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Tin and fluoride uptake in human enamel in situ: electron probe and chemical microanalysis
B ila te ra lly opposed c lin ic a lly norm al te e th procured fro m naval recruits scheduled fo r fu ll-m o u th e x tra ctio n s were tre a te d by thorough p u m icing and topical a p p lica tio n o f e q u im o la r solutions o f stannous flu o rid e and sodium chloride (c o n tro l). A fte r e x tra c tio n , tin c o n te n t was determ ined in the enamel by electron probe m icroanalysis. Fluoride c o n te n t was determ ined ch e m ica lly in the fir s t 25 to 50 n o f enam el. T in was d is trib u te d random ly on tre a te d surfaces in circum scribed areas o f e n rich m e n t o f a b o u t 125 d ia m e te r and 20 v- deep. Fluoride was generally increased a b o u t 20 percent in tre a te d enamel and appeared also io be dispersed random ly a t depths o f a b o u t 25 a*.
Tin and fluoride uptake in human enamel in situ: electron probe and chemical microanalysis
Kirk C. Hoerman, DDS, MSD, Bethesda; James E. Klima, DDS, Great Lakes, III.; L. S. Birks, MS; David J. Nagel, BS; William E. Ludwick, DDS, Washington, D. C., and Harvey W. Lyon, DDS, PhD, Chicago
Systematic application of fluoride compounds to tooth surfaces reduces the incidence of dental caries. The process by which surface-applied flu oride and an accompanying cation obtain anticariogenic action is not clearly understood.1-3 Re gardless of divergent opinions as to mode of action, choice of medicaments, and clinical effec tiveness,4'8 deriving an optimum effect must surely be a function of detail and not of kind. The mode of incorporating tin and fluoride into clinically sound enamel is not well understood. Brudevold and others9 reported that enamel treated for 20 minutes with 1.86 percent stannous fluoride obtained up to 640 ppm of tin in outer layers, while the fluoride content reached 1,200 ppm. Although it appeared that tin uniformly
penetrated sound enamel along the surface, the possibility remained that circumscribed regions of enrichment occurred that were not detectable by chemical analysis. The present study was designed to obtain fur ther information about the penetration, especially the distribution, of stannous fluoride in apparently sound tooth enamel in situ. A relatively new tech nic was used for the determination of tin in tooth structures. Electron probe microanalysis detects elemental components of metals, alloys, or certain biologic tissues with qualitative and quantitative reliability. In addition, the method reveals the specific region or volume fraction occupied by the element of interest. Such versatility is desir able and was therefore applied in the present study 1301
of the distribution and concentration of tin along an enamel surface. The applicability of electron probe microanalysis to mineralized tissues was first shown by Boyde, Switsur and Fearnhead.10 Subsequent use of the instrument for analysis in dental structures was limited. The equipment used for the present study was a Phillips (Norelco) Electron Probe Microanalyzer. The electron probe directs an electron beam on a small area of the specimen surface (about 1 ¡x. in diameter). The beam excites atoms and causes the emission of X rays that have wave frequencies characteristic of the elements residing in the specimen. Spectrographic analysis of the X-ray emission from the tissue or specimen makes possible the qualitative identification of the elements present. Also, quan tities may be calculated from the intensity of the characteristic X rays. The distribution of an ele ment over an entire region of interest, for example a tin-enriched region, may be determined by elec trostatically deflecting the electron beam in a scanning action across the specimen surface while displaying the X-ray intensity on a cathode-ray screen. Birks11 offers a more detailed description of the methods and equipment.
Materials and methods Twelve naval recruits, 17 to 24 years of age, who were reporting for primary training at the U.S. Naval Training Center, Great Lakes, 111., were selected for this study. Those chosen to participate in the study were recruits who eventually would require complete extractions and subsequent re placement by appropriate prostheses. Initially, however, each subject was required to have a lat erally opposed tooth pair free of large carious lesions or restorations or both in the remaining natural dentition. The designation of the control and experimental teeth was at random assuming bilateral symmetry of carious lesions. Three tooth pairs were selected for electron probe micro analysis for tin incorporation. These teeth were also included in the 12 tooth pairs submitted to fluoride analysis.
Treatment ■ Control quadrant: First, the entire dental arch containing the laterally opposed experimental teeth was cleaned by scaling. A chemically pure 1302 ■ JA D A , V o l. 7 3 , Dec. 1966
sodium chloride-pumice mixture (1.62 Gm. of sodium chloride, 6 Gm. of pumice, and 3 ml. of distilled water) was applied to each tooth surface for 10 seconds by use of a rubber cup rotating in a dental handpiece. Each tooth was burnished for about 10 seconds. After pumicing, the quad rant was isolated with cotton rolls and dried. The surfaces were then swabbed with 1.2 normal so dium chloride solution and, after 30 seconds, den tal floss was used in interproximal spaces. The patient was instructed not to rinse his mouth or swallow for 30 minutes; however, he was advised to expectorate as needed. After rinsing, a local anesthetic was administered. The control tooth was subsequently extracted, freed of vascular and fibrous debris, rinsed briefly in distilled water, blotted, and placed in a test tube and was frozen immediately in dry ice until prepared for electron probe or chemical microanalysis. ■ Experimental quadrant: Four days after the extraction of the control tooth, the subject re turned and the teeth in the experimental quadrant were burnished with a 9.09 percent stannous fluoride-pumice mixture (0.9 Gm. of SnF2, 6 Gm. of pumice, and 3 ml. of distilled water). This treatment was followed by the topical application of 1.28 normal SnF2 solution (10 percent) to all enamel surfaces including the experimental tooth. After 30 minutes, the experimental tooth was ex tracted and prepared for analysis.
Experimental probe microanalysis
Six teeth (3 paired specimens) were cross-sec tioned perpendicular to the long axis through the thickest enamel portion of the crown. These teeth were mounted, 3 teeth at a time, to one side in a 1-inch diameter Bakelite piece. Standards of pure tin were similarly mounted. Both teeth and stand ards were separately abraded with successively finer papers, through no. 4-0, and then polished with diamond paste on paper and Linde “B” powder on wet felt. The Bakelite mounts were next halved, and two final sample mounts were reconstructed for microprobe analysis. Each sam ple had 3 teeth on one side and the tin standard on the other. This separate handling of samples and standard precluded the possibility of smear ing tin on the teeth during polishing. The probe operator was not told which teeth were treated and which were control teeth.
Fig. 1 ■ C a th o d e -ra y screen presenta tio n ( le ft) and dia g ra m (rig h t) o f X -ra y e m issivity o f tin in enriched region of enamel tre a te d w ith stannous flu o rid e . X -ra y emission fo llo w s electron b o m bard m ent o f enam el using P hillip s Electron Probe M icro a n a lyze r
Fluoride determinations Fluoride determinations were made by use of the microdiffusion methods described by W harton.12 Modifications in technic were made according to Weatherell and Hargreaves13 so that in 7 tooth pairs fluoride determinations could be made in the first 25 /j, layer of enamel. Specimens were coded before chemical analysis so that control and treat ed teeth were not identifiable.
intensity (ordinate) was recorded against the dis tance traversed by the exciting electron beam (abscissa). The data confirm the presence of tin and also indicate the depth of penetration and the concentration of tin through the enrichment band. The effective depth of penetration was about 20 ¡j, in the experiment shown in Figure 2. y
SPEC MEN 711
5 0 0 'M
R esults and discussion ■ Tin: Electron probe microanalysis revealed tin enrichment in each of three pairs of the experi mental teeth treated with stannous fluoride. Fig ure 1 is a photograph of the oscilloscope screen that displays an X-ray intensity map from tin accumulation in treated enamel. A diagrammatic presentation of the photograph is also shown in Figure 1. On the photograph, there are large white blobs of X-ray emission. These white blobs were caused by Bakelite-embedding artifacts. The X-ray emissivity caused by tin enrichment is seen as tiny white pips along a diagonal line consistent with the enamel surface. The penetra tion of tin in treated teeth appeared confined to depths from 5 to 20 p. and extended, in most instances, along the surface from 100 to 200 ¡x. These tin-enriched regions occurred in enamel so as to suggest a localized element predilection. Figure 2 shows a line scan across the depth of one of the typical tin-enriched regions. The elec tron probe was set so that only tin X-ray emission
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Fig. 2 ■ Recorder tra cin g o f X - r a y emission in te n sity from tin versus distance in m icrons traversed by h ig h in te n sity electron beam across section o f enam el con ta in in g tin -e n ric h e d region subsequent to tre a tm e n t w ith SnFj. (Same enamel section as shown in Figure 1 )
Hoerm an and others— T IN A N D FLUORIDE UPTAKE IN H U M A N E N A M E L ■ 1303
The content of tin in the enriched region was calculated from X-ray emission assuming tooth enamel to be hydroxylapatite and allowing for X-ray absorption. A t the highest concentration, tin was at a level of 37,000 ppm. If a further cor rection were made, assuming that rounding of the edges occurred during polishing, the tin concen tration would not be less than 20,000 ppm in the enriched regions. The limit of detectability of the electron probe microanalysis in this experiment was calculated from Figure 2. Counting times for each region were 300 seconds. Thus, background counts in this period amounted to 3,000. Three standard deviations of this number equaled 165 counts. From this value, the limit of detection of tin emission intensities near those shown in Figure 2 was calculated to be about 100 ppm. Since it has been shown by chemical methods that tin up take was about 500 ppm along the entire intact enamel surface,9 a fact not confirmed by us by use of electron probe microanalysis, it appears that this result could have been due, in fact, to incorporation of tin in entrapped regions. U su ally, an electron probe scan of the periphery of a treated specimen revealed from 3 to 5 tin-enriched regions. One may calculate an approximate enamel tin content o f about 220 ppm from our data, and this value is reasonable when compared with that obtained chemically by Brudevold and others.9 The fact that tin uptake by enamel occurs in circumscribed trapping areas is grounds for specu lation that these regions are the same as the “white spots” reported by Gray14 or the prime tin uptake sites postulated by Muhler.15 An interesting experiment would be to deter mine the calcium:phosphorus ratio in tin-enriched regions. This ratio might indicate whether the tin replaces either calcium or phosphorus or whether it simply diffuses into the regions. The distribu tion of tin throughout the tin-enriched zones ap peared from the oscilloscope tracings to be uni form; however, uniformity was not verified since only one scan was made across the width. Repet itive quantifying scans would verify the concen tration distribution in one enriched region.
Fluoride levels The table shows the results o f fluoride determina tions in control and treated experimental teeth after in situ application of stannous fluoride to the enamel surfaces. The values shown have been 1304 ■ J A D A , V o l. 7 3 , Dec. 1966
Table ■ The presence o f tin and flu o rid e in hum an enam el a fte r to p ica l a p p lica tio n in situ o f 10 percent stannous flu o rid e solution Enamel Paired specim en No. C ontrol
Tooth
Treated
Sample w e ig h t (m g.)
Fluoride (ppm )
T in *
t
C oronal powdered enam el 701 711
Lower la te ra l incisor
10.55 10.30
105.5 136.0
710
U pper second p rem olar
10.29 10.59
41.0 32.8
725
Upper second m olar
10.04 10.25
27.1 32.8
728
U pper la te ra l incisor
10.94 10.53
203.4 182.2
+
Upper canine
10.87 10.11
70.7 100.1
+
U pper c e ntra l incisor
15.90 59.50
3 4 8 .2 f 503.4
Upper canine
26.80 21.65
784.8 1065.2
727
Upper central incisor
17.20 7.25
393.9 403.8
703
Upper la te ra l incisor
11.15 9.55
198.5 234.9
716
Lower central incisor
10.00 9.50
372.0 324.9
704
Upper central incisor
10.05 16.95
472.3 576.3
705
Upper central incisor
7.35 9 .40
176.4 437.1
724 726 715 717 •
734
P erchloric a cid -e tch e d enamel 745 708 747 743 750 732 702 735 744
*D e te rm in e d by e le ctro n probe m icroanalysis tV a lu e s corrected to a basis o f 10 m g. o f surface enam el, e q u iv a le n t to 25 fi depth
corrected so that they express fluoride concentra tion in a 10-mg. specimen of enamel. These re sults have been divided into two major groups: 5 pairs of teeth in which fluoride levels were de termined by using an aliquot of the total coronal powdered enamel, and 7 pairs in which fluoride levels were determined on the first 25 ¡j. of per chloric acid-dissolved enamel surfaces. In the powdered-enamel fluoride determina tions, it was not possible to detect significant ele vations caused by the stannous fluoride treatment. These findings were consistent with those of others16 in that fluoride content of enamel is in creased only in the first few microns beneath the surface. Use o f total powdered enamel would mask real differences between control and treated teeth by the diluent effect of normal deeper en amel. The reliability and reproducibility o f the technic, however, were confirmed, and it was shown that fluoride content tended to follow the principle of bilateral symmetry. Measurement of total enamel fluoride levels was usually in agree ment with measurements reported by others.16 In
posterior teeth, the fluoride content of enamel was lower by about half. Surface fluoride determinations revealed signifi cant increases of fluoride in 6 of the 7 treated teeth. Values up to 1,065 ppm were found in the first 25 ¡J. of treated enamel. Fluoride levels at the same depth in controls were usually compa rable with those reported by others using similar quantitative procedures.13 In 2 treated lower cen tral incisors, the fluoride levels were the same as in their untreated laterally opposed controls. This finding could be explained if the surface7applied fluoride was distributed along the enamel surface in a similar fashion as tin is distributed, that is, principally in trapping regions instead of uniform ly dispersed along the first few micron depths. Accordingly, it would be possible, where enamel was free of trapping regions, to find no differences after application of the fluoride compound.
Conclusions Tin, and possibly fluoride, topically applied to human enamel surfaces in situ is incorporated in circumscribed trapping regions of about 125 p diameter and 20 p. deep. This conclusion was made using an electron probe microanalyzer, which scans the intact enamel cross section re vealing regions of tin enrichment. The apparent concentration of tin found in trapping regions is about 2 0 ,000 ppm. Assuming a regular distribution of trapping regions on intact enamel surfaces, this value is consistent with that derived by chemical analysis. Fluoride applied in a stannous compound ap parently reaches a level in the first few microns of enamel at about 500 ppm. This level consti tutes a 20 percent increase over normal observed enamel levels. Current preventive procedures using topical ap plication of stannous fluoride compounds, as de scribed in this study, effectively incorporate tin and fluoride into tooth enamel structure.
The opinions a n d /o r assertions contained herein are those o f th e authors and are n o t to be construed as o ffic ia l or re fle ctin g the views o f the N avy D epartm ent or the naval service a t large. T he authors th a n k M r. John M oskal o f the Phillips Electronics Instrum ents Laboratories, M o u n t Vernon, N .Y ., fo r operating the electron probe analyzer, and acknow ledgem ent is made to M r. V . J. Berzinskas, M r. A. Y . B ale kjia n , and M r. Robert Caldw ell fo r th e ir assist
ance in the d e te rm in a tio n o f enamel flu o rid e levels. This study was supported by N a vy M ed ica l Research Projects M R 0 0 5 .1 2 -5 0 0 0 .0 2 and M R 0 0 5 .1 2 -5 0 0 4 .1 4 . Doctor Hoerm an is a ca p tain in th e Dental Corps, U.S. Navy, and head o f the chem istry division o f th e dental departm ent, N aval M ed ica l Research In stitu te , N a tio n a l N aval M ed ica l Center, Bethesda, 2 0 0 1 4 . Doctor Lyon (C apt, DC, USN, Ret) was fo rm e rly d ire cto r o f the den ta l departm ent, N a va l M ed ica l Research In stitu te , Bethesda. He is presently senior research associate a t the A m erican Dental A ssociation, 211 E. Chicago Ave., Chicago, 6 0 6 1 1 . Doctor K lim a is a lie u te n a n t com m ander in the Dental Corps, U.S. Navy, and head of the preventive d e n tistry section, dental d e p artm en t, U.S. Naval T ra in in g Center, Great Lakes, III. M r. B irk's and M r. N agel's address is U.S. N a va l Research Laboratory, W ashington, D.C., 2 0 3 9 0 , Code 7 3 2 0 . Doctor Ludw ick is a ca p ta in and s ta ff d e ntal o ffic e r in the Dental Corps, U.S. Navy, U.S. M a rin e Corps Headquarters, W a s h in g ton, D.C. 1. Knutson, J. W ., and A rm stro n g , W . D. E ffe ct of to p ic a lly applied sodium flu o rid e on dental caries e x perience. Public H ealth Rep 5 8 :1 7 0 1 N ov., 1943. 2. H owell, C. L., and others. E ffe ct o f to p ic a lly applied stannous flu o rid e on dental caries experience in children. JA D A 5 0 :1 4 Jan., 1955. 3. Scola, F. P., and Ostrom , C. A . C lin ica l eva lu a tion o f the use o f stannous flu o rid e in naval personnel. JA D A 7 3 :1 3 0 6 Dec., 1966. 4. N e vitt, George A .; W itte r, David H., and Bowman, W in sto n D. T opical a p p lica tio ns o f sodium flu o rid e and stannous flu o rid e . Public H e alth Rep 7 3 :8 4 7 , 1958. 5. M u h le r, J. C.; Stookey, G. K., and Bixler, D. Effect o f d iffe re n t p h o sph a te -fluo rid e systems on dental caries in children. IADR P reprinted A b stracts, 4 9 , 1965. 6. W e llo ck, W . D.; M a itla n d , A ., and Brudevold, F. Caries increments, tooth discoloration, and state o f oral hygiene in children given single annual a p p lica tio ns of acid p h o sph a te -fluo rid e and stannous flu o rid e . A rch O ral Biol 1 0 :4 5 3 , 1965. 7. Hodge, H. C. Reactions between flu o rid e and hyd ro xyla p a tite . J D ent Res 3 9 :1 1 1 4 N o v.-D ec., 1960 A bstract. 8. Cooley, W . E. Reactions o f tin ( I I ) and flu o rid e ions w ith etched enam el. J D ent Res 4 0 :1 1 9 9 N o v.Dec., 1961. 9. Brudevold, Finn, and others. U ptake o f tin and flu o rid e by in ta c t enam el. JA D A 5 3 :1 5 9 A u g ., 1956. 10. Boyde, A .; Switsur, V . R., and Fearnhead, R. W. A p p lic a tio n o f the scanning e le ctro n -p ro b e x -ra y m ic ro a n a lyze r to dental tissues. J U ltra s tru c t Res 5:201 June, 1961. 11. Birks, L. S. Electron probe m icroanalysi§. New Y o rk, John W ile y £r Sons, 1963. 12. W h a rto n , H. W . Isolation and d e term in a tion of m icrogram am ounts o f flu o rid e in m aterials co n taining ca lcium and orthophosphate. A n a l Chem 3 4 :1 2 9 6 , 1962. 13. W e a th ere ll, J. A ., and Hargreaves, J. A . The m icro -sa m p lin g o f enamel in th in layers by means of strong acids. A rch OraJ Biol 1 0 :1 3 9 Jan.-F eb., 1965. 14. Gray, J. A . K in e tics o f enam el dissolution d u ring fo rm a tio n o f in cip ie n t c a rie s-like lesions. IADR P re p rin t ed Abstracts, 105, 1965. 15. M uh le r, J. C. Stannous flu o rid e enamel p ig m e n ta tio n — evidence o f caries arrestm ent. J D ent C h ild 27:1 57 3rd q u a rt., 1960. 16. Brudevold, F.; Gardner, D. E., and Sm ith, F. A. D istrib u tio n o f flu o rid e in hum an enam el. J Dent Res 3 5 :4 2 0 June, 1956.
H oerm an and others— T IN A N D FLUORIDE UPTAKE IN H U M A N E N A M E L ■ 1305
Tin and fluoride uptake in human enamel in situ: electron probe and chemical microanalysis
Kirk C. Hoerman, DDS, MSD, Bethesda; James E. Klima, DDS, Great Lakes, III.; L. S. Birks, MS; David J. Nagel, BS; William E. Ludwick, DDS, Washington, D. C., and Harvey W. Lyon, DDS, PhD, Chicago
Systematic application of fluoride compounds to tooth surfaces reduces the incidence of dental caries. The process by which surface-applied flu oride and an accompanying cation obtain anticariogenic action is not clearly understood.1-3 Re gardless of divergent opinions as to mode of action, choice of medicaments, and clinical effec tiveness,4'8 deriving an optimum effect must surely be a function of detail and not of kind. The mode of incorporating tin and fluoride into clinically sound enamel is not well understood. Brudevold and others9 reported that enamel treated for 20 minutes with 1.86 percent stannous fluoride obtained up to 640 ppm of tin in outer layers, while the fluoride content reached 1,200 ppm. Although it appeared that tin uniformly
penetrated sound enamel along the surface, the possibility remained that circumscribed regions of enrichment occurred that were not detectable by chemical analysis. The present study was designed to obtain fur ther information about the penetration, especially the distribution, of stannous fluoride in apparently sound tooth enamel in situ. A relatively new tech nic was used for the determination of tin in tooth structures. Electron probe microanalysis detects elemental components of metals, alloys, or certain biologic tissues with qualitative and quantitative reliability. In addition, the method reveals the specific region or volume fraction occupied by the element of interest. Such versatility is desir able and was therefore applied in the present study 1301
of the distribution and concentration of tin along an enamel surface. The applicability of electron probe microanalysis to mineralized tissues was first shown by Boyde, Switsur and Fearnhead.10 Subsequent use of the instrument for analysis in dental structures was limited. The equipment used for the present study was a Phillips (Norelco) Electron Probe Microanalyzer. The electron probe directs an electron beam on a small area of the specimen surface (about 1 ¡x. in diameter). The beam excites atoms and causes the emission of X rays that have wave frequencies characteristic of the elements residing in the specimen. Spectrographic analysis of the X-ray emission from the tissue or specimen makes possible the qualitative identification of the elements present. Also, quan tities may be calculated from the intensity of the characteristic X rays. The distribution of an ele ment over an entire region of interest, for example a tin-enriched region, may be determined by elec trostatically deflecting the electron beam in a scanning action across the specimen surface while displaying the X-ray intensity on a cathode-ray screen. Birks11 offers a more detailed description of the methods and equipment.
Materials and methods Twelve naval recruits, 17 to 24 years of age, who were reporting for primary training at the U.S. Naval Training Center, Great Lakes, 111., were selected for this study. Those chosen to participate in the study were recruits who eventually would require complete extractions and subsequent re placement by appropriate prostheses. Initially, however, each subject was required to have a lat erally opposed tooth pair free of large carious lesions or restorations or both in the remaining natural dentition. The designation of the control and experimental teeth was at random assuming bilateral symmetry of carious lesions. Three tooth pairs were selected for electron probe micro analysis for tin incorporation. These teeth were also included in the 12 tooth pairs submitted to fluoride analysis.
Treatment ■ Control quadrant: First, the entire dental arch containing the laterally opposed experimental teeth was cleaned by scaling. A chemically pure 1302 ■ JA D A , V o l. 7 3 , Dec. 1966
sodium chloride-pumice mixture (1.62 Gm. of sodium chloride, 6 Gm. of pumice, and 3 ml. of distilled water) was applied to each tooth surface for 10 seconds by use of a rubber cup rotating in a dental handpiece. Each tooth was burnished for about 10 seconds. After pumicing, the quad rant was isolated with cotton rolls and dried. The surfaces were then swabbed with 1.2 normal so dium chloride solution and, after 30 seconds, den tal floss was used in interproximal spaces. The patient was instructed not to rinse his mouth or swallow for 30 minutes; however, he was advised to expectorate as needed. After rinsing, a local anesthetic was administered. The control tooth was subsequently extracted, freed of vascular and fibrous debris, rinsed briefly in distilled water, blotted, and placed in a test tube and was frozen immediately in dry ice until prepared for electron probe or chemical microanalysis. ■ Experimental quadrant: Four days after the extraction of the control tooth, the subject re turned and the teeth in the experimental quadrant were burnished with a 9.09 percent stannous fluoride-pumice mixture (0.9 Gm. of SnF2, 6 Gm. of pumice, and 3 ml. of distilled water). This treatment was followed by the topical application of 1.28 normal SnF2 solution (10 percent) to all enamel surfaces including the experimental tooth. After 30 minutes, the experimental tooth was ex tracted and prepared for analysis.
Experimental probe microanalysis
Six teeth (3 paired specimens) were cross-sec tioned perpendicular to the long axis through the thickest enamel portion of the crown. These teeth were mounted, 3 teeth at a time, to one side in a 1-inch diameter Bakelite piece. Standards of pure tin were similarly mounted. Both teeth and stand ards were separately abraded with successively finer papers, through no. 4-0, and then polished with diamond paste on paper and Linde “B” powder on wet felt. The Bakelite mounts were next halved, and two final sample mounts were reconstructed for microprobe analysis. Each sam ple had 3 teeth on one side and the tin standard on the other. This separate handling of samples and standard precluded the possibility of smear ing tin on the teeth during polishing. The probe operator was not told which teeth were treated and which were control teeth.
Fig. 1 ■ C a th o d e -ra y screen presenta tio n ( le ft) and dia g ra m (rig h t) o f X -ra y e m issivity o f tin in enriched region of enamel tre a te d w ith stannous flu o rid e . X -ra y emission fo llo w s electron b o m bard m ent o f enam el using P hillip s Electron Probe M icro a n a lyze r
Fluoride determinations Fluoride determinations were made by use of the microdiffusion methods described by W harton.12 Modifications in technic were made according to Weatherell and Hargreaves13 so that in 7 tooth pairs fluoride determinations could be made in the first 25 /j, layer of enamel. Specimens were coded before chemical analysis so that control and treat ed teeth were not identifiable.
intensity (ordinate) was recorded against the dis tance traversed by the exciting electron beam (abscissa). The data confirm the presence of tin and also indicate the depth of penetration and the concentration of tin through the enrichment band. The effective depth of penetration was about 20 ¡j, in the experiment shown in Figure 2. y
SPEC MEN 711
5 0 0 'M
R esults and discussion ■ Tin: Electron probe microanalysis revealed tin enrichment in each of three pairs of the experi mental teeth treated with stannous fluoride. Fig ure 1 is a photograph of the oscilloscope screen that displays an X-ray intensity map from tin accumulation in treated enamel. A diagrammatic presentation of the photograph is also shown in Figure 1. On the photograph, there are large white blobs of X-ray emission. These white blobs were caused by Bakelite-embedding artifacts. The X-ray emissivity caused by tin enrichment is seen as tiny white pips along a diagonal line consistent with the enamel surface. The penetra tion of tin in treated teeth appeared confined to depths from 5 to 20 p. and extended, in most instances, along the surface from 100 to 200 ¡x. These tin-enriched regions occurred in enamel so as to suggest a localized element predilection. Figure 2 shows a line scan across the depth of one of the typical tin-enriched regions. The elec tron probe was set so that only tin X-ray emission
o
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L .
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•a
s
t
' r> ..... ô , K «s. cn 2T O o o
Q
5
»,
§
........ o.n , .. : .."8.....
* Ï;
1 *
É flM É K S N '
:
Fig. 2 ■ Recorder tra cin g o f X - r a y emission in te n sity from tin versus distance in m icrons traversed by h ig h in te n sity electron beam across section o f enam el con ta in in g tin -e n ric h e d region subsequent to tre a tm e n t w ith SnFj. (Same enamel section as shown in Figure 1 )
Hoerm an and others— T IN A N D FLUORIDE UPTAKE IN H U M A N E N A M E L ■ 1303
The content of tin in the enriched region was calculated from X-ray emission assuming tooth enamel to be hydroxylapatite and allowing for X-ray absorption. A t the highest concentration, tin was at a level of 37,000 ppm. If a further cor rection were made, assuming that rounding of the edges occurred during polishing, the tin concen tration would not be less than 20,000 ppm in the enriched regions. The limit of detectability of the electron probe microanalysis in this experiment was calculated from Figure 2. Counting times for each region were 300 seconds. Thus, background counts in this period amounted to 3,000. Three standard deviations of this number equaled 165 counts. From this value, the limit of detection of tin emission intensities near those shown in Figure 2 was calculated to be about 100 ppm. Since it has been shown by chemical methods that tin up take was about 500 ppm along the entire intact enamel surface,9 a fact not confirmed by us by use of electron probe microanalysis, it appears that this result could have been due, in fact, to incorporation of tin in entrapped regions. U su ally, an electron probe scan of the periphery of a treated specimen revealed from 3 to 5 tin-enriched regions. One may calculate an approximate enamel tin content o f about 220 ppm from our data, and this value is reasonable when compared with that obtained chemically by Brudevold and others.9 The fact that tin uptake by enamel occurs in circumscribed trapping areas is grounds for specu lation that these regions are the same as the “white spots” reported by Gray14 or the prime tin uptake sites postulated by Muhler.15 An interesting experiment would be to deter mine the calcium:phosphorus ratio in tin-enriched regions. This ratio might indicate whether the tin replaces either calcium or phosphorus or whether it simply diffuses into the regions. The distribu tion of tin throughout the tin-enriched zones ap peared from the oscilloscope tracings to be uni form; however, uniformity was not verified since only one scan was made across the width. Repet itive quantifying scans would verify the concen tration distribution in one enriched region.
Fluoride levels The table shows the results o f fluoride determina tions in control and treated experimental teeth after in situ application of stannous fluoride to the enamel surfaces. The values shown have been 1304 ■ J A D A , V o l. 7 3 , Dec. 1966
Table ■ The presence o f tin and flu o rid e in hum an enam el a fte r to p ica l a p p lica tio n in situ o f 10 percent stannous flu o rid e solution Enamel Paired specim en No. C ontrol
Tooth
Treated
Sample w e ig h t (m g.)
Fluoride (ppm )
T in *
t
C oronal powdered enam el 701 711
Lower la te ra l incisor
10.55 10.30
105.5 136.0
710
U pper second p rem olar
10.29 10.59
41.0 32.8
725
Upper second m olar
10.04 10.25
27.1 32.8
728
U pper la te ra l incisor
10.94 10.53
203.4 182.2
+
Upper canine
10.87 10.11
70.7 100.1
+
U pper c e ntra l incisor
15.90 59.50
3 4 8 .2 f 503.4
Upper canine
26.80 21.65
784.8 1065.2
727
Upper central incisor
17.20 7.25
393.9 403.8
703
Upper la te ra l incisor
11.15 9.55
198.5 234.9
716
Lower central incisor
10.00 9.50
372.0 324.9
704
Upper central incisor
10.05 16.95
472.3 576.3
705
Upper central incisor
7.35 9 .40
176.4 437.1
724 726 715 717 •
734
P erchloric a cid -e tch e d enamel 745 708 747 743 750 732 702 735 744
*D e te rm in e d by e le ctro n probe m icroanalysis tV a lu e s corrected to a basis o f 10 m g. o f surface enam el, e q u iv a le n t to 25 fi depth
corrected so that they express fluoride concentra tion in a 10-mg. specimen of enamel. These re sults have been divided into two major groups: 5 pairs of teeth in which fluoride levels were de termined by using an aliquot of the total coronal powdered enamel, and 7 pairs in which fluoride levels were determined on the first 25 ¡j. of per chloric acid-dissolved enamel surfaces. In the powdered-enamel fluoride determina tions, it was not possible to detect significant ele vations caused by the stannous fluoride treatment. These findings were consistent with those of others16 in that fluoride content of enamel is in creased only in the first few microns beneath the surface. Use o f total powdered enamel would mask real differences between control and treated teeth by the diluent effect of normal deeper en amel. The reliability and reproducibility o f the technic, however, were confirmed, and it was shown that fluoride content tended to follow the principle of bilateral symmetry. Measurement of total enamel fluoride levels was usually in agree ment with measurements reported by others.16 In
posterior teeth, the fluoride content of enamel was lower by about half. Surface fluoride determinations revealed signifi cant increases of fluoride in 6 of the 7 treated teeth. Values up to 1,065 ppm were found in the first 25 ¡J. of treated enamel. Fluoride levels at the same depth in controls were usually compa rable with those reported by others using similar quantitative procedures.13 In 2 treated lower cen tral incisors, the fluoride levels were the same as in their untreated laterally opposed controls. This finding could be explained if the surface7applied fluoride was distributed along the enamel surface in a similar fashion as tin is distributed, that is, principally in trapping regions instead of uniform ly dispersed along the first few micron depths. Accordingly, it would be possible, where enamel was free of trapping regions, to find no differences after application of the fluoride compound.
Conclusions Tin, and possibly fluoride, topically applied to human enamel surfaces in situ is incorporated in circumscribed trapping regions of about 125 p diameter and 20 p. deep. This conclusion was made using an electron probe microanalyzer, which scans the intact enamel cross section re vealing regions of tin enrichment. The apparent concentration of tin found in trapping regions is about 2 0 ,000 ppm. Assuming a regular distribution of trapping regions on intact enamel surfaces, this value is consistent with that derived by chemical analysis. Fluoride applied in a stannous compound ap parently reaches a level in the first few microns of enamel at about 500 ppm. This level consti tutes a 20 percent increase over normal observed enamel levels. Current preventive procedures using topical ap plication of stannous fluoride compounds, as de scribed in this study, effectively incorporate tin and fluoride into tooth enamel structure.
The opinions a n d /o r assertions contained herein are those o f th e authors and are n o t to be construed as o ffic ia l or re fle ctin g the views o f the N avy D epartm ent or the naval service a t large. T he authors th a n k M r. John M oskal o f the Phillips Electronics Instrum ents Laboratories, M o u n t Vernon, N .Y ., fo r operating the electron probe analyzer, and acknow ledgem ent is made to M r. V . J. Berzinskas, M r. A. Y . B ale kjia n , and M r. Robert Caldw ell fo r th e ir assist
ance in the d e te rm in a tio n o f enamel flu o rid e levels. This study was supported by N a vy M ed ica l Research Projects M R 0 0 5 .1 2 -5 0 0 0 .0 2 and M R 0 0 5 .1 2 -5 0 0 4 .1 4 . Doctor Hoerm an is a ca p tain in th e Dental Corps, U.S. Navy, and head o f the chem istry division o f th e dental departm ent, N aval M ed ica l Research In stitu te , N a tio n a l N aval M ed ica l Center, Bethesda, 2 0 0 1 4 . Doctor Lyon (C apt, DC, USN, Ret) was fo rm e rly d ire cto r o f the den ta l departm ent, N a va l M ed ica l Research In stitu te , Bethesda. He is presently senior research associate a t the A m erican Dental A ssociation, 211 E. Chicago Ave., Chicago, 6 0 6 1 1 . Doctor K lim a is a lie u te n a n t com m ander in the Dental Corps, U.S. Navy, and head of the preventive d e n tistry section, dental d e p artm en t, U.S. Naval T ra in in g Center, Great Lakes, III. M r. B irk's and M r. N agel's address is U.S. N a va l Research Laboratory, W ashington, D.C., 2 0 3 9 0 , Code 7 3 2 0 . Doctor Ludw ick is a ca p ta in and s ta ff d e ntal o ffic e r in the Dental Corps, U.S. Navy, U.S. M a rin e Corps Headquarters, W a s h in g ton, D.C. 1. Knutson, J. W ., and A rm stro n g , W . D. E ffe ct of to p ic a lly applied sodium flu o rid e on dental caries e x perience. Public H ealth Rep 5 8 :1 7 0 1 N ov., 1943. 2. H owell, C. L., and others. E ffe ct o f to p ic a lly applied stannous flu o rid e on dental caries experience in children. JA D A 5 0 :1 4 Jan., 1955. 3. Scola, F. P., and Ostrom , C. A . C lin ica l eva lu a tion o f the use o f stannous flu o rid e in naval personnel. JA D A 7 3 :1 3 0 6 Dec., 1966. 4. N e vitt, George A .; W itte r, David H., and Bowman, W in sto n D. T opical a p p lica tio ns o f sodium flu o rid e and stannous flu o rid e . Public H e alth Rep 7 3 :8 4 7 , 1958. 5. M u h le r, J. C.; Stookey, G. K., and Bixler, D. Effect o f d iffe re n t p h o sph a te -fluo rid e systems on dental caries in children. IADR P reprinted A b stracts, 4 9 , 1965. 6. W e llo ck, W . D.; M a itla n d , A ., and Brudevold, F. Caries increments, tooth discoloration, and state o f oral hygiene in children given single annual a p p lica tio ns of acid p h o sph a te -fluo rid e and stannous flu o rid e . A rch O ral Biol 1 0 :4 5 3 , 1965. 7. Hodge, H. C. Reactions between flu o rid e and hyd ro xyla p a tite . J D ent Res 3 9 :1 1 1 4 N o v.-D ec., 1960 A bstract. 8. Cooley, W . E. Reactions o f tin ( I I ) and flu o rid e ions w ith etched enam el. J D ent Res 4 0 :1 1 9 9 N o v.Dec., 1961. 9. Brudevold, Finn, and others. U ptake o f tin and flu o rid e by in ta c t enam el. JA D A 5 3 :1 5 9 A u g ., 1956. 10. Boyde, A .; Switsur, V . R., and Fearnhead, R. W. A p p lic a tio n o f the scanning e le ctro n -p ro b e x -ra y m ic ro a n a lyze r to dental tissues. J U ltra s tru c t Res 5:201 June, 1961. 11. Birks, L. S. Electron probe m icroanalysi§. New Y o rk, John W ile y £r Sons, 1963. 12. W h a rto n , H. W . Isolation and d e term in a tion of m icrogram am ounts o f flu o rid e in m aterials co n taining ca lcium and orthophosphate. A n a l Chem 3 4 :1 2 9 6 , 1962. 13. W e a th ere ll, J. A ., and Hargreaves, J. A . The m icro -sa m p lin g o f enamel in th in layers by means of strong acids. A rch OraJ Biol 1 0 :1 3 9 Jan.-F eb., 1965. 14. Gray, J. A . K in e tics o f enam el dissolution d u ring fo rm a tio n o f in cip ie n t c a rie s-like lesions. IADR P re p rin t ed Abstracts, 105, 1965. 15. M uh le r, J. C. Stannous flu o rid e enamel p ig m e n ta tio n — evidence o f caries arrestm ent. J D ent C h ild 27:1 57 3rd q u a rt., 1960. 16. Brudevold, F.; Gardner, D. E., and Sm ith, F. A. D istrib u tio n o f flu o rid e in hum an enam el. J Dent Res 3 5 :4 2 0 June, 1956.
H oerm an and others— T IN A N D FLUORIDE UPTAKE IN H U M A N E N A M E L ■ 1305