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Isomorphous Substitution in Enamel Apatite
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Isom orphous substitution in enam el ap atite Aaron S. Posner,* M.S., Washington, D. C., and Samuel R. Stephenson ,f B.S., Birmingham, Ala.
Human tooth enamel is found by chemical analysis to be about 95 per cent inorganic. Understanding of dental enamel requires a knowledge of the struc ture of the mineral-like portion of this tissue. The inorganic material in enamel is essentially a calcium phosphate con taining about 3 per cent carbonate ions. The molar ratio of calcium to phosphorus has been found to range from 1.571 to 1.662 per cent, the carbonate content from 2 to 4 per cent. This chemical variance does not result in any ap preciable change in the roentgen-ray diffraction data, the pattern being essen tially hydroxyapatite in all cases. In order to understand how it is possible to vary a substance chemically without varying a fundamental crystallographic property such as its roentgen-ray pattern, it is necessary to examine more closely the crystal chemistry o f the apatite series. Before doing this, it is well to review briefly the basic theory of roentgen-ray diffraction.
T H E O R Y O F R O E N T G E N -R A Y D IF F R A C T IO N
A crystal is a regular, three-dimensional assemblage of some fundamental repeat unit. In the case of hydroxyapatite this repeat unit is Ca10(P O 4) ,(O H ) 2, which is contained in a parallelepiped known as the unit cell, shown in Figure 1, left, in a stylized fashion. This unit translates itself through space in three directions, form ing a massive crystal in much the same way as bricks form a wall. The axes, or cell edges, of the unit cell can be calcu lated from the roentgen-ray diffraction
Presented before the Section on Research, ninetythird annual session o f the A m e rica n Dental Asso ciation , St. Louis, Se p te m b er 9, 1952. •Research associate, A m e rica n Dental A sso ciation Research Fellow ship at the N a tio n al Bureau o f Standards, tD e partm e nt of chem istry, U niversity of A la b a m a . 1. A rm stro n g, W . D., and Brekhus, P. J . C h e m ical constitution of enam el a^d dentin: p rin c ip a l com ponents. J . B io l. C h e m . 120:677, Sep t., 1937. 2. M urray, M. M., and Bowes, J . H . Com p osition of enam el, dentin and root in caries and pyorrhea. Brit. D. J . 61:473, O c t. 15, 1936.
258 • THE J O U R N A L O F THE A M E R IC A N DENTAL A S S O C IA T IO N
Fig. I • L eft: Stylized view of unit cell of hy droxyapatite. Right: Pro jection on basal plane of unit cell of fluorapatite showing ionic positions. This represents hydroxyapatite structure also if hydroxyl ions are put in place of all fluoride ions
\
data; and these values then become a means of identifying the compound in question. A more detailed view of the unit cell of apatite can be seen in Figure 1, right. When monochromatic roentgen rays are passed through apatite, each ion of the structure scatters roentgen rays in a pattern dependent on the electron cloud of that ion. The fact that the various ions are periodically spaced in a threedimensional arrangement, the spacing being of the order of magnitude of the wave length of roentgen rays, enables the scattering from the various ions to inter act and to cause constructive and de structive interference at certain critical angles, resulting in roentgen-ray maxima and minima respectively. The ionic spac ing and arrangement are quantitatively related to the angles and intensities of the roentgen-ray diffraction maxima. Figure 2 shows roentgen-ray diffraction diagrams of powdered human enamel and powdered hydroxyapatite. IS O M O R P H O U S
S U B S T IT U T IO N
Klement3 found that the mineral pyro morphite, a phosphate which contains
bivalent lead but no calcium, gave a roentgen-ray pattern characteristic of hydroxyapatite. As calcium ions and biva lent lead ions have the same valence and are of the same order of size,4 it is not surprising to find a hydroxyapatite in which all the calcium is supplanted by lead ions. As the lead ion is slightly larger than the calcium ion, the unit cell of pyromorphite measured from the roent gen-ray diagram shows a corresponding increase in volume, approximately 5 per cent. This replacement of one ion for another in a structure is called isomorphous substitution. In general, one ion can substitute for another when it is very similar in its chemical properties and similar enough in size so that the struc ture symmetry is not disrupted. Such sub stitution is not always complete as in the case of pyromorphite; often there is only a partial replacement of one ion by an other. The amount of an ion which can be supplanted by one or more ions is
3. Klem ent, R. Basische Phosphat zw eiw ertiger M e ta lle . II. B le i-h yd ro xy la p atit. Z e it, an o rg. C h e m . 237: 161, 1938. 4. P au lin g, L. The sizes o f ions and the structure of the ig n ic crysta ls. A m . CH em . Soc. 49:765, 1927.
PO SN ER— ST E P H E N S O N . . . V O L U M E 46, M A R C H 1953 • 259
determined by the physical and chemical conditions o f the substitution as well as by the relative stability of the various intermediate compounds. S U B S T IT U T IO N
IN
A P A T IT E
There is no doubt that ions such as mag nesium, potassium and sodium can proxy for the calcium positions in the structure of hydroxyapatite.5 Some investigators believe that at least 6 per cent of the positions normally occupied by calcium in apatite are occupied by magnesium, potassium and sodium in enamel apatite.6 In the case of bone apatite, however, Logan and Taylor7 have presented evi dence that these ions are adsorbed on readily available surfaces rather than sub stituted in the apatite structure. They found that sodium and magnesium ions are preferentially dissolved by acid dis proportionately to their stoichiometric ratios in the solid substance. The problem of fluoride ions entering the structure of enamel apatite is of im portance to the clinician as well as the research worker. Whether or not the
Fig. 2 • Roentgen-ray dif fraction diagrams of pow dered human enamel and powdered hydroxyapatite. Enamel has been treated in a hydrothermal bomb at 300° C., 2,000 p.s.i., for two days to sharpen originally broad maxima
fluoride ions actually enter into the struc ture or lodge on the surfaces of the tiny enamel apatite crystals is not yet under stood. The structure of a completely fluoride-substituted apatite, fluorapatite, as it is called, was worked out in detail by Mehmel8 and St. Naray-Szabo9 (Fig. 1, right). They demonstrated that fluor apatite has the same structure as hydroxy apatite with the fluoride ions substituting for all the hydroxyl ions. The unit cell size of fluorapatite is slightly smaller than that o f hydroxyapatite; and this fact, coupled with chemical analysis, has been used to identify fluorapatite. Perdok10
5. M cC o n n ell, D. The nature of rock phosphates, teeth, and bones. J . W ash in gto n A c a d . S c . 42:36, 1952. 6. Lo ga n , M. A ., and Taylor, H . L . S o lu b ility of bone salt; p a rtia l solution of bone and carbonate-co nta in in g calciu m phosphate p re cip itate s. J . Biol. C h e m . 125:391, Se p t., 1938. 7. Lo g a n , M. A ., and Taylor, H . L. So lu b ility of bone sa lt; facto rs affectin g its form ation . J . Biol. C h e m . 125:377, Se p t., 1938. 8. M ehm el, M. Ü b er d ie Struktur des A p a tits. Z e it. f. K rist. 75:323, 1930. 9. St. N a ray-Szab o , S. The structure o f ap atite . Z e it, f. Krist. 75:387, 1930. 10. Perdok, W . Die Aufnahm e von Fluorienen durch Zahnschm elz. Schw eiz. M onatschr. f. Zah n h eilku n d e und Zahntechnik 62:269, 1952.
260 • THE J O U R N A L P F THE A M E R IC A N DENTAL A S S O C IA T IO N
found no evidence of the presence of fluorapatite in dental enamel which con tained fluoride ions after treatment with potassium fluoride. Trautz,11 on the other hand, found some shark’s teeth which analyzed as fluorapatite and gave the characteristic fluorapatite rocntgen-ray pattern. It remains to be proved whether or not the fluoride can definitely enter the structure of human enamel apatite and form fluorapatite. There is evidence against isomorphous replacement of hydroxyl by fluoride ions when the fluoride solution is topically ap plied to the enamel. Gerould12 and later Scott, Picard and YVyckoff13 demon strated that calcium fluoride was de posited on the enamel surface on topical application of sodium fluoride. It was shown that the electron diffraction pat tern of the surface of the enamel changed from an apatite pattern to a calcium fluoride pattern. The use of electron dif fraction technics as opposed to roentgenray diffraction was most expedient, since the electron beam does not penetrate the enamel more than about 100 A. Thus, this surface phenomenon is not lost in the bulk diffraction by the rest of the enamel apatite. There are experiments in which fluoride neither enters the structure of enamel apatite nor deposits on the surface as calcium fluoride. Volker and his co workers14 showed that powdered enamel adsorbs fluoride ions very much as char coal adsorbs gases. There is a great deal of interest in the ability of enamel apatite to adsorb various ions from solution, and many workers are investigating this phenomenon. C A R B O N A T E IN A P A T IT E
Another controversial subject is the carbonate content of enamel. Here, too, the argument hinges on the existence of an ion as an integral part of the apatite structure or as an admixed or adsorbed material. The advocates of the theory of
isomorphous substitution of carbonate in enamel apatite have attempted to prove their assumption by showing evidence of carbonate substitution in the structure of francolite, a carbonate-containing fluor apatite. Proof by analogy is attempted on account of the difficulty of obtaining sharp roentgen-ray diffraction patterns from tooth mineral because of the fine ness of the enamel apatite crystals. M cConnell15 compared francolite with fluorapatite and showed minor differences in their roentgen-ray patterns. He pointed out also that francolite has a higher optical birefringence than fluor apatite and in a paper with Gruner16 demonstrated that the mean index of re fraction of francolite is less than that of fluorapatite. In addition he suggested15 a substitution mechanism for carbonate in francolite based on the powder roent gen-ray data and a series of careful chemical analyses. T o make an accurate study of the roentgen-ray diffraction diagrams and the index of refraction of enamel apatite, it is necessary to have crystals of at least 10,000 A. in diameter. Since the crystals of enamel apatite are very small, about 500 A., it is necessary to grow them larger to study them crystallographically. This increase in size was accomplished in our laboratory by treating the material in a hydrothermal bomb according to the method of Morey and Ingerson.17 A
11. Trautz, O . R. Private com m unication. 12. G ero u id , C . H . Electron m icroscop e study of the mechanism of fluorine d ep ositio n in teeth. J . D. Res. 24:223, O c t., 1945. 13. Scott, D. B.; P icard, R. S . , and W yckoff, R. W . S . Studies of the action o f sodium fluoride on human enam el by electron m icroscop y and electron d iffraction . Pub. H ealth Rep. 65:43, Ja n . 13, 1950. 14. Volker, J. F., an d others. A d so rp tio n o f fluorides by enam el, dentin, bone, an d h ydroxyapatite as shown by ra d io a ctive isotopes. J . Biol. C hem . 134:543, Ju ly , 1940. 15. M cC o n n ell, D. The p rob lem s o f the carb on ate ap atites. IV. Structural substitutions in vo lvin g carbonate and hydroxyl ions. (In p rin t.) 16. M cC o n n ell, D., and G ru n er, J . W . The problem of the carb on ate -ap atite s. I I I . C a rb o n a te -a p a tite from M agnet C o ve , A rkan sas. A m . M ineral. 25:157, 1940. 17. Morey, G . W ., and Ingerson, E. Bomb for use in hydrotherm al e xp erim entation. A m . M ineral. 22:1121, 1937.
POSNER— STEPHENSON . . . V O LU M E 46, M A R C H ¡953 • 261
charge was placed in water in a plati num-lined, sealed autoclave or bomb. The system was heated to about 300 de grees C., so that there was an internal pressure of about 2,000 pounds per square inch. This treatment resulted in the growth of the apatite crystals from about 500 to 30,000 A. A chemical anal ysis of the enamel apatite crystals both before and after the bomb treatment, as well as a microanalysis of the bomb water, showed that there is no chemical change during the bomb treatment. With these enlarged crystals, it was possible to obtain better crystallographic data on enamel apatite. When the improved roentgen-ray pat tern of enamel apatite is compared with the pattern of hydroxyapatite (Fig. 2), there does not seem to be enough differ ence in the patterns to say that there is isomorphous substitution of the carbonate ion. The intensities and the positions of the maxima are very similar, and for all practical purposes the roentgen-ray pat terns may be considered identical. At this writing there is no definite crystallo graphic proof of the presence of carbonate in the structure of enamel apa tite or any other apatite mineral. The presence of carbonate in the form of admixed calcium carbonate in dental enamel has not been detected with the electron microscope or by roentgen-ray diffraction methods. Silverman, Fuyat and Weiser18 established the fact that one can detect by roentgen-ray diffrac tion powder methods as little as 3 per cent calcium carbonate admixed physi cally with fluorapatite. These investigators demonstrated also that it is possible to detect by differential thermal analysis 1.6 per cent calcium carbonate mixed with apatite and by differential solubility tests 0.1 per cent calcium carbonate when mixed with apatite. In the latter test the solvent used to differentiate between cal cium carbonate and fluorapatite was 0.5 molar triammonium citrate, in which the former was alrtiost 50 times more soluble
than the latter. The conclusion was that if calcium carbonate were present in dental enamel as a separate phase, it would be possible to detect it by all three methods mentioned, since carbonatc is present in sufficient amount. The arguments for two phases in hu man enamel are just as numerous as those for isomorphous substitution. Preferential solution of carbonate by acid on samples of bone has been interpreted by Dallemagne10 to be evidence for the presence of a separate carbonate phase. Logan and Taylor7 have shown that it is possible to precipitate an apatite from a synthetic plasma solution. This precipitation takes place in the presence o f carbonate, and the apatite on precipitation steadily with draws carbonate ion from the solution. This action indicates the reversibility of “ dissociating” carbonate from the apa tites and lends weight to the idea that the carbonate in apatites exists as a separate phase. Hendricks and Hill20 believe that the carbonate in dental enamel is located on “ entrapped” surfaces. They demonstrate the presence of such surfaces by showing that the surface area, as measured by low temperature nitrogen adsorption, goes up much beyond the expected value when enamel is ground finer and finer. The presence of a high surface area, coupled with the fact that there is a preferential solution of carbonate in enamel apatite, supports the theory that the carbonate is located on entrapped surfaces and not bound structurally. These workers make no definite statement as to whether the carbonate is present as calcium carbonate or as some other compound or ion.
18. Silverm an, S. R.; Fuyat, R. K., and W eiser, J . D. Q u an tita tive determ ination of c a lc ite associated with carb o n a te -b ea rin g ap atites. A m . M ineral. 37:211, M archA p r il, 1952. 19. D allem agn e, M. J . , and Brasseur, H . Un cas d'in te rp rétatio n d iffic ile : la nature du sel p rin cip al de l'os étudiée par la d iffractio n des rayons x. Experientia 3:469, 1947. 20. H en dricks, S. B., and H ill, W . L. The nature or bone and phosphate rock. Proc. N a t. A c a d . Sc. 36:731, Dec., 1950.
262 • THE J O U R N A L O F THE A M E R IC A N DEN T A L A S S O C IA T I O N
SPECIMEN SLIT FILM
Fig. 3 • Small-angle scattering apparatus
E V ID E N C E F O R T W O -P H A S E S Y S T E M
There has been a need for some positive crystallographic evidence for the occur rence of carbonate on entrapped surfaces. A more specific way of describing such a two-phase system is to say there is an electron density inhomogeneity in the enamel apatite. Thus particles of calcium carbonate, too small to be seen in the electron microscope, may be distributed throughout the apatite and result in a two-phase system, each phase having a different average electron density. There is a technic in roentgen-ray crystallog raphy known as low-angle scattering which can be used to distinguish between two substances of different electron densi ties and to determine the particle size distribution of this system. This tool for evaluating the particle size in the range 10 A. to 1,000 A. has been used to de termine the size and shape of such mate rials as carbon black, alumina gels, col lagen fibers and many viruses. This method is not concerned with intensity maxima due to the arrangement of the ions in a structure but with the scatter ing of roentgen rays close to the center of the main, monochromatic beam as it passes through the sample. This scatter ing at small angles is directly related to the particle shape and size of the sample. Figure 3 shows a diagram of the ap paratus used, and Figure 4 shows the re
sults of a low-angle scattering experiment on an apatite mineral. The latter is a trace of intensity of scattering at various angles on either side of the main beam, the more intense scattering appearing closest to the main beam. Guinier21 has shown that there is a relationship between the intensity of scattering, the angle at which the scatter ing o f the roentgen rays occurs, and the radius of the particles doing the scatter ing. According to Guinier, assuming the particles to be spherical, I = M N 2 exp
(2
It Q e ) 2
5 x2
where I = intensity of roentgen rays at angle e, M = number of particles, N = number of electrons per particle, ^ = wave length o f roentgen rays, and q = radius of the particles. By taking the natural logarithm of both sides of this equation and converting to logarithms of the base 10, the following equation is obtained: log I = log M N
/ 4 Jt2Q2 \ \ 11.5 X2 )
21. G u in ie r, A . La d iffractio n des rayons x aux très petits an gle s: ap p lica tio n a l'etude de phenomenes ultram icroscopiques. A n n . de phys. "12:161, 1939.
POSNER— STEPHENSON . . . V O LU M E 46, M A R C H 1953 • 263
This is an equation of the form y — bmx and thus the equation of a straight line. If all the particles are equal and log I versus e2, or some function o f e2, is plotted, a straight line is obtained whose slope is equal to
4 jc2p2
. Since all the
other quantities are known, it is now pos sible to solve for p. the radius of the particles doing the scattering. The plot of log I against R 2 for dental enamel is seen in Figure 5. R, a function of e, is the distance from the beam center measured perpendicular to the main beam. There is a large portion of this curve which approximates a straight line, and the particle size calculated from the slope o f this portion is 48 A. The curve changes abruptly from this low slope to a higher slope corresponding to a particle size of about 500 A. This abrupt change indicates a discontinuity in particle size from about 50 A. to about 500 A. Another method of measuring the fine ness of crystals is the study of the broad ening o f the roentgen-ray maxima. As mentioned before, a substance begins to give broad, rather than sharp, roentgenray maxima when its average crystal size goes below 10,000 A. The effect of crystal lite size in broadening diffraction maxima
is seen in (Jones22) :
the
_
following
equation
x B cos 8
where t = size of crystal, X = wave length of roentgen rays, B =■- broadening of the maxima measured in angular half width, and 6 = angle of diffraction at maxi mum. By this method it is possible to calculate the average crystal size of enamel apatite to be 500 A. In this study of the particle size of enamel apatite, there is discontinuity in the sizes from about 50 A. to about 500 A. It is certain that the 500 A. particles are enamel apatite because these measure ments were made on the broadening of characteristic diffraction maxima. Since 500 A. is a size appearing in both lowangle scattering and roentgen-ray line broadening measurements, the 50 A.
22. Jones, F. W . The m easurement o f p a rticle size by the x-ray m ethod. Proc. Roy. Soc., London A I6 6 :I6 , 1938.
Fig. 4 • Curve showing in tensity of scattering versus angle; obtained on sample of chemically precipitated tricalcium phosphate hy drate
MICROPHOTOMETER CURVE OF DIFFRACTION PATTERN OF TRICALCIUM PHOSPHATE HYDRATE
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1M • THE J O U R N A L OF THE A M E R IC A N DENTAL A S S O C IA T IO N
ever, that' such small particles do exist somewhere in the body of enamel apatite. SU M M ARY
Fig. 5 • Plot of logarithm of scattered, inten sity versus square of a function of the scatter ing angle; obtained from low-angle scattering data on human enamel
particles are probably not apatite. If they were, the average crystal size as measured by line broadening would be lower than 500 A. Certainly more data are needed to say definitely whether the 50 A. particles are admixed carbonate or merely apatite particles. It is interesting, how
The crystal chemistry of the inorganic portion o f the enamel of human teeth is still under dispute. A general background of the crystallography involved is neces sary for an understanding of arguments for and against the substitution of ions such as carbonate, fluoride, magnesium and others in the apatite structure. In re gard to the matter of carbonate substitu tion, although facts have been presented on both sides, the data seem to indicate that the carbonate is included as a separate phase which is too small to be seen by the electron microscope or with normal roentgen-ray diffraction but which can be seen by low-angle scatter ing of roentgen rays. It is hoped that some day a better understanding of such funda mental problems as the structure o f dental enamel will aid the clinician in dealing with many of his practical problems.
Health Education • The object of all health education is to change the conduct of individual men, women, and children by teaching them to care for their bodies well, and this instruction should be given throughout the entire period of their educational life. C. H. M ayo, " When
Does Disease Begin? Can This Be Determined by Health Examinations?” Minnesota Medicine ¡5 :4 0 , January, 1932.
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Isom orphous substitution in enam el ap atite Aaron S. Posner,* M.S., Washington, D. C., and Samuel R. Stephenson ,f B.S., Birmingham, Ala.
Human tooth enamel is found by chemical analysis to be about 95 per cent inorganic. Understanding of dental enamel requires a knowledge of the struc ture of the mineral-like portion of this tissue. The inorganic material in enamel is essentially a calcium phosphate con taining about 3 per cent carbonate ions. The molar ratio of calcium to phosphorus has been found to range from 1.571 to 1.662 per cent, the carbonate content from 2 to 4 per cent. This chemical variance does not result in any ap preciable change in the roentgen-ray diffraction data, the pattern being essen tially hydroxyapatite in all cases. In order to understand how it is possible to vary a substance chemically without varying a fundamental crystallographic property such as its roentgen-ray pattern, it is necessary to examine more closely the crystal chemistry o f the apatite series. Before doing this, it is well to review briefly the basic theory of roentgen-ray diffraction.
T H E O R Y O F R O E N T G E N -R A Y D IF F R A C T IO N
A crystal is a regular, three-dimensional assemblage of some fundamental repeat unit. In the case of hydroxyapatite this repeat unit is Ca10(P O 4) ,(O H ) 2, which is contained in a parallelepiped known as the unit cell, shown in Figure 1, left, in a stylized fashion. This unit translates itself through space in three directions, form ing a massive crystal in much the same way as bricks form a wall. The axes, or cell edges, of the unit cell can be calcu lated from the roentgen-ray diffraction
Presented before the Section on Research, ninetythird annual session o f the A m e rica n Dental Asso ciation , St. Louis, Se p te m b er 9, 1952. •Research associate, A m e rica n Dental A sso ciation Research Fellow ship at the N a tio n al Bureau o f Standards, tD e partm e nt of chem istry, U niversity of A la b a m a . 1. A rm stro n g, W . D., and Brekhus, P. J . C h e m ical constitution of enam el a^d dentin: p rin c ip a l com ponents. J . B io l. C h e m . 120:677, Sep t., 1937. 2. M urray, M. M., and Bowes, J . H . Com p osition of enam el, dentin and root in caries and pyorrhea. Brit. D. J . 61:473, O c t. 15, 1936.
258 • THE J O U R N A L O F THE A M E R IC A N DENTAL A S S O C IA T IO N
Fig. I • L eft: Stylized view of unit cell of hy droxyapatite. Right: Pro jection on basal plane of unit cell of fluorapatite showing ionic positions. This represents hydroxyapatite structure also if hydroxyl ions are put in place of all fluoride ions
\
data; and these values then become a means of identifying the compound in question. A more detailed view of the unit cell of apatite can be seen in Figure 1, right. When monochromatic roentgen rays are passed through apatite, each ion of the structure scatters roentgen rays in a pattern dependent on the electron cloud of that ion. The fact that the various ions are periodically spaced in a threedimensional arrangement, the spacing being of the order of magnitude of the wave length of roentgen rays, enables the scattering from the various ions to inter act and to cause constructive and de structive interference at certain critical angles, resulting in roentgen-ray maxima and minima respectively. The ionic spac ing and arrangement are quantitatively related to the angles and intensities of the roentgen-ray diffraction maxima. Figure 2 shows roentgen-ray diffraction diagrams of powdered human enamel and powdered hydroxyapatite. IS O M O R P H O U S
S U B S T IT U T IO N
Klement3 found that the mineral pyro morphite, a phosphate which contains
bivalent lead but no calcium, gave a roentgen-ray pattern characteristic of hydroxyapatite. As calcium ions and biva lent lead ions have the same valence and are of the same order of size,4 it is not surprising to find a hydroxyapatite in which all the calcium is supplanted by lead ions. As the lead ion is slightly larger than the calcium ion, the unit cell of pyromorphite measured from the roent gen-ray diagram shows a corresponding increase in volume, approximately 5 per cent. This replacement of one ion for another in a structure is called isomorphous substitution. In general, one ion can substitute for another when it is very similar in its chemical properties and similar enough in size so that the struc ture symmetry is not disrupted. Such sub stitution is not always complete as in the case of pyromorphite; often there is only a partial replacement of one ion by an other. The amount of an ion which can be supplanted by one or more ions is
3. Klem ent, R. Basische Phosphat zw eiw ertiger M e ta lle . II. B le i-h yd ro xy la p atit. Z e it, an o rg. C h e m . 237: 161, 1938. 4. P au lin g, L. The sizes o f ions and the structure of the ig n ic crysta ls. A m . CH em . Soc. 49:765, 1927.
PO SN ER— ST E P H E N S O N . . . V O L U M E 46, M A R C H 1953 • 259
determined by the physical and chemical conditions o f the substitution as well as by the relative stability of the various intermediate compounds. S U B S T IT U T IO N
IN
A P A T IT E
There is no doubt that ions such as mag nesium, potassium and sodium can proxy for the calcium positions in the structure of hydroxyapatite.5 Some investigators believe that at least 6 per cent of the positions normally occupied by calcium in apatite are occupied by magnesium, potassium and sodium in enamel apatite.6 In the case of bone apatite, however, Logan and Taylor7 have presented evi dence that these ions are adsorbed on readily available surfaces rather than sub stituted in the apatite structure. They found that sodium and magnesium ions are preferentially dissolved by acid dis proportionately to their stoichiometric ratios in the solid substance. The problem of fluoride ions entering the structure of enamel apatite is of im portance to the clinician as well as the research worker. Whether or not the
Fig. 2 • Roentgen-ray dif fraction diagrams of pow dered human enamel and powdered hydroxyapatite. Enamel has been treated in a hydrothermal bomb at 300° C., 2,000 p.s.i., for two days to sharpen originally broad maxima
fluoride ions actually enter into the struc ture or lodge on the surfaces of the tiny enamel apatite crystals is not yet under stood. The structure of a completely fluoride-substituted apatite, fluorapatite, as it is called, was worked out in detail by Mehmel8 and St. Naray-Szabo9 (Fig. 1, right). They demonstrated that fluor apatite has the same structure as hydroxy apatite with the fluoride ions substituting for all the hydroxyl ions. The unit cell size of fluorapatite is slightly smaller than that o f hydroxyapatite; and this fact, coupled with chemical analysis, has been used to identify fluorapatite. Perdok10
5. M cC o n n ell, D. The nature of rock phosphates, teeth, and bones. J . W ash in gto n A c a d . S c . 42:36, 1952. 6. Lo ga n , M. A ., and Taylor, H . L . S o lu b ility of bone salt; p a rtia l solution of bone and carbonate-co nta in in g calciu m phosphate p re cip itate s. J . Biol. C h e m . 125:391, Se p t., 1938. 7. Lo g a n , M. A ., and Taylor, H . L. So lu b ility of bone sa lt; facto rs affectin g its form ation . J . Biol. C h e m . 125:377, Se p t., 1938. 8. M ehm el, M. Ü b er d ie Struktur des A p a tits. Z e it. f. K rist. 75:323, 1930. 9. St. N a ray-Szab o , S. The structure o f ap atite . Z e it, f. Krist. 75:387, 1930. 10. Perdok, W . Die Aufnahm e von Fluorienen durch Zahnschm elz. Schw eiz. M onatschr. f. Zah n h eilku n d e und Zahntechnik 62:269, 1952.
260 • THE J O U R N A L P F THE A M E R IC A N DENTAL A S S O C IA T IO N
found no evidence of the presence of fluorapatite in dental enamel which con tained fluoride ions after treatment with potassium fluoride. Trautz,11 on the other hand, found some shark’s teeth which analyzed as fluorapatite and gave the characteristic fluorapatite rocntgen-ray pattern. It remains to be proved whether or not the fluoride can definitely enter the structure of human enamel apatite and form fluorapatite. There is evidence against isomorphous replacement of hydroxyl by fluoride ions when the fluoride solution is topically ap plied to the enamel. Gerould12 and later Scott, Picard and YVyckoff13 demon strated that calcium fluoride was de posited on the enamel surface on topical application of sodium fluoride. It was shown that the electron diffraction pat tern of the surface of the enamel changed from an apatite pattern to a calcium fluoride pattern. The use of electron dif fraction technics as opposed to roentgenray diffraction was most expedient, since the electron beam does not penetrate the enamel more than about 100 A. Thus, this surface phenomenon is not lost in the bulk diffraction by the rest of the enamel apatite. There are experiments in which fluoride neither enters the structure of enamel apatite nor deposits on the surface as calcium fluoride. Volker and his co workers14 showed that powdered enamel adsorbs fluoride ions very much as char coal adsorbs gases. There is a great deal of interest in the ability of enamel apatite to adsorb various ions from solution, and many workers are investigating this phenomenon. C A R B O N A T E IN A P A T IT E
Another controversial subject is the carbonate content of enamel. Here, too, the argument hinges on the existence of an ion as an integral part of the apatite structure or as an admixed or adsorbed material. The advocates of the theory of
isomorphous substitution of carbonate in enamel apatite have attempted to prove their assumption by showing evidence of carbonate substitution in the structure of francolite, a carbonate-containing fluor apatite. Proof by analogy is attempted on account of the difficulty of obtaining sharp roentgen-ray diffraction patterns from tooth mineral because of the fine ness of the enamel apatite crystals. M cConnell15 compared francolite with fluorapatite and showed minor differences in their roentgen-ray patterns. He pointed out also that francolite has a higher optical birefringence than fluor apatite and in a paper with Gruner16 demonstrated that the mean index of re fraction of francolite is less than that of fluorapatite. In addition he suggested15 a substitution mechanism for carbonate in francolite based on the powder roent gen-ray data and a series of careful chemical analyses. T o make an accurate study of the roentgen-ray diffraction diagrams and the index of refraction of enamel apatite, it is necessary to have crystals of at least 10,000 A. in diameter. Since the crystals of enamel apatite are very small, about 500 A., it is necessary to grow them larger to study them crystallographically. This increase in size was accomplished in our laboratory by treating the material in a hydrothermal bomb according to the method of Morey and Ingerson.17 A
11. Trautz, O . R. Private com m unication. 12. G ero u id , C . H . Electron m icroscop e study of the mechanism of fluorine d ep ositio n in teeth. J . D. Res. 24:223, O c t., 1945. 13. Scott, D. B.; P icard, R. S . , and W yckoff, R. W . S . Studies of the action o f sodium fluoride on human enam el by electron m icroscop y and electron d iffraction . Pub. H ealth Rep. 65:43, Ja n . 13, 1950. 14. Volker, J. F., an d others. A d so rp tio n o f fluorides by enam el, dentin, bone, an d h ydroxyapatite as shown by ra d io a ctive isotopes. J . Biol. C hem . 134:543, Ju ly , 1940. 15. M cC o n n ell, D. The p rob lem s o f the carb on ate ap atites. IV. Structural substitutions in vo lvin g carbonate and hydroxyl ions. (In p rin t.) 16. M cC o n n ell, D., and G ru n er, J . W . The problem of the carb on ate -ap atite s. I I I . C a rb o n a te -a p a tite from M agnet C o ve , A rkan sas. A m . M ineral. 25:157, 1940. 17. Morey, G . W ., and Ingerson, E. Bomb for use in hydrotherm al e xp erim entation. A m . M ineral. 22:1121, 1937.
POSNER— STEPHENSON . . . V O LU M E 46, M A R C H ¡953 • 261
charge was placed in water in a plati num-lined, sealed autoclave or bomb. The system was heated to about 300 de grees C., so that there was an internal pressure of about 2,000 pounds per square inch. This treatment resulted in the growth of the apatite crystals from about 500 to 30,000 A. A chemical anal ysis of the enamel apatite crystals both before and after the bomb treatment, as well as a microanalysis of the bomb water, showed that there is no chemical change during the bomb treatment. With these enlarged crystals, it was possible to obtain better crystallographic data on enamel apatite. When the improved roentgen-ray pat tern of enamel apatite is compared with the pattern of hydroxyapatite (Fig. 2), there does not seem to be enough differ ence in the patterns to say that there is isomorphous substitution of the carbonate ion. The intensities and the positions of the maxima are very similar, and for all practical purposes the roentgen-ray pat terns may be considered identical. At this writing there is no definite crystallo graphic proof of the presence of carbonate in the structure of enamel apa tite or any other apatite mineral. The presence of carbonate in the form of admixed calcium carbonate in dental enamel has not been detected with the electron microscope or by roentgen-ray diffraction methods. Silverman, Fuyat and Weiser18 established the fact that one can detect by roentgen-ray diffrac tion powder methods as little as 3 per cent calcium carbonate admixed physi cally with fluorapatite. These investigators demonstrated also that it is possible to detect by differential thermal analysis 1.6 per cent calcium carbonate mixed with apatite and by differential solubility tests 0.1 per cent calcium carbonate when mixed with apatite. In the latter test the solvent used to differentiate between cal cium carbonate and fluorapatite was 0.5 molar triammonium citrate, in which the former was alrtiost 50 times more soluble
than the latter. The conclusion was that if calcium carbonate were present in dental enamel as a separate phase, it would be possible to detect it by all three methods mentioned, since carbonatc is present in sufficient amount. The arguments for two phases in hu man enamel are just as numerous as those for isomorphous substitution. Preferential solution of carbonate by acid on samples of bone has been interpreted by Dallemagne10 to be evidence for the presence of a separate carbonate phase. Logan and Taylor7 have shown that it is possible to precipitate an apatite from a synthetic plasma solution. This precipitation takes place in the presence o f carbonate, and the apatite on precipitation steadily with draws carbonate ion from the solution. This action indicates the reversibility of “ dissociating” carbonate from the apa tites and lends weight to the idea that the carbonate in apatites exists as a separate phase. Hendricks and Hill20 believe that the carbonate in dental enamel is located on “ entrapped” surfaces. They demonstrate the presence of such surfaces by showing that the surface area, as measured by low temperature nitrogen adsorption, goes up much beyond the expected value when enamel is ground finer and finer. The presence of a high surface area, coupled with the fact that there is a preferential solution of carbonate in enamel apatite, supports the theory that the carbonate is located on entrapped surfaces and not bound structurally. These workers make no definite statement as to whether the carbonate is present as calcium carbonate or as some other compound or ion.
18. Silverm an, S. R.; Fuyat, R. K., and W eiser, J . D. Q u an tita tive determ ination of c a lc ite associated with carb o n a te -b ea rin g ap atites. A m . M ineral. 37:211, M archA p r il, 1952. 19. D allem agn e, M. J . , and Brasseur, H . Un cas d'in te rp rétatio n d iffic ile : la nature du sel p rin cip al de l'os étudiée par la d iffractio n des rayons x. Experientia 3:469, 1947. 20. H en dricks, S. B., and H ill, W . L. The nature or bone and phosphate rock. Proc. N a t. A c a d . Sc. 36:731, Dec., 1950.
262 • THE J O U R N A L O F THE A M E R IC A N DEN T A L A S S O C IA T I O N
SPECIMEN SLIT FILM
Fig. 3 • Small-angle scattering apparatus
E V ID E N C E F O R T W O -P H A S E S Y S T E M
There has been a need for some positive crystallographic evidence for the occur rence of carbonate on entrapped surfaces. A more specific way of describing such a two-phase system is to say there is an electron density inhomogeneity in the enamel apatite. Thus particles of calcium carbonate, too small to be seen in the electron microscope, may be distributed throughout the apatite and result in a two-phase system, each phase having a different average electron density. There is a technic in roentgen-ray crystallog raphy known as low-angle scattering which can be used to distinguish between two substances of different electron densi ties and to determine the particle size distribution of this system. This tool for evaluating the particle size in the range 10 A. to 1,000 A. has been used to de termine the size and shape of such mate rials as carbon black, alumina gels, col lagen fibers and many viruses. This method is not concerned with intensity maxima due to the arrangement of the ions in a structure but with the scatter ing of roentgen rays close to the center of the main, monochromatic beam as it passes through the sample. This scatter ing at small angles is directly related to the particle shape and size of the sample. Figure 3 shows a diagram of the ap paratus used, and Figure 4 shows the re
sults of a low-angle scattering experiment on an apatite mineral. The latter is a trace of intensity of scattering at various angles on either side of the main beam, the more intense scattering appearing closest to the main beam. Guinier21 has shown that there is a relationship between the intensity of scattering, the angle at which the scatter ing o f the roentgen rays occurs, and the radius of the particles doing the scatter ing. According to Guinier, assuming the particles to be spherical, I = M N 2 exp
(2
It Q e ) 2
5 x2
where I = intensity of roentgen rays at angle e, M = number of particles, N = number of electrons per particle, ^ = wave length o f roentgen rays, and q = radius of the particles. By taking the natural logarithm of both sides of this equation and converting to logarithms of the base 10, the following equation is obtained: log I = log M N
/ 4 Jt2Q2 \ \ 11.5 X2 )
21. G u in ie r, A . La d iffractio n des rayons x aux très petits an gle s: ap p lica tio n a l'etude de phenomenes ultram icroscopiques. A n n . de phys. "12:161, 1939.
POSNER— STEPHENSON . . . V O LU M E 46, M A R C H 1953 • 263
This is an equation of the form y — bmx and thus the equation of a straight line. If all the particles are equal and log I versus e2, or some function o f e2, is plotted, a straight line is obtained whose slope is equal to
4 jc2p2
. Since all the
other quantities are known, it is now pos sible to solve for p. the radius of the particles doing the scattering. The plot of log I against R 2 for dental enamel is seen in Figure 5. R, a function of e, is the distance from the beam center measured perpendicular to the main beam. There is a large portion of this curve which approximates a straight line, and the particle size calculated from the slope o f this portion is 48 A. The curve changes abruptly from this low slope to a higher slope corresponding to a particle size of about 500 A. This abrupt change indicates a discontinuity in particle size from about 50 A. to about 500 A. Another method of measuring the fine ness of crystals is the study of the broad ening o f the roentgen-ray maxima. As mentioned before, a substance begins to give broad, rather than sharp, roentgenray maxima when its average crystal size goes below 10,000 A. The effect of crystal lite size in broadening diffraction maxima
is seen in (Jones22) :
the
_
following
equation
x B cos 8
where t = size of crystal, X = wave length of roentgen rays, B =■- broadening of the maxima measured in angular half width, and 6 = angle of diffraction at maxi mum. By this method it is possible to calculate the average crystal size of enamel apatite to be 500 A. In this study of the particle size of enamel apatite, there is discontinuity in the sizes from about 50 A. to about 500 A. It is certain that the 500 A. particles are enamel apatite because these measure ments were made on the broadening of characteristic diffraction maxima. Since 500 A. is a size appearing in both lowangle scattering and roentgen-ray line broadening measurements, the 50 A.
22. Jones, F. W . The m easurement o f p a rticle size by the x-ray m ethod. Proc. Roy. Soc., London A I6 6 :I6 , 1938.
Fig. 4 • Curve showing in tensity of scattering versus angle; obtained on sample of chemically precipitated tricalcium phosphate hy drate
MICROPHOTOMETER CURVE OF DIFFRACTION PATTERN OF TRICALCIUM PHOSPHATE HYDRATE
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nb|>
1M • THE J O U R N A L OF THE A M E R IC A N DENTAL A S S O C IA T IO N
ever, that' such small particles do exist somewhere in the body of enamel apatite. SU M M ARY
Fig. 5 • Plot of logarithm of scattered, inten sity versus square of a function of the scatter ing angle; obtained from low-angle scattering data on human enamel
particles are probably not apatite. If they were, the average crystal size as measured by line broadening would be lower than 500 A. Certainly more data are needed to say definitely whether the 50 A. particles are admixed carbonate or merely apatite particles. It is interesting, how
The crystal chemistry of the inorganic portion o f the enamel of human teeth is still under dispute. A general background of the crystallography involved is neces sary for an understanding of arguments for and against the substitution of ions such as carbonate, fluoride, magnesium and others in the apatite structure. In re gard to the matter of carbonate substitu tion, although facts have been presented on both sides, the data seem to indicate that the carbonate is included as a separate phase which is too small to be seen by the electron microscope or with normal roentgen-ray diffraction but which can be seen by low-angle scatter ing of roentgen rays. It is hoped that some day a better understanding of such funda mental problems as the structure o f dental enamel will aid the clinician in dealing with many of his practical problems.
Health Education • The object of all health education is to change the conduct of individual men, women, and children by teaching them to care for their bodies well, and this instruction should be given throughout the entire period of their educational life. C. H. M ayo, " When
Does Disease Begin? Can This Be Determined by Health Examinations?” Minnesota Medicine ¡5 :4 0 , January, 1932.