 37% H3PO4 60-second etch
27.1
4.3
28.0
5.1
B: 37% H3PO4 30-second etch
16.7
1.0
16.4
2.2
C: 37% H~PO4 15-second etch
8.9
0.6
8.8
1.1
D" 15% H~PO4 60-second etch
21.0
3.2
22.8
4.8
E: 15% H3PO4 30-second etch
16.1
1.7
13.8
2.5
F: 15% H~PO4 15-second etch
7.6
1.2
9.4
0.9
G:
5% H3PO4 60-second etch
9.1
1.0
9.8
1.8
H:
5% H~POa 30-second etch
6.2
0.5
6.0
1.4
I:
5% H~PO4 15-second etch
3.5
0.3
4.7
0.7
r = 0.959, p = 0.0001.
from 14 I.tm with 20% HaPO4 to 2 I.tm with 70% H3PO4. RetieP 9 etched enamel surfaces ground on 600-grit silicon carbide paper with from 10% to 85% HaPO4 solutions for 60 seconds and recorded depths of etch ranging from 15 p.m to 0.8 i.tm. Etching unground enamel surfaces with 37% H3PO4 for 90 seconds resulted in a mean loss of 6.9 p.m of surface enamel.2° The unground facial surfaces of premolar teeth were etched with three
commercially available etching solutions for 60 seconds and a mean depth of etch of 22.5 I.tm was found. 2~ The depths of etch ranged from 16.6 ism to 29.5 t.tm, depending on the etchant used. These findings can readily be explained by the phase diagram developed by Chow and Brown 22 for the ternary system of calcium hydroxide, phosphoric acid, and water. Etching enamel surfaces with solutons containing more than 27% H3PO4
Volume98 Number2
Effects of H3P04 concentration and etch duration 159
Table IV. Ra values of etched enamel surfaces (~m) listed in descending order of magnitude
Ra values (fun)
of specimens
Mean*
A: 37% tt3PO4 60-second etch
5
1.7
0.5
0.9-2.2
D: 15% H~PO~ 60-second etch
5
1.2
0.2
0.9-1.5
B: 37% H~PO~ 30-second etch
5
1.0
0.3
0.6-1.4
E" 15% H3PO~ 30-second etch
5
0.8
0. I
0.6-0.9
C: 37% H3PO~ 15-second etch
5
0.5
0.1
0.4-0.6
F: 15% H~PO4 i 5-second etch
5
0.5
0.1
0.4-0.6
G:
5% H3PO4 60-second etch
5
0.4
0.1
0.4-0.6
H:
5% H~PO4 30-second etch
5
0.4
0.1
0.4-0.5
5
0.2
0.0
0.2-0.2
Procedure
I:
5% H~PO~ 15-second etch
No.
I
SD
[
Range
*Means linked by vertical lines were not significantly different.
results in the formation of monocalcium phosphate monohydrate (MCPM), while etching with more dilute H3PO~ solutions results in the formation of dicalcium phosphate dihydrate (DCPD). Because MCPM is more soluble than DCPD, more concentrated H a P O 4 solutions are used clinically. The phase diagram clearly indicates that the amount of enamel dissolved will increase with acid concentrations up to 27% H3PO4. With acid concentrations greater than 27% H3PO4, the amount of enamel dissolved will decrease with an increase in acid concentration. The present study determined calculated and measured depths of etch on enamel surfaces ground on 600grit silicon carbide paper. It was not possible to record the surface profiles of the demarcated biopsy sites on unground enamel surfaces because of the curvature of unground enamel surfaces. Orthodontic attachments are bonded to unground enamel surfaces, and the depths of etch on such surfaces are less than the depths recorded in this study. The depth of etch or the amount of surface enamel removed during the etching procedure is dependent on the type of acid used, the concentration of the acid, the duration of etching, and the chemical composition of the enamel. 4 It was shown that etching high-fluoride unground enamel surfaces with perchloric acid resulted in a significantly decreased depth of etch compared with that obtained by grinding. It is interesting to note that, given the same acid
concentrations and durations of etching, significant differences in surface roughness of tooth enamel did not result in significant differences in the shear bond strengths of an orthodontic bonding resin .4 In the study that produced these findings a scanning electron microscope was used to observe great variations in the surface morphology of enamel surfaces etched with the nine etching procedures. These observations suggest that the depth of resin penetration into the etched enamel surfaces is not of prime importance. Etching enamel surfaces with orthophosphoric acid results in a significant increase in the surface free energy of the enamel surfaces, 24 producing greater wettability of the etched enamel surfaces and subsequent penetration of the curing resin into the subsurface porous zones. It is interesting to note that the current study found a highly significant correlation between the calculated and measured depths of etch (Table III). Silverstone ~6 has demonstrated that etching of enamel surfaces with H3PO4 results in a superficial etched zone and subsurface qualitative and quantitative porous zones. Only the superficial etched zone is measured from the surface profilometer tracings, while the calculated depths of etch are obtained from the total calcium (enamel) dissolved during the etching procedures. The results of our study suggest that the amount of calcium (enamel) dissolved from the subsurface zones is minimal. Enamel decalcification occurring during orthodontic
160 Legler, Retief, and Bradley
treatment is reduced b y a p p r o x i m a t e l y 25% b y topical fluoride treatment, z5 H o w e v e r , it has b e e n d e m o n s t r a t e d that m o r e than 5 0 % o f subjects c o m p l y p o o r l y with a p r e v e n t i v e fluoride m o u t h rinse program w h i l e underg o i n g orthodontic treatment. 2s A n o t h e r w a y e n a m e l decalcification during orthodontic treatment m a y be red u c e d is b y reduction o f the phosphoric acid c o n c e n tration and the duration o f etching. A m a j o r c o n c e r n is the effect o f these parameters on the retention o f b o n d e d attachments in the clinical situation. P r e l i m i n a r y studies, however, h a v e s h o w n that reducing the acid c o n centration and duration o f etching had no adverse effect on the clinical retention o f b o n d e d attachments. 27"2s We are indebted to Ms. S. Roper for typing the manuscript. REFERENCES 1. Buonocore MG. A simple method of increasing the adhesion of acrylic filling materials to enamel surfaces. J Dent Res 1955;38:849-53. i 2. Retief DH, Dreyer CJ, Gavron G. The direct bonding of orthodontic attachments to teeth by means of an epoxy adhesive. AM J ORTHOD1970;58:21-40. 3. Ceen RF, Gwinnett AJ. White spot formation associated with sealants in orthodontics. Pediatr Dent 1981;3:174-8. 4. Legler LR, Retief DH, Bradley EL, Denys FR, Sadowsky PL. Effects of phosphoric acid concentration and etch duration on the shear bond strength of an orthodontic bonding resin to enamel: an in vitro study. AM J ORTHODDENTOFACORTHOP1989; 96:485-92. 5. Srremark R, Samsahl K. Gamma-ray spectrophotometric analysis of elements in normal human enamel. Arch Oral Biol 1961 ;6:275-83. 6. Mazily RS, Hodge HS, Ange LE. Density and refractive index studies of dental hard tissues. II. Density distribution curves. J Deni Res 1939;19:203-11. 7. Retief DH, Busscher HJ, de Boer P, Jongebloed WL, Arends J. A laboratory evaluation of three etching solutions. Dent Mater 1986;2:202-6. 8. Ostle B, Mensing R. Statistics in research. 3rd ed. Iowa City, Iowa: Iowa State University Press, 1975:452-61. 9. Steele R , Torrie J. Principles and procedures in statistics. New York: McGraw-Hill, 1960:99-160. 10. Snedecor GW, Cochran WG. Statistical methods. 7th ed. Ames, Iowa: Iowa State University Press, 1980:175-91. 11. SAS/STAT guide for personal computers, version 6 ed. Gary, North Carolina: SAS Institute, 1985:235-6. 12. Gorelick L, Geigei" AM, Gwinnett AJ. Incidence of white spot formation after bonding and banding. AM J OR~IOD 1982;81: 93-8.
Am. J.
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13. Mizrahi E. Surface distribution of enamel opacities following orthodontic treatment. AM J ORrHOD 1983;84:323-31. 14. O'Reilly MM, Featherstone JDB. Demineralization and remineralization around orthodontic appliances: an in vivo study. AM J ORTtIODDENTOFACORTHOP 1987;92:33-40. 15. Brudevold F, Gardner DE, Smith FA. The distribution of fluoride in human enamel. J Dent Res 1956;35:420-9. 16. Silverstone LM. The acid etch technique: in vitro studies with special reference to the enamel surface and enamel-resin interface. In: Silverstone LM, Dogon IL. Proceedings of an international symposium on the acid etch technique. St. Paul: North Central, 1975:13-39. 17. Lee H, Ocumpaugh DE, Shaffer J, Sheble AM. Sealing of developmental pits and fissures. IV. Measurements of in vivo fluoride pickup by electron microprobe x-ray spectrophotometry. J Dent Res 1972;51:634-9. 18. Silverstone LM. Fissure sealants: Laboratory studies. Caries Res 1974;8:2-26. 19. Retief DH. The use of 50 per cent phosphoric acid as an etching agent in orthodontics: a rational approach. AM J ORTHOD 1875;68:165-78. 20. Pus MD, Way 1)(2. Enamel loss due to orthodontic bonding with filled and unfilled resins using various clean-up techniques. AM J ORa'HOD1980;77:269-83. 21. SheyZ, BrandtS. Enamel loss due to acid treatment for bonding. J Clin Orthod 1982;16:338-40. 22. Chow LC, Brown WE. Phosphoric acid conditioning of teeth for pit and fissure sealants. J Dent Res 1973;52:1158. 23. Thornton JB, Retief DH, Bradley EL, Denys FR. The effect of fluoride in phosphoric acid on enamel fluoride uptake and the tensile bond strength of an orthodontic bonding resin. AM J ORTHODDErZrOFACORT~OP 1986;90:91-101. 24. BusscherHJ, RetiefDH, Arends J. Relationship between surface free energies of dental resins and bond strengths. Dent Mater 1987;3:60-3. 25. Saloum IS, Sondhi A. Preventing enamel decalcification after orthodontic treatment. J Am Dent Assoe 1985;115:257-61. 26. Gejger AM, Gorelick L, Gwinnett AJ, Griswold PG. The effect of a fluoride progi'am on white spot formation during orthodontic treatment. AM J ORTHODDENTOFACORTHOP1988;93:29-37. 27. Sadowsky PL, Retief DH, Cox PR, Hemandez R, Rape G. Effect of etchant concentration and duration of etching on retention of orthodontic attachments [Abstract]. J Dent Res 1988;67:361. 28. ViljoenWP, Swb.nepoel F, Pretorius LM, DuPlessis LS, Smit HJ, De Mflelenaere JJGG. Shorter etching times in orthodontic bonding: an in vitro study [Abstract]. J Dent Res 1988;67:776. Reprint requests to: Dr. D.H. Retief Department of Biomaterials University of Alabama School of Dentistry University Station Birmingham, AL 35294
