Four analytical methods show differences and/or similarities in the atom ic and the crystalline nature of acid-resistant and acid-susceptible dental enamel specimens. Both groups of samples contained the same elements as detected by ion probe analysis. However, there was a convincing quantitative atom ic difference between groups of most but not all elements. The structure type of the crystalline material was similar in both types of specimens, but crystallite size differed between the two groups.
C o m p o s i t i o n e l e m e n t a l s t r u c t u r e t o
its
a n d
s t r u c t u r e
c o m p o s i t i o n o f
d e n t a l
a n d
e n a m e l
o f
d e n t a l
e n a m e l :
c r y s t a l l i n e a s
t h e y
r e la t e
s o lu b ilit y
F. C. Besic, DDS M. Bayard M. R. W iem ann, Jr., SB K. H. Burrell, DDS, MS, Chicago
Several different analytical m ethods w ere used in this study to obtain data related to the solu bility of intact dental enamel surfaces. D ental caries is a multifactorial process. One com ponent in the disintegration of enamel by caries is its resistance or susceptibility to dis solution. This study is concerned with the atom ic species and th eir concentrations and som e crys talline characteristics in enamel specim ens clas sified as acid resistant or acid susceptible. Since caries is a com plex process (including salivary and m icrobial factors, diet, tooth resistance, oral hygiene, and so forth), it is inappropriate to determ ine enam el solubility on the basis of caries scores or lesions present in specim ens. T he dis solution o f enamel is not synonym ous with the carious process. Investigators have used several m ethods in th e in vitro dissolution of enamel to sim ulate w hite spot décalcifications produced by dental caries: the proteolytic and proteolysis chelating m ethods described by L u ra ,1 and the m ore widely accepted acid dissolution m ethod. T he latter has w ithstood the test of time, and our own stu d ies2'4 have supported the acid dissolution hypothesis in caries. 594 ■ JADA, Vol. 91, September 1975
M anson-H ing and co-w orkers5 have pro duced carieslike décalcifications in highly vis cous acid solutions. Johnson, Poole, and T y ler6 state “ the pattern of tissue destruction seen in enam el caries could be explained on the basis of acid dissolution alone, or by a m ixture of acid dissolution and calcium com plexation.” We have observed that with proper acid-mineral m ixtures, enamel décalcifications in vitro pro duce a recrystallized zone in the affected area that is similar in refractive index to the white spot enamel décalcification produced by caries.3'4 Such recrystallization has not been dem onstrated by chelation m ethods o f dem ineralization nor has the characteristic zone of positive birefring ence7 in carious enamel (as show n in polarized light m icroscopy studies2 4) been observed in the chelation process. Until recently, precise m ethods for quantitat ing more than a few trace elem ents in enamel have been difficult and perhaps unsatisfactory, if not impossible. T he recent developm ent and use of the ion m icroprobe* has perm itted us to m ake qualitative and quantitative determ ina tions of 21 elem ents in enamel specim ens clas sified as acid resistant or acid susceptible in vitro. T he nature of the enamel matrix does not per mit a reliable analysis for nitrogen and sulfur. Electronegative elem ents, except fluorine, can not be quantitated precisely in parts per million, but the abundance of each such atom is reliable on a relative basis (from one specim en to an other). Fluorine, though electronegative, can be quantitated precisely and, for som e presently
unexplained reason, produces a p ositive signal w hen analyzed.
Method R eliable standards for the exact quantitation o f som e elem ents present in the com plex enam el matrix are not available; how ever, the standards used in this study w ere C a F 2 , fluorapatite, and “ d op e” glasses contam inated with trace el em ents (from the N ational Bureau o f Standards). For the major elem ents analyzed (calcium , phos phorus, oxygen, hydrogen, and carbon), spec im en R -l w as used as a standard for the relative abundance o f each elem ent in the other sam ples. T h e ion probe is related to its precursor, the electron microprobe. T he ion m icroprobe has sensitivities o f 10 parts per billion (ppb) for el em ents such as sodium , Ca, and so forth (50 to 500 ppb for F , O , and H) and for m any elem ents is 1,000 tim es more sen sitive than the electron m icroprobe. It can analyze all elem ents and their isotopes (all excep t N and S in enam el) in any selected area, 1/xm2 to 2ju.m2. Perm anent teeth w ere selected on the basis o f being free o f any filling material and with suffi cient sound enamel suitable for acid susceptibility pretesting, ion probing, electron and X-ray dif fraction, and infrared absorption spectrum de term inations. T he sam e tooth specim ens w ere used for all the analytical m ethods in this study. Specim ens w ere not separated as to patient age, sex , or tooth exposure to the oral environm ent. Our purpose was simply to determ ine w hat el em ents (and their concentrations) are present in acid-resistant and acid-susceptible enam el. The eight teeth reported on here w ere selected from a group o f more than 1,800 specim ens. They w ere selected on the basis o f being either the m ost acid resistant or the m ost acid susceptible within that group. A ll 1,800 specim ens, after extraction, w ere w iped clean o f blood and debris with a gauze sponge and distilled water and then placed in a dry, clean, stoppered receptacle. T he crow n o f each specim en was divided in tw o by m eans o f a m otor-driven diamond disk. O ne part o f each specim en was used for the acid susceptibility test and the other half was retained for analyses. F or enam el surface acid testing, each speci m en w as covered with D ental Sticky W a x t ex cept a w indow area about 3 m m 2. T w enty such specim ens were tested at a time by being placed
in the sam e container with an acid solution pre viously prepared by adding e x ce ss tricalcium phosphate to 0.2 N hydrochloric acid. A fter agi tation and filtration, the filtrate w as diluted with an equal volum e o f distilled deionized water. T he final pH w as approxim ately 3.5. Such solutions contained about 1.0 mg o f C a and 1.0 mg o f P per milliliter o f solution. C oolidge and co-w orkers2 described in more detail the production of caries like décalcifications. After eight hours o f exposure to the acid solu tion, the specim ens w ere rem oved, blotted dry with absorbent paper, and exam ined grossly by reflected nonpolarized w hite light, using a stereobinocular m icroscope provided with tungsten top lighting for the presence or absence o f w hite spot “ lesio n s.” Eight total specim ens w ere sel ected as being either acid susceptible or acid res istant, respectively; o f th ose eight, four show ed a w hite spot on the entire w indow area and four were com pletely free o f any w hite spots. B y this method each selected specim en w as, under th ese conditions, either extrem ely acid susceptible or extrem ely acid resistant. T he retained half o f each o f these eight teeth was used for analytical testing (ion probing and electron and X -ray dif fraction determinations) conducted at M cC rone Laboratories by one o f us (M.B.). T w o separate types o f probing analyses w ere performed on each o f the eight teeth: an analy sis o f uncrushed tooth sam ples and an analysis o f crushed portions. In the first analysis, the sam ples w ere thoroughly cleaned, m ounted in epoxy $ with part o f the enam el extending above the ep oxy, and then carbon coated for conduc tivity purposes. During probe analysis, a small w indow was etched through the carbon on the enam el surface. It is extrem ely difficult to make a precise surface analysis since a finite am ount o f time is required to obtain a steady-state em is sion from the probe and therefore a finite am ount o f material is sputtered aw ay. This m eans that the “ surface” analyzed is, o f n ecessity, at least 100 Angstrom units below the true starting sur face. For probing deeper layers, below the surface, the specim ens w ere polished flat at right angles to the surface, and analyses w ere made at 100/xm, 200/u.m, and 300/u.m from the surface. T he second type o f analysis w as done on pow dered tooth material from each specim en. Por tions o f chipped enam el from the gross sam ples w ere crushed in an agate mortar to a particle size o f approximately 5/xm. In the crushing o f the enam el sample into a fine pow der, w e hoped to Besic—others: COMPOSITION AND STRUCTURE OF ENAMEL ■ 595
-4«
fractu re at least part o f the specim en along the lines o f least resistance betw een the crystals. T h is w as done in an effort to determ ine w hether the elem ents w ere uniform ly distributed through out the cry stallites or w hether som e elem ents w ould be co ncentrated either on the su rface or internally. S in ce the particles are insulators, they act as an odd form o f electrostatic lens in com bination with the co llecto r electrod e. T h erefo re, the crushing m ethod allow s an enhancem ent o f the peak-to-backgroun d ratio by as much as a fa cto r o f 100. A ls o , with the particles spread out on a co n d u ctiv e tantalum substrate, it is possible to bleed o ff the electrostatic ch arge more e ffe c tiv ely than with the w hole uncrushed specim en. T h is perm its the use o f a p o sitive prim ary beam w hich again en han ces the sensitivity.
Both p o sitive and negative beam s w e re used on the bulk sam ples. A lth ough the n egative beam g av e the m ost consistent results, the p ositive beam sh ow ed greater sensitivity. T h e beam sp ecies used w ere O 2 for the positive and O ” fo r the negative. E lectro n diffraction and X -ra y diffraction data w ere obtained from the sam e specim ens used for probing. E lectron diffraction w as done by co n ventional electron transm ission m icro scop e tech n iqu es, and p o w d er X -ray diffraction data w ere obtained b y the use o f a low angle scatter ing cam era. Inform ation on the infrared absorption sp e c trum w as obtained from tw o o f the acid-resistant and tw o o f the acid-susceptible sam ples. W ith a tungsten carbide scriber, small particles o f en-
-4 -4
T ab le 1 ■ Relative abundance of major elements.
Acid Resistant Specimens
Element
Ca
P
0
H
C
Relative abundance Spc.No. (surface) R -1 R -2 R -3 R -4
1.0 0 1.05 1.13 .9 8
ACID R E SIS TA N T SPECIM ENS ppm
Acid Susceptible Specimens
Spc.No. S -1 S -2 S"3 S -4
Relative abundance (surface)
Element
0.71
0.68 0.65 0.71
V
Spc. No.
7
3
111
28
15
average
7
3
2.6
R- 1 R -2 R -3 R -4
800 620 900 680
1 2 ,0 0 0 * * * 540 600 1 ,2 4 0
S -1 S -2 S -3 S -4
200 180 70 190
130 160 65 270
190 29 0 130
750
average
160
156
202
600
1,200
650 170 2 10 730
600 630 290 460
1,032
440
495
70
2,100
45 1 ,8 0 0 2 ,4 0 0 3 ,9 0 0
2 ,5 0 0 3 ,7 0 0
1,870
2 ,0 3 6
2,080 14 65 29 35
0.69
S -2 S -3 S -4
average
1.04
average
0 .87
average R -1 R -2 R -3 R -4
R- 1 R -2 R -3 R -4
1.00 0 .9 3 0 .9 5 1.03
S-1 S -2 S -3 S -4
1.2 1.1 1.2 1.1
average
0 .9 8
average
1.15
average
R- 1 R -2 R -3 R -4
1.00 1.05 0 .9 1 0 .9 7
S-1 S -2 S -3 S -4
1.3 1.4 1.3 1.5
R -1 R -2 R -3 R -4
average
0 .9 8
average
1.4
average
R- 1 R -2 R -3 R -4
1.00 0 .9 5 0 .8 9 1.0
S-1 S -2 S -3 S -4
1.4 1.3 1.3 1.4
average
0 .9 6
average
1.35
1,400 3 ,5 0 0
600 390 800 2 ,1 5 0
7 9 3 *** *
985
1 5 - -
-
200
1,600 1,640 2 ,6 0 0 1,900
1 ,8 0 0 1 ,600 700 1 ,6 0 0
S-1 S -2 S -3 S -4
4 ,3 0 0 **
1,935
1,425
average
3 ,0 0 0 6 ,3 0 0 2 ,7 0 0 3 ,6 0 0
3 ,1 0 0 3 ,7 0 0 3 ,0 0 0 3 ,9 0 0
3 ,3 0 0 3 ,6 5 0 3 ,4 0 0
S -1 S -2 S -3 S -4
3,900
3,4 25
3,1 12
average
R -1 R -2 R -3 R -4
120 1 10
15 17 5
S -1 s -2 S -3 S -4
60 42
90 130
70 30 35 60
70
75 110 28 130
average
112
49
9
average
48
86
36 I
R- 1 R -2 R -3 R -4
600 1 ,900 900 900
800 1,200 600
1,200
800 780 800 780
S -1 S -2 S -3 S -4
500 650 240 580
300 630 3 80 2 50
580 I 490 1 310 I 500
average
1,075
9 50
790
average
492
3 90
470 1
R- 1 R -2 R -3 R -4
900 15 550 640
10 5 5 5
5 12 5 7
S -1 S -2 S -3 S -4
93 80 45 130
3 5 31 5
5 I 24 1 45 ® 7
average
526
6
7
average
86
11
20
8,000 3 1 ,0 0 0 *
‘ Potassium rich inclusion
596 ■ JADA, Vol. 91, Septem ber 1975
0.5I 7
average
0.91 0.85 0.87 0.85
Zn
( 300/am i depth)
5 3 1 18
s- 1
Fe
(100/j.m depth)
S-1 S -2 S -3 S -4
average
Specimen R -l given base value of 1.00 surface determinations only. H and 0 lost in dehydration of the specimen would not be represented. N and S in the enamel matrix are indeterminable with the ion probe presently.
(surface)
16 5 3 35
1.00 1.05 1.07 1.03
Cu
Spc. No.
10 80 3 20
1.04
F
(300/¿m depth)
90 1 10 75 170
R- 1 R -2 R -3 R -4
K
(100/Am depth)
R- 1 R -2 R -3 R -4
average
Al
(surface)
ACID S U S C EP T IB LE SPECIMENS ppm
2,100
* R- 4 not included
1
2,100 230
2,200 3 ,1 0 0
20
***A lum inum rich inclusion
20 2,100
* R- 1 not included
i
j
j
hydration in the process o f specim en prepara tion for probing obviously are not indicated. T w o elem ents, N and S , which are a part o f enam el protein, could not be determ ined in the enam el by the ion probe. Table 2 gives data for 13 trace elem ents in each sam ple at three sites: the enam el surface and 100/Mm and 300jU,m beneath the surface. In m ost instances, the elem ents show n are m ore co n cen trated at the enam el surface than internally, with a trend toward decreasing abundance from lOOpim to 300^im beneath the surface. T he table show s potassium - and aluminum-rich inclusions that were not included in the average counts. Similar chlorine-rich inclusions were encountered in another study8 using the electron m icroprobe for elem ental analyses.
amel w ere scraped from the surfaces o f the sp ec im ens and the infrared absorption spectrum ob tained.
Results T h e tables show data obtained from four acidresistant and four acid-susceptible tooth enamel specim ens. Table 1 show s the relative abundance o f five elem ents. D ata obtained from specim en R -1 w ere used as a base for com parative values for each o f the five elem ents. Ca and P w ere 35% and 17% greater resp ectively in acid-resistant specim ens, w hereas O, H , and C w ere 6%, 42%, and 39% greater respectively in the acid-sus ceptible sam ples. D ata for H and O lost by de
AC ID S U S C E P T IB L E SP EC IM EN S
AC ID R E S IS T A N T S P E C IM E N S
ppm
ppm
E lem ent
Sr
Mo
Pb
S p c. No.
(su rfa ce )
(surface)
(300yu.m depth)
38 35 85 40
29 25 60 19
S-1 S -2 S -3 S- 4
15 15 90 10
18 21 1 10 22
32 70 25 10
average
39
50
33
average
32
43
34
R -1 R -2 R -3 R -4
35 51 65 38
10 48 40 26
2
S-1
39 22 5
s -2
10 15 5 10
5 12
S-3 S -4
60 75 — 35
average
47
31
17
average
42
10
R -1 R -2 R -3 R -4
R -1 R -2 R -3 R -4
2 — 4 1.5
0.5 — 0.3 3 0 .9 5
1.9 0.4 1.2 1
1.2
_
1
S-1 S -2 S -3 S -4
0.2
0.5
0.1
0 .4
average
0.3 —
— —
1 7 — —
average S -1 S -2 S -3 S -4
0 .6
5 — — —
0.5 2 — —
2 _ —
9 6 .5 2 1 — —
0 .6
—
3
2
-
-
0.3
0.1
0.1
38 26 28 19
S -1 S -2 S -3 S -4
25 35 40 25
21 28 31 31
20 31 45 35
average
30
28
28
average
3 1
28
33
3
3 2 1 2
S -1 S -2 S -3 S -4
15 18 65 38
22 27 15 10
5 32 27 21
2 .2
2
average
34
18
21
average
3
5 1 —
b e n e a th s u r
0.1
— -
32 37 27 17
3 2 5 1
D a ta f o r 1 3 t r a c e e le m e n ts a t e n a m e l
fa c e .
28 35 35 2 1
-1 R -2 R- 3 R -4
■
0 .8
0.5
1.2
Table 2
s u r f a c e a n d a t 1 0 0 /^ .m a n d 3 0 0 / j. m
R -1 R -2 R -3 R -4
r
B
Spc. No.
(100/j.m depth)
30 26 80 21
average
Ti
(3 0 0 /i.m d ep th)
R -1 R- 2 R -3 R -4
average
!_i
(100/xm dep th)
Besic— others: COMPOSITION AND STRUCTURE OF ENAMEL ■ 597
Table 3 a Data for sodium, magnesium, and
Table 4
chlorine at enam el surfaces.
o f 1 6 tr a c e e le m e n ts p r e s e n t a t e n a m e l s u r fa c e
Acid Resistant Group Element
Na
Spc.No.
ppm (surface)
Spc.No.
ppm (surface)
R-1
4 100
S-1
2150
R -2 R -3 R -4
3600 3100
S-2 S -3 S -4
2900 2600
average
Mg
Cl*
Acid Susceptible Group
3 500 3575
average
( lis te d
■ A v e r a g e c o u n t s ( in p a r t s p e r m i l l i o n )
in o r d e r o f a b u n d a n c e in
each g ro u p of
s p e c im e n s ).
Acid Resistant Group
2500 2538
Acid Susceptible Group
K
ppm 4300
ppm Na 2 538
F
3900
F
1870
Na 3 5 7 5
K
1032
Fe
492
Fe
1075
R- 1
280
S-1
2 10
Al
750
Mg
260
R -2
3 10
S -2
260
Zn
526
Al
160
R -3 R -4
S -3 S -4
320
Cl
1 38
Cl
1 32
Zn
86
average
350 2 20 260
Mg
average
3 70 3 20 3 20
Cu
1 12
Cu
48
R- 1
1 30
s- 1
140
V
111
Mo
42
47
B
34
R -2
170
S -2
110
Mo
R -3
120
S -3
160
Sr
39
Sr
32
R -4
1 10 1 32
S -4
140
Ti
30
Ti
31
average
138
B
3
V
7
Pb
1.9
Pb
2
Li
0.6
Li
2
average
*CI quantity in ppm is not precise relotive to the counts of other elements. Ci abundance is precise in relative amounts in the two groups of specimens.
Table 3 show s data for sodium , m agnesium, and Cl at enam el surfaces only. Cl counts in parts per m illion are not precise since electro negative elem ents present this problem in ion m icroprobing. N ev erth eless, the relative values o f Cl betw een specim ens are precise. D ata for Cl by electron microprobe m ethods8 indicate Cl counts should approxim ate those o f N a. T able 4 lists the average counts (parts per mil lion) o f each o f the 16 trace elem ents in each group. In the acid-resistant sam ples K, F , and N a show ed the highest counts. In decreasing order w ere iron, aluminum, zinc, m agnesium, chlorine, copper, and so forth. In the acid-sus ceptible group, N a w as greater in abundance, F was second , and follow ed by K , F e, Mg, and so on. T he concentrations o f the last six elem ents w ere significantly low er in both groups o f sam ples. T able 5 sh ow s the intergroup ratios for each elem ent. T he count for vanadium w as sevenfold greater in the acid-resistant sam ples, follow ed in decreasing order by Z n, A l, Cu, F e, K, F , N a, strontium , and Mg. T h e next five elem ents had approxim ately the sam e concentrations in both groups. T he last elem ent, boron, had a tenfold greater value in the acid-susceptible sam ples. A sum m ation o f all trace elem ents found in each specim en at the surface produced a count o f 14,172 ppm per acid-resistant sam ple and 6,773 ppm per acid-susceptible sample. M issing 598 ■ JADA, Vol. 91, Septem ber 1975
in the counts o f both groups are N and S and H and O lost by dehydration o f the specim ens. In Table 6 electron diffraction data indicate that the structure types o f the crystallites in the acid-resistant and acid-susceptible sam ples do not differ and that the substance is the sam e in both groups. H ow ever, the X-ray diffraction data show a difference in crystallite size in the tw o groups. T he crystallites in the acid-resistant sam ples were larger by an order o f more than tw o. D eterm inations by R. Z. Muggli o f M cC rone A sso cia tes, C hicago, o f the infrared absorption spectrum o f the particles from acid-resistant and acid-susceptible sam ples o f enam el revealed spectra o f apatite only. N o bands correspondTable 5
■
R a tio s
of
c o n c e n tr a t io n s
of
e a c h e l e m e n t in b o t h g r o u p s o f s p e c i m e n s (a v e ra g e by
in
a v e ra g e
a c id - r e s is ta n t e n a m e l d iv id e d in
a c id - s u s c e p t ib le
Enamel Surface Determinations 7.4
Sr 1.2
Zn 6.1
Mg 1.2
Al 4.7
Mo 1. 1
Cu 2.3
Ti 0 .9 6
V
Fe 2.2
Pb 0.95
F
2.1
Cl 0.95
K
2.1
Li 0.3
Na 1.4
B
0 .0 9
e n a m e l) .
Table 6
■ D iffr a c tio n
l i t e s iz e , b a s e d o n
d a ta g iv in g
lin e b r o a d e n in g
r e la t iv e
c r y s t a l
a t tr ib u ta b le t o
p a r t i c l e s iz e .
S h o rt term order of
Long term order in
spacing w ith in c ry s ta ls
s m all c ry s ta llite s ; determ ined by x -ra y d iffra c tio n , 2 0 0 to 1 ,0 0 0 Â.
(la ttic e ) ; determ ined by electron d iffra c tio n , several ACID RESISTANT R -1
1 .0 0
1 .0 0
R -2
0 .8
1.2
R -3
0 .9
1.3
R -4
1 .0
1.1
ACID S U S C E P T IB LE S -1
0 .9
0 .4
S- 2
0 .9
0 .6
S -3
0 .8
0 .5
S -4
0 .9
0 .4
Specimen R-1 given base value of 1.00 for relative determinations
ing to other materials w ere apparent. In a previous report4 it was show n that enamel solubility increases progressively from the en amel surface (highest refractive index and den sity) toward the dentinoenam el junction (low est refractive index and density).
Discussion T ables 1 , 2 , and 3 show that the sam e elem ents were found in both acid-resistant and acid-susceptible groups; only the concentrations o f these elem ents varied. O f the 16 trace elem ents de tected, only 1 elem ent, B, was more con cen trated in acid-susceptible specim ens. O f the other 15 trace elem ents, 4 show ed no differenc es in concentrations betw een the two groups o f sam ples. T he remaining 11 elem ents all had higher concentrations in the acid-resistant en amel. Included in this group was F. T he elem ents that show ed the greatest ratio differences in concentrations in the tw o groups were V > Z n > A l ; how ever, their concentrations in parts per million w ere substantially low er than those for K, F , N a , and Fe. Since apatite crystallites w ere found to be the sam e in both enam el typ es, but crystallite size varied, it appears as though trace elem ent abun dance (either singly or in mutual multiple a sso ciations) or total trace elem ent count is related to enam el solubility. All the trace elem ents found in enam el may have served only as “ foreign par
ticles” in apatite crystallization and by their greater concentrations in acid-resistant enam el w ere responsible for the increased size o f apatite crystallites and probably greater enam el density. Such speculation is in agreem ent with studies by K ham skii,9 w ho reported similar findings re garding the presence o f foreign trace elem ents in most all crystallization processes. H e and others listed in his bibliographies report the physical characteristic o f large crystallites (relatively small surface areas) as being less readily soluble than small crystallites (large surface areas). In solutions saturated with respect to large crystal lites, small crystallites w ould d issolve. R ecrys tallization accom panies dissolution in saturated and supersaturated sta tes.9 That recrystalliza tion occurs in enam el caries was indicated in a previous paper.3 T he refractive index o f carious enam el w as higher than that o f unaltered sub surface enam el adjacent to the lesion; this indi cates a dissolution o f small enam el crystallites with larger ones remaining but more likely hav ing grown in size by recrystallization in the dis solving medium saturated or supersaturated with elem ents present in calcified enam el. One should also consider the data in Table 1 show ing that the percentages o f Ca and P in acidresistant teeth are much higher than th ose in acid-susceptible teeth (by at least 20%). T he C, H, and O percentages are correspondingly high er for the acid-susceptible teeth. A cid-resistant teeth, therefore, contain much less organic ma terial— more calcium phosphate. This is con sist ent with our unpublished findings that acid-resis tant teeth are more dense. T he difference in the ratio o f calcium ph os phate to organic material betw een acid-resistant and acid-susceptible teeth is certainly significant and may w ell be related to the observed trace elem ent differences. R esistance to caries may be a function of organic content w hich, in turn, may be a function o f trace elem ent content. T he ion probe data (Table 7) obtained from crushed particles identifies Z n > B > F > V > C u > K (acid-resistant group) (Table 8) as being more concentrated on crystallite surfaces than intern ally. T he data presented in this report support the works o f O ck erse1012 and o f oth ers13'17 w ho found caries scores in natives o f several g e o graphic areas to be in inverse proportion to the concentration o f minerals found in soil, food, and water. T he abundance o f trace and major elem ents in Besic— others: COMPOSITION AND STRUCTURE OF ENAMEL ■ 599
T a b le 7 ■ S u rfa c e c o u n ts vs in te r n a l c o u n ts o f c ru s h e d p a rtic le s .
Acid Resistant Specimens
Acid Susceptible Specimens
Specimen No. Element
Specimen No.
R-1
R -2
R -3
Li
- 1 .0
-
-
—
B
~ 3 .0
- 3 .0
- 2 .0
- 3 .0
F
1.9
2.1
2.3
1.7
Al
-1 .0
- 1 .0
- 1 .0
K
1.1 1.1
Ti
0 .9
- 0 .8
1.7
1.9
V
1.2
1.3 - 1 .0
1.6
R -4
1.1 - 0 .9
2.0
S '1 - 1.0 -1 .0
1.1
S‘ 2
S "3
—
-
1.3
2 .5
1.4
1.2
- 1 .0 1.3
-1 .0
1.0
S '4
Zn 2 .3 5
B
2 .7 5
V
1.6
1.3
F
B
1.45
- 1 .0
V
2.00 1.8
1.1
-1 .1
-0 .9
-0 .9
- 2 .5
-1 .3
-0 .9
- 1.7
1.0 1.6 2.0
- 1 .0
- 1 .0
- 1 .0
- 1 .0
- 1.0
Cu
1.4
1.3
1.5
- 1 .0
1.4
1.1
1.7
Zn
4.5
5.0
4 .3
3.7
2 .5
3.1
1.8 0.6
-1 .0
-
-
Sr
- 1 .0
- 1 .0
- 1 .0
- 1 .0
~ 1.0
-1 .0
Mo
-1 .0
- 1 .0
- 1 .0
- 1 .0
0 .4
0.7
-
-
-
-1 .0 ?
-
0 .5
- 1 .0
-
A cid S u s c e p tib le Group
- 1 .0
—0 .7
0 .9
A cid R e s is ta n t Group Zn 4 .4
—
Fe
P6
Table 8 ■ Surface counts vs internal counts of crushed particles (averages of ratios listed for each element in each specimen).
-
Cu 1.45
Cu
1.3
F
1.3
K
1.17
K
Al
Al
1.12 1.00 1.00
Ti
0 .9
Mo
1.02 1.00 1.00 1.00
Ti
0 .9
Fe Sr
Pb Li
tra c e am ts . H h
Fe
S r 0 .9 Mo Pb Li
tra c e am ts . h .i «
»
Data is given as a ratio of surface counts to infernal counts, C g /C j.
ingested food and liquids no doubt is o f import an ce, but the effective assim ilation (biological factors) must also play a major role in the chem ical com position and structure o f enamel.
Summary and conclusion T h e sam e elem en ts, 21 in number, were found in both acid-resistant and acid-susceptible en am el specim ens by ion m icroprobe analyses. H o w ev er, there w ere differences in the con cen trations o f 14 elem ents betw een groups. O f the 16 trace elem ents found in enam el, the concen trations o f K, F , and N a in parts per million were the greatest. Enamel surface analyses show ed V and Zn w ere m ore concentrated in acid-resistant sp ec im ens by factors o f 7.4 and 6.1 respectively, fol low ed by nine other elem ents, the last m olyb denum , that were ju st slightly more prevalent in acid-resistant enam el. Four elem ents were equal ly concentrated in both groups o f sam ples, and only one trace elem ent, B, was more abundant in the acid-susceptible sam ples by a factor of ten. Ca and P w ere predom inantly more abundant in acid-resistant specim ens, and C, H , and O were m ore concentrated in acid-susceptible sam ples. T he enam el substance in acid-resistant and acid-susceptible groups was found to be similar by these m ethods. 600 ■ JADA, Vol. 91, Septem ber 1975
— Ion microprobe: the sam e elem ents were detected in both groups. Four trace elem ents (titanium , lead, Cl, and lithium) were equally abundant in the two groups. — Electron diffraction: the crystal structure type was the sam e in both groups o f sam ples. — Infrared absorption: the crystalline sub stance show ed the sam e spectra in both groups. T he enam el substance in the tw o groups o f specim ens was found to be dissim ilar by these m ethods. — Ion microprobe: the concentrations o f 11 trace elem ents as well as Ca and P w ere greater in acid-resistant enam el. T he concentrations of C, H , O, and B were greater in acid-susceptible enam el. — X-ray diffraction: crystallites in the acidresistant specim ens were larger by a factor of more than two. Enam el solubility appears not to be related to atom ic sp ecies but associated with one, som e, or all elem ents quantitatively and also with its phys ical form (crystallite size and probably density).
The authors thank the W alter G. Z oller Memorial Dental Clin ic, University of C h icago ; M cCrone A sso ciate s and M cCrone R esea rch Institute, C h icago , and the LaR ab id a C hildren’s H os pital and R esea rch Center, University of C hicago, for the su p port of this p roject; Dr. R. Z. Muggli for the infrared absorption spectrum d ata; Drs. H. T. Betz, A. V. C rew e, I. N. Hill, W. C. M cCrone, and P. B. M oore for helpful su g g e stio n s and review of this m an uscript; and Drs. F. R. Christopher, A. A. D ahlberg, I. N. Hill, J. H. Keith, B. Ritchey, R. R o ss, P. L. Vukovich, and J . K. W heeler, Jr., and the oral surgery and oral pathology serv ice s at the Zoller C linic for providing tooth sp ecim en s used in this research.
Dr. Besic is professor emeritus and Mr. Wiemann is biological research microscopist for the W. G. Zoller Memorial Dental Clin ic, University of Chicago, 950 E 59th St, Chicago, 60637. Dr. Bayard is senior research microscopist with Walter C. McCrone Associates, and instructor for McCrone Research Institute, Chicago. Dr. Burrell is assistant professor and head of the den tal service, La Rabida Children's Hospital and Research Center, University of Chicago. Address requests for reprints to Dr. Besic. ‘ Applied Research Laboratories, Glendale, Calif 91200. tKerr Mfg. Co., Romulus, Mich 48174. iWard's Natural Science Establishment, Inc., Rochester, NY 14603. 1 Lura, H.E. De cariespatogenetiske teorier i dag. Tandlaegebladet 73.283 April 1969. 2. Coolidge, T.B.; Besic, F.C.; and Jacobs, M.H. A microscopic comparison of clinically and artificially produced changes in enamel. Oral Surg 8:1204 Nov 1955. 3. Besic, F.C., and Wiemann, M.R., Jr. Dispersion staining, dispersion and refractive indices in early enamel caries. J Dent Res 51:973 July-Aug 1972. 4 Wiemann, M.R., Jr., and Besic, F.C. Dissolution and recrys tallization in dental enamel. The Microscope 21:81 April 1973. 5. Manson-Hing, L.R., and others. Microradiographic com parison of artificial caries systems J Dent Res 51 923 July-Aug 1972. 6. Johnson. N.W.; Poole, D.F ; and Tyler, J.E. Factors affecting the differential dissolution of human enamel in acid and EDTA. A s c a n n in g electron microscope study Arch Oral Biol 16385 April !9 ’ 1
7. Nishimura, T. Histologische untersuchungen iiber die aufange der zahnkaries, speziell de karies des schmelzes. Schweiz Monatsschr Zahnheilkd 36:491; 1926. 8. Besic, F.C., and others. Electron probe microanalysis of noncarious enamel and dentin and calcified tissues in mottled teeth. J Dent Res 48:131 Jan-Feb 1969. 9. Khamskii, E.V. Crystallization from solutions, translated from the Russian by Albin Tybulcwicz. New York, Consultants Bureau, 1969. 10. Ockerse, T. The chemical composition of enamel and dentin in high and low caries areas of South Africa. J Dent Res 22:441 Dec 1943. 11. Ockerse, T. Report on the incidence of dental caries among school children in South Africa. Pretoria, Dept of Public Health, Union of South Africa, 1944, p 23. 12. Ockerse, T. The relationship of fluorine content, hardness and pH values of drinking-water and the incidence of dental caries. S African Med J 18:255 Aug 12, 1944. 13. Forberg, E. Study of the teeth of children in the various schools of Sweden. Dent Cosmos 43:360, 1901. 14. Rose, C. Erdsaltzarmut und Entartung. Dtsch Monatsschr Zahnheilkd 26:1 (and subsequent articles), 1908. 15. Ludwig, T.G.; Healy, W.B.; and Losee, F.L. An association between dental caries and certain soil conditions in New Zealand. Nature 186:695 May 28, 1960. 16. Barmes, D.E. Caries etiology in Sepik villages—trace element, micronutrient and macronutrient content of soil and food. Caries Res 3:44, 1969. 17. Rothman, K.J., and others. Dental caries and soil content of trace metals in two Colombian villages. J Dent Res 51:1686 Nov-Dee 1972.
B esic—others: COMPOSITION AND STRUCTURE OF ENAMEL ■ 601