1030 Volume 56, Number 8, 2002 APPLIED SPECTROSCOPY0003-7028 / 02 / 5608-1030$2.00 / 0q 2002 Society for Applied Spectroscopy

Human Tooth Enamel: A Raman Polarized Approach

G. LEROY, G. PENEL,* N. LEROY, and E. BRESL.B.M. Raman Faculte d’Odontologie, place de Verdun 59045 Lille, France (G.L., G.P., N.L); and L.S.P.E.S. C.N.R.S. E.S.A. 8008,U.S.T.L. Batiment C6, 59655 Villeneuve d’Ascq, France (N.L., E.B.)

Despite numerous studies of human biominerals, some problemsstill remain concerning the relationship between their compositionand their structure. For a better understanding of this problem, fullspectra of the internal vibrations of human tooth enamel crystalliteswere obtained through polarized Raman microspectrometry andthese are published for the � rst time. The micro-Raman techniqueis nondestructive and enables micrometric-scale examination of allthe samples with a minimum of artifacts. The spectra show somevariation from the predicted bands and many similarities with� uorapatite spectra. The mineral part of enamel is initiated in anorganic environment and contains carbonate ions. Despite the car-bonation, the crystal structure is preserved. Based on these results,a new description of the structure of apatite crystal is proposed. Abox of Ca21 ions surrounds and isolates the PO 4

32 ions from oneanother, decreasing the in� uence of substitutions.

Index Headings: Enamel; Apatite; Polarization; Raman.

INTRODUCTION

Apatites are phospho-calcium compounds of funda-mental importance to several � elds, speci� cally, to biol-ogy. The roles of and applications for biological apatites,the main compounds of mineralized hard tissue, are well-known, but their structure still presents some unresolvedproblems. Different analytical techniques, such as X-rays,electron or neutron diffraction, and Raman or IR spec-troscopy,1–9 have been used to provide information on thestructure of apatite, but these studies were not exhaustiveand need to be completed. In the study of calci� ed tissue,the mineral and organic phases are often examined sep-arately, neglecting the interactions that could exist be-tween the two phases. The analysis of molecular bondspermits a global approach to these structures in order toobtain new and complementary information.

Human tooth enamel is a convenient model for vibra-tional studies of biological apatites because of the largesize of the enamel crystallites and the large proportion ofthe inorganic phase, which makes up 95% wt of the tis-sue.2 Enamel crystallites were described as a carbonatedapatite, belonging to the P63 /m space group.2,10 They es-sentially present a Type B substitution of one PO 4

32 ionwith one CO3

22 ion, with the loss of one Ca 21 and oneOH2 ion to preserve electric neutrality, and a smallamount of Type A substitution, where an OH2 ion isreplaced with an X2 ion. A small proportion of the enam-el crystallites are randomly orientated but the majorityare highly orientated within the sub-surface area, withtheir c-axis perpendicular to the outer surface.2,11 Chem-ical analysis of the mineral part of enamel shows that theoccurrence of various compounds (PO 4

32, Ca 21, CO322,

HPO 422, F2, OH2, Cl2, Mg 21, Cu 21, etc.) and traces of

Received 23 October 2001; accepted 15 March 2002.* Author to whom correspondence should be sent.

different atoms (La, Ce, Ag, Sn21, Mn, etc.) are includedin the apatite structure in varying degrees according tothe various substitutions. Chemical and structural defectsproduce residual background noise and in� uence thebroadness of the Raman bands, speci� cally the internalvibrational modes that are mainly composed of PO4

32 ionvibration modes.

The free PO432 ion has four internal vibration modes:

n1, n2, n3, and n4, all associated with a symmetry species.In an apatite crystal, PO 4

32 ions are distorted under theeffect of the crystal � eld and degenerate vibration modesoccur, of which only some are Raman- and/or IR-active.Two theoretical models have been used to represent theeffects of crystal � elds and to determine the activity ofbands: group analysis and the Halford site method.6,12 Infact, the Raman signal is dependent upon the double in-� uence of the composition and the structure of the sam-ple. But only polarized Raman acquisition is able to sep-arate all the active bands of the spectrum. From theseassays the complete attribution of the spectrum is possi-ble.13

To our knowledge, there has been no report of a fullRaman polarized spectrum of the internal modes of hu-man tooth enamel. In the present work, we studied humantooth enamel crystallites by means of polarized Ramanmicrospectrometry. We also compared the band attribu-tions obtained on the polarized Raman spectra to the pre-dicted assignments. The aim of this study is to obtainnew and complementary information on the relationshipbetween the composition and the structure of humanenamel.

MATERIALS AND METHODS

Micro-Raman Spectrometry. We used an OMARS89microspectrometer from DILOR (Lille, France) with twodifferent excitation sources reaching the sample, a heli-um–neon laser (632.8 nm, 3 mW) and an argon-ion laser(514 nm, 10 mW). The overall spectral resolution was 2cm21. A micrometric-sized probe was obtained with a1003 (for the HeNe laser) or 503 (for the Ar-ion laser)microscope objective used in a confocal con� guration.The magni� cation of the lens was 503 with the Ar-ionlaser and the depolarization caused was negligible.14 Inthe present work, no signi� cant differences were foundbetween the two lenses used. The main information wasobtained on the four internal vibration modes of PO4

32

ions in the 400–1120 cm21 region. With the Ar-ion lasersource, we also studied the OH2 bands in the 3470–3680cm21 region for enamel samples.

Polarization. The P63 /m space group enables the FApcrystal 15 and enamel crystallite structures to be de-scribed.10 They both present some symmetry-species ac-tivity in Raman and IR spectroscopy, presented in the

APPLIED SPECTROSCOPY 1031

TABLE I. Correlation table for the PO432 ions (R: Raman activity, IR: infrared activity) and compounds of the tensor of polarizability

enabled isolation of Raman active symmetry species.

FIG. 1. Polarization experiments in transverse excitation/detection ar-rangement with human enamel crystallites, according to Tsuda and Ar-ends.11 (A) Ag species; (B) E1g species; (C ) Ag 1 E2g species. LP : linearpolarizer; Obj: objective lens; IF : interference � lter; Bs : beam splitter.

character table of the factor group (C 6h for the FAp).Three symmetry-species, called Ag, E1g, and E2g, are ac-tive in Raman spectroscopy and two species, Au and E1u,are IR-active. According to the components of the Ramanpolarizability tensor and with the appropriate polarizationdirection, we can isolate the vibrational wavenumbers as-sociated with the activity of one symmetry-species on thespectra obtained.

The polarization studies were performed according to

the arrangements described in previous publications.11,16

Brie� y, each polarization direction can be associated witha component of the polarizability tensor of the crystalspace group. In the case of FAp, space group P6 3 /m, onlyfour polarization directions (zz, zx, xy, and xx) are suf-� cient for studying all polarization states. Three orien-tations of the polarization direction are enough to isolateall the Raman-active symmetry species: zz for Ag, zx forE1g, and xy for E2g (see Table I).16

The � rst experiments were made following the samearrangements as those described by Tsuda and Arends11

on hydroxyapatite (HAp) single crystals and on enamelcrystallites. The spectral variations were checked as afunction of the angle between the c-axis and the inci-dence polarization. A 08 angle gives the Ag species, anda 458 angle, the E1g species. When the c-axis and theincidence polarization are perpendicular, the E2g speciesappear in combination with the Ag species (see Fig. 1).11

In order to strictly isolate the zx and yx polarizationdirections (respectively, E1g and E2g symmetry species),we must modify the optical arrangement (see Fig. 2).16

The microscopic observations show the orientation ofenamel prisms, which are parallel to the c-axis.2,11 Theorientation of the enamel prisms gives us a good esti-mation of the direction of the z-axis.

Human Enamel Samples. For enamel samples, sound� rst premolars extracted for orthodontic reasons from 12-

1032 Volume 56, Number 8, 2002

FIG. 2. Polarization experiments in new transverse excitation/detectionarrangement with human enamel crystallites. (A) E1g species; (B) E2g

species. LP : linear polarizer; Obj: objective lens; IF : interference � lter;Bs : beam splitter.

FIG. 3. Raman spectra of human enamel crystallites, as a function ofthe angle of the c-axis and incidence polarization, according to thearrangement of Tsuda and Arends11 and with the HeNe laser. The n1

mode is divided into 10 parts.

TABLE II. Band positions (in cm21) and symmetry assignments inRaman and IR spectroscopy for FAp and human enamel samples.

Vibrationmodes andsymmetry

species

Fluorapatite

a b c d e, f e, g

Enamel

f g

n3 PO432 Ag

E2g

Ag

E1g

E2g

10821060105410411034

10821060105410411034

10781059105110401033

10801058105110401033

10811059105310411033

10821060105410421034

107010501043n.o.

1024

10711053104610441027

n1 PO432 Ag

E2g

966966

966 963963

963965

965965

965 959959

959

n4 PO432 E2g

Ag

E1g

Ag

E2g

617605592590582

617605592590582

615606591591580

617607591591581

614606591591581

615606590590581

615607590590577

615609590590580

n2 PO432 Ag

E2g

E1g

Ag

n.m.n.m.n.m.

446440427

451446429

452445431

451445431

451447431431

453446430430

450446433433

OH2 Ag

Ag

o.s.o.s.

35733513

n1 CO322 § 1070 1071

a Ref. 5.b Ref. 7.c Ref. 18.d Ref. 1.e Ref. 16.f Results obtained with the arrangement from Tsuda and Arends, Ref.

11.g Results obtained with the new arrangement.n.o. 5 Not observed.n.m. 5 Not mentioned.o.s. 5 Out of scale.§ 5 main polarization direction 5 z(xx)z.

year-old children were used. The root was cut off with ahigh-speed dental burr under water spray. The crown wasfractured along the longitudinal axis with a surgical in-strument placed between the cusps. The enamel crystal-lites near the surface are known to be highly orientated,perpendicular to the intact outer surface axis.2,11 Then theenamel samples were oriented on the microscope slidesin transverse or normal (for zx or xy polarization direc-tion) excitation/detection arrangements, with the c-axisperpendicular to the excitation laser beam, as describedby Tsuda and Arends.11

RESULTS

Figure 3 presents Raman spectra of enamel samples,obtained as a function of the angle between the c-axisand the incidence polarization, according to the arrange-ment of Tsuda and Arends.11 The results are similar tothose obtained by Tsuda and Arends and are listed inTable II (column 1).

Spectra obtained with the new arrangement are pre-sented in Fig. 4. The OH2 stretching bands were ob-served at 3573 and 3513 cm21. The main bands due toPO 4

32 ions and the n1 CO3 Type B seen at 1071 cm21

were detected.17 A single band made up the n1 mode ofPO 4

32 ions at 959 cm21, which corresponds to the Ag

symmetry species. In the n2 mode, four bands can benoted: 433 (A g), 433 (E1g), 446 (E2g), and 450 (A g) cm21.The n3 mode presented � ve bands with an approximate10 cm21 shift from FAp: 1027 (E2g), 1044 (E1g), 1046(A g), 1053 (E2g), and 1071 (Ag) cm21. Five bands consti-tuted the n4 mode: 580 (E2g), 590 (A g), 590 (E1g), 609(A g), and 615 (E2g) cm21. All the assignments are detailedin Table II (column 2).

DISCUSSION

The n1 mode was expected to be two bands attributedto the Ag and E2g symmetry-species, but we observed asingle band at 959 cm21 (see Table II). The E2g symmetry-species might exist at a very weak intensity; however, theimportance of the residual background noise did not al-low us to observe it in the n1 mode, whereas the Ag bandwas clearly detected. In the n2 mode, four bands were

observed while only three bands were predicted by grouptheory. Two of these bands were attributed to the Ag sym-metry species (450 and 433 cm21), which is incompatiblewith group theory predictions (see Tables I and II). Tsudaand Arends11 observed the same phenomenon in HApspectra and we � nd the same assignments in FAp spectra(see Table II).16 Moreover, in the n4 and the n2 modes weobserved two symmetry species, Ag and E1g, that vibratedat the same wavenumbers: 590 cm21 in the n4 mode and433 cm21 in n2. Species Ag and E1g derive from two dif-

APPLIED SPECTROSCOPY 1033

FIG. 4. Raman spectra of human enamel crystallites, with the newarrangement16 and the Ar-ion laser. (A) OH2 band (3680–3470 cm21).(B) n3 and n1 modes (1120–930 cm21). (C ) n4 and n2 modes (650–400cm21).

FIG. 5. FAp and human enamel spectra according to the polarizationdirection. (A) zz (Ag): the enamel spectrum was magni� ed 103 (*10).(B) zx (E1g) direction. (C ) xy (E2g) direction. (D ) z(xx)z polarizationdirection. The n1 mode of the FAp spectrum was divided by 10 (1/10).The n3 mode of the enamel spectrum was magni� ed 23 (*2).

ferent species (A 9 for Ag and A 0 for E1g) and statisticallyshould have two different vibration wavenumbers.

The n3 mode presented � ve bands (see Table II). Theonly particularity observed concerned the intensity ratioof these � ve bands between FAp and the enamel spectra.

Raman spectra of enamel presented the same bands asfor FAp spectra (see Fig. 5 and Table II),1,5,16,18 except forthe n1 mode of the CO3

22 ion at 1071 cm21, which ob-viously cannot be observed in FAp spectra (see Table II).The symmetry-species assignments of enamel spectrawere very similar to the FAp assignments. The main var-iation concerned the n2 mode, more precisely, the two Ag

bands observed in this mode. On enamel spectra, we ob-served that the 433 cm21 band was stronger than that at450 cm21. On FAp spectra, this intensity was completelyinverted (see Fig. 5). This surprising observation was alsomade in the HAp spectra.11 This con� guration can alsobe observed in the n3 mode, in the z(xx)z polarizationdirection, and for the E2g symmetry species.

On all the spectra, the bands were broader and weakerthan those of FAp, and some band shifts were observed

between the two compounds. They were probably due tothe difference in crystallinity between the perfectly crys-tallized FAp and the enamel, where the crystallinity pre-sented some defects and illustrated the in� uence of car-

1034 Volume 56, Number 8, 2002

FIG . 6. Schema of the PO432 environment.

bonation on apatite spectra. The major difference wasobserved in the n3 mode of PO4

32 with a 10 cm21 shiftbetween � uorapatite and enamel, although the number ofbands was preserved.

A previous study17 showed that a low level of carbon-ate substitution induced large modi� cations in HAp spec-tra. Increasing the substitution ratios did not bring aboutany further alterations to the spectra. Other studies19,20 onmagmatic carbonated FAp crystals, with between 0 and7% weight CO3

22, showed spectra similar to those ofenamel. Despite the loss of symmetry due to the substi-tution of one PO 4

32 with one CO322, carbonation pre-

serves the P63 /m space group of the enamel crystallites.The loss of OH2 ions observed upon carbonation permitsa new ionic distribution, especially along the c-axis, andgives an ionic rearrangement with lower lattice strength.

Despite these discrepancies, the internal modes ofPO 4

32 ions remain quite stable upon apatite substitution.Only the n1 and n3 modes are slightly affected. This phe-nomenon could be explained by the PO4

32 short-rangeenvironment, exclusively composed of nine Ca 21 ions,four CaI

21 and � ve CaII21 (see Fig. 6), surrounding the

PO 432 ion, and where each O atom is correlated with three

of these Ca21 ions. This box isolates one PO 432 ion from

the other PO432 ions of the apatite crystal and so decreas-

es the in� uence of the disturbances. The substitution ofa PO 4

32 with a CO322 implies the loss of a Ca 21 and an

OH2 ion, which means that the PO432 short-range envi-

ronment is now composed of eight Ca 21 ions. The struc-ture of the box surrounding the PO4

32 ions does not un-dergo any major change, which is observed on the Ramanspectra.

In the present work, we have studied all the polariza-

tion directions of Raman spectra of enamel to obtain abetter understanding of the in� uence of carbonation onbiominerals. The full study of the fundamental vibrationsof enamel was compared to the FAp spectra. The enameland the FAp presented certain differences between thepredictions of group theory and the P63 /m space groupbased on diffraction data. The structural differences be-tween FAp and enamel crystallites, due to the substitutionof some PO 4

32 ions with CO322, did not cause any major

disturbance of the spectra, supporting the notion that thePO 4

32 ions are isolated from each other by a box of Ca 21

ions, which should decrease the in� uence of all the sub-stituants on the PO4

32 ion spectra.

ACKNOWLEDGMENTS

These studies were carried out at the ‘‘Federation des Biomateriaux’’with the � nancial support of the ‘‘Region Nord-Pas-de-Calais’’, the EU,and the MENRT.

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