Improved Fluoride Incorporation and New In Vitro Fluorine Elemental Determination K. S. RAJAN*, ELLIOTT RAISEN, JOHN W. MANDLERt, DENNIS L. RILEYI:, and RICHARD A. SEMMLER Illinois Institute of Technology Research Institute, Chicago, Illinois 60616, USA A new, nondestructive technique, charged particle activation, was used to determine the fluorine concentration as a function of depth at 0.1-micrometer (,um) increments in the enamel of human teeth treated in vitro with three different fluorides. Twenty-fourhour treatment with K,,ZrF6 showed a 24% fluorine concentration in the first 0.1-p.m depth and 14% at 2.5 ,um compared, respectively, with 4 and 2.5% concentrations after NaF treatments.
On the basis of a review of the topical fluorides investigated so far,1-13 it appears that improved incorporation of fluoride and more efficacious cariostatic activity are achieved by using polyanionic complex fluorides such as stannous hexafluorozirconate and sodium monofluorophosphate than by using the simple compounds such as stannous fluoride and sodium fluoride. However, a major disadvantage of the complex fluorides thus far examined is their solution instability, the results of which, toxic tissue irritations and lowered efficiencies, have been reported. Furthermore, there is much to be learned about the distribution of the levels of the fluorides at different depths of the enamel surface that might have a significant relationship with the most effective protection. Also, quantitative information on the depths of penetration of the different topical fluorides will be very valuable. Therefore, it was the objective of this exploratory research to incorporate simple This investigation was supported by Illinois Institute of Technology Research Institute in-house funds. Received for publication February 18, 1975. Accepted for publication January 20, 1976. 0 For reprints. t Present address: Aeroject Nuclear Corp., Idaho Falls, Idaho. $ Present address: National Accelerator-Laboratories, Batavia, Ill.
and complex fluorides (in vitro) into the enamel of extracted teeth and to determine the fluoride levels at different depths of the enamel surface. Since the presently available methods for determining the concentration profile of the incorporated fluoride are extremely laborious and destroy the sample,'-18 the feasibility of applying nuclear techniques of charged particle activation (CPA) and proton stimulated X-ray fluorescence (PSF) to nondestructively determine the fluoride levels was undertaken. These techniques have the potential of much finer spatial resolution than previous techniques. The CPA technique determines the concentration of fluorine as a function of depth by following the natural surface irregularities of a tooth. In a single analysis, the PSF method determines the concentration of all elements heavier than Mg within a short depth from the surface. Potassium hexafluorozirconate was chosen as the model complex fluoride for this study because of its high solution stability, binegative charge, and aqueous solubility characteristics.
Materials and Methods Samples of extracted human teeth were freed of accretions and adhering soft tissues by brushing which was followed by rinsing with distilled water. After a one-minute application of 0.01 M H3PO4 (by means of a swab), the tooth samples were thoroughly rinsed with distilled water. One half of each of the cleaned teeth was coated with wax and the remaining half subjected to treatments with the different fluorides, that is, K2ZrF6, NaF, and acidulated phosphate fluoride (APF) solution. The treatment consisted of equilibrating the individual tooth samples with aqueous solutions of the fluorides for different periods of time at 25 C. 671
Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 19, 2015 For personal use only. No other uses without permission.
672
RAJAN ET AL
J Dent Res July-August 1976
After the treatment, the samples were rinsed with distilled water and analyzed by CPA and PSF techniques. The treated surface was analyzed first. After removing the wax by means of organic solvents, the untreated surface was analyzed. CPA.-The CPA technique consists of bombarding a sample with charged particles (protons in our case) and observing the radiations produced by the charged particleinduced nuclear reactions. The emitted radiations are characteristic of the emitting isotope together with the energy and the type of the incident-charged particles. These nuclear reactions occur only within very narrow, specific (resonance) energy bands. If the energy of the incident-charged particle beam is greater than the resonance energy, the charged particles enter the surface of the sample without initiating the reaction. They penetrate deeper, continually losing energy, until the resonance energy is reached and the nuclear reaction occurs. The depth at which the reaction occurs can be determined from the difference between the initial beam energy and the resonance energy, whereas the elemental concentrations at that depth are determined from the spectrum of the emitted radiations. We used the F19 (p, ay) 016 resonance reaction, which occurs when fluorine is placed in a beam of protons, to determine the fluorine concentration. In this reaction, a- and y-rays are emitted. Although either radiation can be used in the measurement of fluorine, we used the y-rays because they have a greater penetrability than do a-rays. This reaction has been shown to provide a sensitive method for detecting fluorine.19-21
It has a strong resonance at a proton energy of 672 kev. This is the first strong resonance that results in the emission of copious 6.13 mev y-rays. Although a number of resonances occurs for proton energies lower than 672 kev, the flux of 6.13 mev y-rays produced by any of these resonances is weak compared with that produced by the 672 kev resonance. Thus, as the energy of the proton beam is increased, no 6.13 mev y-rays will be emitted until the proton energy reaches 672 kev. At a proton energy of 672 kev, the reaction occurs and 6.1 3-mev -y-rays are produced. The intensity of this 6.1 3-mev -y-ray flux yields the concentration of fluorine on the surface of the samples. As the proton energy is raised to more than 672 kev, the protons enter the sample without initiating a reaction. They penetrate deeper, continuously losing energy (at a rate of about 70 kev/ micrometer (gm) in calcium hydroxylapatite,22 the major component of tooth enamel), until an energy level of 672 mev is reached. The y-ray flux produced at that point is then used to determine the concentration of fluorine at the depth where the reaction takes place. The next resonance greater than 672 kev occurred at a proton energy of 835 kev. An incident 835 kev proton traverses about 2.5 /m in calcium hydroxylapatite before its energy is reduced to 672 kev. Therefore, by incrementing the proton energy from 672 kev to just less than 835 kev and by measuring the intensity of the 6.13-mev -y-rays at each step, the fluorine concentration was determined as a function of depth to a depth of 2.5 ym. A schematic of the experimental setup is shown in Figure 1.
.-Collimator
Proton Beam
el
0
FIG 1.-Experimental setup for charged-particle activation analysis. Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 19, 2015 For personal use only. No other uses without permission.
Vol 55 No. 4
FLUORIDE INCORPORATION & F DETERMINATION
673
Proton Beam
FIG 2.-Illustration of depth penetration
followving contour of tooth surface in CPA technique.
It is interesting to note that because of the nature of this technique, the depth level detected followed the contour of the tooth including any defects originally associated with the tooth as shown in Figure 2. The depth resolution obtainable using the CPA technique depends on various para-
meters, namely, energy spread in the proton beam, the width of the resonance, and the range straggling of the charged particles. In general, the depth resolution is better than 10%. In the case of the measurement of fluorine in tooth enamel, the proton beam energy and resonance had a half width of 3
Sample
r
Proton Beam
.0
FIG 3.-Block diagram of electronics used in CPA and PSF techniques. A, preamplifier; B, Ge (Li) detector; C, Canberra 1413 amplifier; D, HP 5401 multichannel height analyzer; E, Si (Li) detector; F, preamplifier; G, 343 pileup rejector; H, TC 304 linear gate; I, Canberra 1413 amplifier; J, TC 601 strobed pulse stretcher; and K, ND 2200 multichannel pulse height analyzer. Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 19, 2015 For personal use only. No other uses without permission.
674
J Dent Res July-August 1976
RAJAN ET AL
kev and the depth resolution was about 0.07 ym at the surface of the tooth and about 0.15 um at a depth of 2 tum below the surface. A 2-mev Van de Graaff accelerator with an analyzing magnet was used with a Ge (Li) y-ray detector that has a resolution of 1.8 kev at 1.33 mev and a detection efficiency of 6% (relative to a 7.6 x 7.6 cm NaI (TI) detector) and with multichannel pulse height analyzers, and associated electronics as shown in Figure 3. The energy stability of the Van de Graaff machine is better than 0.5%. The beam current is variable from about 0.5 namp to about 150 yta and beam sites are from 0.5 to 1 cm in diameter. Beam currents of 50 namp and a beam width of 5 mm were used with counting times of 20 minutes. This gives a lower detection limit of about 1,000 ppm fluoride. This could be reduced to about 100 ppm fluoride by using higher currents and longer counting times. Charging effects were eliminated by coating the teeth with a 2,000-A thick layer of gold. This was done after the PSF work on the sample. The absolute fluorine concentration obtained by comparing measured y-ray intensities with data obtained on calibrations were also used to account for the weak resonances that were less than 672 kev. PSF.-The PSF technique was done with the same apparatus using proton beam energy of 1.8 mev and a beam diameter of 5 mm. A 10-mm2 Si (Li) detector that has a resolution of 170 ev at 5.9 kev was used. The beam penetration is about 20 tum which is therefore the depth of the analysis. Charging was a severe problem and limited the beam current to less than 3 namps. As the beam current was increased to a level greater than 3 namps, charging caused the background continuum to increase in intensity, thereby masking small peaks and making analysis more difficult. At beam currents of less than 3 namps, no increase in background intensity was observed. Although a thin carbon coating would have alleviated the charging problem, it would have interfered with the F19 (p, cay) 016 results. Calibration was done with a pure calcium hydroxylapatite pellet and an epoxy standard that contains 12 elements. X-ray peak intensities were compared with the intensities obtained using the standards, and semiquantitative results were obtained. Corrections for matrix effects were not included.
Results The fluorine concentration profiles for the teeth treated with K2KzF6, NaF, and APF are shown in Figure 4. The first two are for teeth that were treated for 24 hours. The tooth treated with the K2ZrF6 showed a very high concentration of fluoride, that is, 24% at the surface ( that is, within the first 0.07 ,um) with a sharp decrease to a still very high level of 14%, which it retained to at least a depth of 2.1 ,um. The tooth treated with sodium fluoride showed a concentration of 4.3% at the surface. This decreased gradually to 2.5% at a depth of 2.1 um. Tooth no. 3 treated for four minutes with the standard APF solution had a concentration of 0.4% at the surface and 0.25% at the 2.1 /m depth. (Note the fourfold change in the fluoride concentration scale in Fig 4.) Teeth numbered 4 and 5 were treated for four minutes with K2ZrF6 and NaF, respectively. They had similar profiles except that the surface concentrations were 1.4 and 0.8%, respectively, as given in Table 1. The fluorine concentrations in the untreated portions of teeth no. 4 and 2 were 0.42 and 0.14%. The X-ray emission spectra of a tooth that had been treated with K2ZrF6 is shown in
5_
Tooth No. 2 Tooth No.3
0.4
0 0
0.5
.0
i.5
2.0
25
Depth (jsm)
FIG 4.-Fluorine concentration profiles in three treated teeth using CPA. Teeth no. 1, 2, and 3 were treated with K2ZrF,, NaF, and APF, respectively (note four-fold scale expansion for tooth 3.)
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FLUORIDE INCORPORATION & F DETERMINATION
Vol 55 No. 4
675
Figure 5. Each of the peaks represents the X-ray emission of a single element, except -| qO e - oin the instance of peaks 2 and 7 where there was an overlapping of the X-ray energies. oo 2l +It+l +1 +1 Identification of the peaks is given in Tao oq o m ble 2. Hq; _ 4 o o Discussion This work was a preliminary study to examine the effects of polyanionic complex fluorides as topical agents and to explore the c cy e ofeasibility of applying the nuclear techl qj Z @g ooooo niques, that is, CPA for the determination < lc Itl00W of fluoride and PSF for the determination n o. am oo of other elements heavier than Mg. Alcli though a small number of tooth samples was 0
-4
used
for
each
test,
the
differences
in
the
significantly greater
z
the normal
o
..o . .
*=
W
oo
H
experimental
variations.
The 24-
K2ZrF6
hour treatment, for instance, incorporated five times as much fluorine on the surface and internally for the first 2.1 ytm, .as in the instance of the NaF treatment, and more than 50 times as much as in the APF treatment. It is not likely that variability in tooth samples or in the experimental procedures
X *c
On the basis of a review of the topical fluorides investigated so far,1-13 it appears that improved incorporation of fluoride and more efficacious cariostatic activity are achieved by using polyanionic complex fluorides such as stannous hexafluorozirconate and sodium monofluorophosphate than by using the simple compounds such as stannous fluoride and sodium fluoride. However, a major disadvantage of the complex fluorides thus far examined is their solution instability, the results of which, toxic tissue irritations and lowered efficiencies, have been reported. Furthermore, there is much to be learned about the distribution of the levels of the fluorides at different depths of the enamel surface that might have a significant relationship with the most effective protection. Also, quantitative information on the depths of penetration of the different topical fluorides will be very valuable. Therefore, it was the objective of this exploratory research to incorporate simple This investigation was supported by Illinois Institute of Technology Research Institute in-house funds. Received for publication February 18, 1975. Accepted for publication January 20, 1976. 0 For reprints. t Present address: Aeroject Nuclear Corp., Idaho Falls, Idaho. $ Present address: National Accelerator-Laboratories, Batavia, Ill.
and complex fluorides (in vitro) into the enamel of extracted teeth and to determine the fluoride levels at different depths of the enamel surface. Since the presently available methods for determining the concentration profile of the incorporated fluoride are extremely laborious and destroy the sample,'-18 the feasibility of applying nuclear techniques of charged particle activation (CPA) and proton stimulated X-ray fluorescence (PSF) to nondestructively determine the fluoride levels was undertaken. These techniques have the potential of much finer spatial resolution than previous techniques. The CPA technique determines the concentration of fluorine as a function of depth by following the natural surface irregularities of a tooth. In a single analysis, the PSF method determines the concentration of all elements heavier than Mg within a short depth from the surface. Potassium hexafluorozirconate was chosen as the model complex fluoride for this study because of its high solution stability, binegative charge, and aqueous solubility characteristics.
Materials and Methods Samples of extracted human teeth were freed of accretions and adhering soft tissues by brushing which was followed by rinsing with distilled water. After a one-minute application of 0.01 M H3PO4 (by means of a swab), the tooth samples were thoroughly rinsed with distilled water. One half of each of the cleaned teeth was coated with wax and the remaining half subjected to treatments with the different fluorides, that is, K2ZrF6, NaF, and acidulated phosphate fluoride (APF) solution. The treatment consisted of equilibrating the individual tooth samples with aqueous solutions of the fluorides for different periods of time at 25 C. 671
Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 19, 2015 For personal use only. No other uses without permission.
672
RAJAN ET AL
J Dent Res July-August 1976
After the treatment, the samples were rinsed with distilled water and analyzed by CPA and PSF techniques. The treated surface was analyzed first. After removing the wax by means of organic solvents, the untreated surface was analyzed. CPA.-The CPA technique consists of bombarding a sample with charged particles (protons in our case) and observing the radiations produced by the charged particleinduced nuclear reactions. The emitted radiations are characteristic of the emitting isotope together with the energy and the type of the incident-charged particles. These nuclear reactions occur only within very narrow, specific (resonance) energy bands. If the energy of the incident-charged particle beam is greater than the resonance energy, the charged particles enter the surface of the sample without initiating the reaction. They penetrate deeper, continually losing energy, until the resonance energy is reached and the nuclear reaction occurs. The depth at which the reaction occurs can be determined from the difference between the initial beam energy and the resonance energy, whereas the elemental concentrations at that depth are determined from the spectrum of the emitted radiations. We used the F19 (p, ay) 016 resonance reaction, which occurs when fluorine is placed in a beam of protons, to determine the fluorine concentration. In this reaction, a- and y-rays are emitted. Although either radiation can be used in the measurement of fluorine, we used the y-rays because they have a greater penetrability than do a-rays. This reaction has been shown to provide a sensitive method for detecting fluorine.19-21
It has a strong resonance at a proton energy of 672 kev. This is the first strong resonance that results in the emission of copious 6.13 mev y-rays. Although a number of resonances occurs for proton energies lower than 672 kev, the flux of 6.13 mev y-rays produced by any of these resonances is weak compared with that produced by the 672 kev resonance. Thus, as the energy of the proton beam is increased, no 6.13 mev y-rays will be emitted until the proton energy reaches 672 kev. At a proton energy of 672 kev, the reaction occurs and 6.1 3-mev -y-rays are produced. The intensity of this 6.1 3-mev -y-ray flux yields the concentration of fluorine on the surface of the samples. As the proton energy is raised to more than 672 kev, the protons enter the sample without initiating a reaction. They penetrate deeper, continuously losing energy (at a rate of about 70 kev/ micrometer (gm) in calcium hydroxylapatite,22 the major component of tooth enamel), until an energy level of 672 mev is reached. The y-ray flux produced at that point is then used to determine the concentration of fluorine at the depth where the reaction takes place. The next resonance greater than 672 kev occurred at a proton energy of 835 kev. An incident 835 kev proton traverses about 2.5 /m in calcium hydroxylapatite before its energy is reduced to 672 kev. Therefore, by incrementing the proton energy from 672 kev to just less than 835 kev and by measuring the intensity of the 6.13-mev -y-rays at each step, the fluorine concentration was determined as a function of depth to a depth of 2.5 ym. A schematic of the experimental setup is shown in Figure 1.
.-Collimator
Proton Beam
el
0
FIG 1.-Experimental setup for charged-particle activation analysis. Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 19, 2015 For personal use only. No other uses without permission.
Vol 55 No. 4
FLUORIDE INCORPORATION & F DETERMINATION
673
Proton Beam
FIG 2.-Illustration of depth penetration
followving contour of tooth surface in CPA technique.
It is interesting to note that because of the nature of this technique, the depth level detected followed the contour of the tooth including any defects originally associated with the tooth as shown in Figure 2. The depth resolution obtainable using the CPA technique depends on various para-
meters, namely, energy spread in the proton beam, the width of the resonance, and the range straggling of the charged particles. In general, the depth resolution is better than 10%. In the case of the measurement of fluorine in tooth enamel, the proton beam energy and resonance had a half width of 3
Sample
r
Proton Beam
.0
FIG 3.-Block diagram of electronics used in CPA and PSF techniques. A, preamplifier; B, Ge (Li) detector; C, Canberra 1413 amplifier; D, HP 5401 multichannel height analyzer; E, Si (Li) detector; F, preamplifier; G, 343 pileup rejector; H, TC 304 linear gate; I, Canberra 1413 amplifier; J, TC 601 strobed pulse stretcher; and K, ND 2200 multichannel pulse height analyzer. Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 19, 2015 For personal use only. No other uses without permission.
674
J Dent Res July-August 1976
RAJAN ET AL
kev and the depth resolution was about 0.07 ym at the surface of the tooth and about 0.15 um at a depth of 2 tum below the surface. A 2-mev Van de Graaff accelerator with an analyzing magnet was used with a Ge (Li) y-ray detector that has a resolution of 1.8 kev at 1.33 mev and a detection efficiency of 6% (relative to a 7.6 x 7.6 cm NaI (TI) detector) and with multichannel pulse height analyzers, and associated electronics as shown in Figure 3. The energy stability of the Van de Graaff machine is better than 0.5%. The beam current is variable from about 0.5 namp to about 150 yta and beam sites are from 0.5 to 1 cm in diameter. Beam currents of 50 namp and a beam width of 5 mm were used with counting times of 20 minutes. This gives a lower detection limit of about 1,000 ppm fluoride. This could be reduced to about 100 ppm fluoride by using higher currents and longer counting times. Charging effects were eliminated by coating the teeth with a 2,000-A thick layer of gold. This was done after the PSF work on the sample. The absolute fluorine concentration obtained by comparing measured y-ray intensities with data obtained on calibrations were also used to account for the weak resonances that were less than 672 kev. PSF.-The PSF technique was done with the same apparatus using proton beam energy of 1.8 mev and a beam diameter of 5 mm. A 10-mm2 Si (Li) detector that has a resolution of 170 ev at 5.9 kev was used. The beam penetration is about 20 tum which is therefore the depth of the analysis. Charging was a severe problem and limited the beam current to less than 3 namps. As the beam current was increased to a level greater than 3 namps, charging caused the background continuum to increase in intensity, thereby masking small peaks and making analysis more difficult. At beam currents of less than 3 namps, no increase in background intensity was observed. Although a thin carbon coating would have alleviated the charging problem, it would have interfered with the F19 (p, cay) 016 results. Calibration was done with a pure calcium hydroxylapatite pellet and an epoxy standard that contains 12 elements. X-ray peak intensities were compared with the intensities obtained using the standards, and semiquantitative results were obtained. Corrections for matrix effects were not included.
Results The fluorine concentration profiles for the teeth treated with K2KzF6, NaF, and APF are shown in Figure 4. The first two are for teeth that were treated for 24 hours. The tooth treated with the K2ZrF6 showed a very high concentration of fluoride, that is, 24% at the surface ( that is, within the first 0.07 ,um) with a sharp decrease to a still very high level of 14%, which it retained to at least a depth of 2.1 ,um. The tooth treated with sodium fluoride showed a concentration of 4.3% at the surface. This decreased gradually to 2.5% at a depth of 2.1 um. Tooth no. 3 treated for four minutes with the standard APF solution had a concentration of 0.4% at the surface and 0.25% at the 2.1 /m depth. (Note the fourfold change in the fluoride concentration scale in Fig 4.) Teeth numbered 4 and 5 were treated for four minutes with K2ZrF6 and NaF, respectively. They had similar profiles except that the surface concentrations were 1.4 and 0.8%, respectively, as given in Table 1. The fluorine concentrations in the untreated portions of teeth no. 4 and 2 were 0.42 and 0.14%. The X-ray emission spectra of a tooth that had been treated with K2ZrF6 is shown in
5_
Tooth No. 2 Tooth No.3
0.4
0 0
0.5
.0
i.5
2.0
25
Depth (jsm)
FIG 4.-Fluorine concentration profiles in three treated teeth using CPA. Teeth no. 1, 2, and 3 were treated with K2ZrF,, NaF, and APF, respectively (note four-fold scale expansion for tooth 3.)
Downloaded from jdr.sagepub.com at The University of Iowa Libraries on June 19, 2015 For personal use only. No other uses without permission.
FLUORIDE INCORPORATION & F DETERMINATION
Vol 55 No. 4
675
Figure 5. Each of the peaks represents the X-ray emission of a single element, except -| qO e - oin the instance of peaks 2 and 7 where there was an overlapping of the X-ray energies. oo 2l +It+l +1 +1 Identification of the peaks is given in Tao oq o m ble 2. Hq; _ 4 o o Discussion This work was a preliminary study to examine the effects of polyanionic complex fluorides as topical agents and to explore the c cy e ofeasibility of applying the nuclear techl qj Z @g ooooo niques, that is, CPA for the determination < lc Itl00W of fluoride and PSF for the determination n o. am oo of other elements heavier than Mg. Alcli though a small number of tooth samples was 0
-4
used
for
each
test,
the
differences
in
the
significantly greater
z
the normal
o
..o . .
*=
W
oo
H
experimental
variations.
The 24-
K2ZrF6
hour treatment, for instance, incorporated five times as much fluorine on the surface and internally for the first 2.1 ytm, .as in the instance of the NaF treatment, and more than 50 times as much as in the APF treatment. It is not likely that variability in tooth samples or in the experimental procedures
X *c
