Basic Science Caries Res 1992;26:77-83
J.H. Meumtanac J.C. Voegelc R. Rauhamaa-Makinena R Gasset* J.M. Thomannc J. Hemmerlec M. Luomanenh I. PaunioH R.M. Frankc
Effects of Carbon Dioxide, Nd:YAG and Carbon Dioxide-Nd:YAG Combination Lasers at High Energy Densities on Synthetic Hydroxyapatite
Departments of 3 Cariology and b Anatomy, University of Helsinki, Finland; c Centre de Recherches Odontologiques, U-157 INSERM, Université Louis-Pasteur. Strasbourg, France
Abstract The aim of this study was to determine the crystalline structure and chemical alterations of synthetic hydroxyapatite after irradiation with either C 0 2, Nd:YAG or C 0 2-Nd:YAG combination lasers at high energy densities of 5003,230 J-cm2. Further, dissolution kinetics of the lased material were analysed and compared with those of unlased apatite. Electron microscopy showed that the lased material consisted of two kinds of crystals. From the micrographs their diameters varied from 6(X) to 1,200 A and from 3,000 to 6,000 A, respec tively. The larger crystals showed 6.9-Angstrom periodic lattice fringes in the transmission electron microscope. a-Tricalcium phosphate (TCP) was identi fied by X-ray diffraction. Selective-area electron diffraction identified the large crystals to consist of tricalcium phosphate while the smaller crystals were probably hydroxyapatite. Assays of dissolution kinetics showed that at these high energy densities lased material dissolved more rapidly than unlased syn thetic hydroxyapatite due to the higher solubility of TCP.
Studies of the effects of laser irradiation on the dental hard tissues have shown that the solubility of the enamel surface can be affected by lasing. Many studies indicate that lasing improves the enamel surface and diminishes its liability to acid dissolution [Stern and Sognnaes, 1965, 1972; Stern et al., 1966, 1972; Yamamoto and Ooya, 1974; Yamamoto and Sato, 1980; Sato, 1983; Lenz et al., 1982; Nelson et al., 1986a, b, 1987; Featherstone and Nelson,
Received: February 12,1991 Accepted after revision: September 13. 1991
1987], but increased enamel permeability after lasing of bovine enamel has also been reported [Borggreven et al., 1980]. Lased enamel has been shown to be softer than un lased one, and the laser effect seems to depend, among other things, on the structure and orientation of enamel prisms [Ferreira et al., 1989], Recently, Oho and Morioka [1990] reported that lasing of enamel with an argon laser of 67 J-cm*2 resulted in microspaces which may entrap the
J.H. Mcurman Department of Cariology. University of Helsinki Mannerhcimintie 172 SF-00300 Helsinki (Finland)
© 1992S.Kargcr AG. Basel 0008-6568/92'0262-4X)77 $2.75/0
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Key Words Laser Hydroxyapatite Tricalcium phosphate
Materials and Methods Synthetic HA Synthetic HA (Biogcl HTP, Bio-Rad Laboratories, Richmond, USA) was used in this study. This crude powder employed contained 9.46 x lO-3 mol-g“1Ca and 5.89 x 10“3 mol-g“1PO] and had thus a Ca/P ratio of 1.61 instead of the theoretical value of 1.667 for an ideal apa tite structure and the value of about 1.65 generally found for sound human enamel [Voegel et al., 1987]. The amount of carbonate within the HA sample was approximately 0.23% [Gramain et al.. 1987]. An equilibration procedure of the powder close to saturation under Ar bubbling did not allow to reduce the amount even after 8 h. This be haviour shows that the carbonate ions are incorporated in the crystal line structure. However, the measured value is inferior to the 4.4% found for enamel powder [Voegel et al.. 1987]. The specific surface area 45 m2-g“' of the unlased sample was determined by N, ad
78
sorption (BET method). A very inprecise specific surface area of 0-0.1 n r • g“1was found with the aid of the same BET method for the lased apatite sample. However since this technique is not appropriate for small surface area determination, the lased sample was subse quently assimilated to spherical heeds. A rough geometrical estima tion led then to a value of 1.14 x 1()~2 m2-g“' for the lased sample. Laser Irradiation The laser device used in the experiments was a multiwavelength laser model 5050 Combo Nd:YAG/CO, laser (Lascrmatic Ltd., Hel sinki, Finland). The beam was focused in a 125-mm handpiece, and the diameter of the spot was 4.7 mm. Aliquots of 20 mg of HA were taken and lased for 20 s by using continuous wave mode on CO, wavelength, CO,-Nd:YAG irradiation wavelengths and plain Nd:YAG irradiation wavelength at the follow ing energy levels: 500,1.000 and 3,230 J-cm ':. The combination laser used 50% CO, irradiation with 50% Nd:YAG irradiation. The apatite powder samples were placed on specimen holders made either of steel or ceramic tile and all the experiments were made in triplicate. Material exposed to 3,230 J -cm“2 irradiation was used for the crystal lographic analyses and assessment of dissolution kinetics. Unlased apatite was used for reference. Scanning and Transmission Electron Microscopy SEM observation of lased and unlased samples was realized with a JEOL 820 scanning microscope operating at 6 kV. Samples were previously covered with a 300-Ângstrôm layer of Au-Pd in a Hummer Junior ion-sputtering device (Siemens AG, Karlsruhe, FRG). For TEM, apatite samples were embedded in Epon 812 and thin sections were prepared with a diamond knife. Sections were floated during sectioning on water (pH » 7.0) saturated with respect to HA (H ,0 equilibrated during at least 2 weeks with the crude HA powder in the presence of 1 mM NaN,). Grids were studied without staining in a JEOL 100B transmission electron microscope operating at 100 kV ac celerating voltage. Electron Diffraction Analysis Selective-area electron diffraction was performed on non-stained sections at 100 kV with a JEO L 100B electron microscope using an in termediary diaphragm of 10 or 20 pm and an anticontamination de vice. The diffracted area under these conditions had a surface of ap proximately 0.1-0.2 g nr. Lithium fluoride was used as control in the same sample holder. Representative crystal areas were studied and photographed. Measurements were then carried out on the photo graphs with the aid of a precision ruler (Siemens). Both unlased and lased materials were studied and the results were compared with the JCPDS reference files 9-432 for hydroxyapatite, 29-359 for a-TCP and 9-169 for |i-TCP [Joint Committee of Powdered Diffraction So ciety, 1989]. These crystalline forms were considered since apatite heating in the presence of COy. HPOy and/or P leads mainly to the formation of these compounds [Fowler and Kuroda, 1986: Apfelbaum et al., 1990], X-Ray Diffraction Analysis Lased apatite samples were pulverized in a Fritsch Pulverisette machine (FRG) and analysed with the aid of a computerized Siemens X-ray Diffractometer D-500 with a Diffrac-Socabim (France) pro gram. The diffraction diagrams were then compared with the JCPDS data bank files (see above). The crude unlased HA has been previ ously shown to possess HA structure [Hemmerle, 1990],
Meurman/Voegel/Rauhamaa-Makinen/ Gasscr/Thomann/Hemmerle/Luomanen/ Paunio/Frank
Effects of Lasers on Hydroxyapatite
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ions released by acid dissolution. This would then explain why enamel lased at a low energy density may become more resistant to acid dissolution than unlased enamel. High-energy lasing (1,000-1(3,000 J-c n r2) causes melt ing of enamel apatite with subsequent formation of mod ified oxyhydroxyapatitc and a-tricalcium phosphate (aTCP), p-tricalcium phosphate (|TTCP) and tetracalcium phosphate [Kuroda and Fowler, 1984; Fowler and Kuroda, 1986], However, it is now widely accepted that heating or pyrolysis of synthetic hydroxyapatite (HA) or tooth enam el causes essentially the formation of a-TCP and [1-TCP [Fowler and Kuroda, 1986; Apfclbaum et al., 1990], These changes were shown either to decrease or increase the sol ubility depending on the Ca/P ratio in the formed miner als. Thus, the used energy density in the laser irradiation may modify the effects on enamel. Low-energy lasing, in particular, has been shown to increase the acid resistance of enamel while laser treatment with a high energy density may decrease it [Fowler and Kuroda, 1986], Lasing of enamel seems to cause an increase in its acid resistance also by enhancing the fluoride uptake in enamel [Tagomori and Morioka, 1989]. The aim of the present study was to analyse the effects of different irradiation processes with C 0 2, Nd:YAG or COr Nd:YAG combination lasers onto crystalline struc ture alteration of synthetic HA. Only high energy densities were used in order to maximize the anticipated crystalline transformation in the apatite. We chose the synthetic HA that is a well-defined compound particularly studied in our laboratory. This apatite was previously found to be a perfect model for describing the aspects of dissolution of synthetic and/or biological apatites such as dental enamel [Thomann et al., 1990a, b, 1991], The effect on apatite of the C 0 2-Nd:YAG combination laser has not been previ ously studied.
Dissolution Experiments The dissolution kinetics at a stirring speed of 1.860 rpm of the lased and unlased apatite samples were analysed by using an auto matic equipment where the proton consumption and calcium release kinetics were continuously followed at constant pH at 37±0.1°C ¡Thomann et al.. 1990a], The equipment comprises a double-walled thermostated reactor of 100 ml capacity (M ettler type 23517), an au toburette (M ettler type DV405), a complete stirring system (Mettler type 23830), a complete titration unit (Mettler type DV702), a ther mostat with ±1°C temperature regulation (Haakc type 000-9601), two titrator units (Mettler type DL21), a combined pH electrode (Mettler type DG111) and a calcium-selective electrode (Orion Research type 93-20). The whole set-up is interfaced with a microcomputer (Victor type V286). Amounts of about 1(H) mg of CO,-Nd:YAG-irradiated materials, 10 mg of plain YAG-irradiatcd material and 10 mg crude unlased HA powder were dissolved in 50 ml of solution. Due to the large specific surface area differences of the samples and in order to detect both proton uptake and calcium release kinetics against time using specific electrodes, 10 times more lased HA than unlased HA were used in the dissolution studies. The dissolution solution con tained initially 1 x HD1M C aC k 5 x 10"4M KH.PO, and 8 x ItU M KCI ensuring a constant ionic strength during the whole dissolution proc ess.
Results Morphological Alterations o f Apatite after Lasing
Fig. 1. Crude unlased HA showing plate let-shaped particles (SEM observation), x 260. Fig. 2. Higher magnification of a crude HA platelet observed by TEM. The particle is composed of the agglomeration of tiny crystallites having a mean diameter of 400 A. x 40.000.
tice planes of a-TCP with an equidistance of 7.31 or 6.85 A, respectively; or to (102) lattice planes of (1-TCP (table 1). This latter compound could be discarded, however, by Xray pattern analysis and selective-electron diffraction study (see below). X-Ray Diffraction a-TCP only was identified in the COr Nd:YAG lased ma terial by X-ray diffraction (table 1). The contribution of any other material, such as unalterated HA, was too small to have an effect on the X-ray diffraction pattern.
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Untransformed HA was composed by platelet-shaped particles of 2.6 ±0.9 pm thickness (fig. 1). At highermagnification (fig. 2) a platelet appeared as composed by many tiny crystallites having an average mean radius of about 400 A [Hemmerle, 1990]. In apatite samples lased with C 0 2 irradiation, melting was observed to begin at 500 J-c n r2 energy level among samples put onto the metallic specimen holder, while 1.000 J-cn r2 was needed for melting of the apatite on the ce ramic tile specimen holder. The effect of the combination COr Nd:YAG laser on the melting of apatite appeared to depend on the energy level of C 0 2 irradiation in the com bination used. In these samples the effect was the same as observed with C 0 2 irradiation alone. NdrYAG laser irra diation alone did not cause melting of the apatite in this study setting. Laser irradiation exceeding 500 J-cm-2 (with COr Nd:YAG laser) transformed the apatite powder into pearl-shaped formations at the focus site (fig. 3). Higher magnification revealed an irregular surface with deep pits and grooves (fig. 4). Thin sections of the lased material showed an arrangement of bigger crystals with a diameter of approximately 3,000-6,000 A adjacent to smaller 600- to 1,200-Angstrom crystals (fig. 5). High-resolution images of the big crystals revealed striations of approximately 6.9 A (fig. 6) corresponding most probably to (031) or (130) lat-
Electron Diffraction
Selective-area electron diffraction also revealed TCP at the bigger, approximately 3,000- to 6,000-Angstrom crystals (fig. 7) while the smaller, approximately 600- to 1,200-Angstrom crystals gave a diffraction pattern more close to HA (fig. 8). Although the smaller crystallites gave rise to Debye-Scherrer diagrams, they represent as de duced from TEM observations, only a few percent of the total amount of available crystals. Their identification was uncertain because the obtained reflections (not given) led to dhki values in the 1.5- to 3-Angstrom domain; this point together with the lower precision connected to DebyeScherrer rings did not allow us to define precisely the com-
Fig. 3. Low-magnification SEM image of a bead formed of syn thetic HA by CO:-Nd:YAG irradiation with 14 W +14 W for 20 s at 7()-mm focus, x 24. Fig. 4. Detail of the surface of the bead in figure 3. x 7,200. Fig. 5. TEM of CO;-Nd:YAG-lascd material showing the ar rangement of small crystals (SC) adjacent to a part of a large crystal (BC). x 150,000. Fig. 6. Periodic lattice fringes visualized at a high magnification by TEM. Equidistances shown are approximately 6.9 Â. x 1,500,000. Fig. 7. Electron diffraction pattern from the large crystal seen in figure 5. A reflection corresponds to (031) and B reflection to (261) of a-TCP. Fig. 8. Polycrystalline electron diffraction pattern obtained from small crystals in figure 5.
Table 1. Most important lattice equidistances and relative intensities with respect to (034) reflection deduced from X-ray diffraction pattern for a C 0 2-Nd:YAG-lased sample in comparison to theoretical values for HA file number 9-432, monoclinic form of a-TCP file number 29-359 and (1-TCP file number 9-169 of the Joint Committee of Powdered Diffraction Society [1989] Monoclinic a-TCP
d À
measured intensity
d A
theoretical intensity
hkl
—
_
7.35 6.33 5.86 5.19 4.00 3.90 3.88 3.69 3.01 2.94 2.91 2.86 2.63 2.60
39 23 19 17 46 52 52 52 26 21 KM) 46 83 58
12.28 7.31 6.31 5.84 5.18 4.01 3.90 3.88 3.69 3.01 2.94 2.91 2.86 2.62 2.60
001 031 201 132 131 201 162 132 261 361 191 034 335 290 400
-
-
-
7 39 15 11 14 24 41 48 33 17 20 1IM) 24 43 29 -
80
HA
-
d A
p-TCP theoretical intensity
8.17
11
-
-
hkl
KM) -
d A
theoretical intensity
8.15
11
-
-
hkl
102 -
-
-
-
6.49
-
-
-
-
-
-
-
-
-
-
-
-
4.07
9
200
-
-
-
5.21 4.06
20 15
110 204
3.88 3.44 3.17 3.08 2.81 2.78 2.72 2.63 2.26
9 KM) 11 17 KM) 60 60 25 30
Ill 211 102 210 211 112 300 202 310
Meurman/Voegcl/Rauhamaa-Makinen/ Gasser/Thomann/Hemmerle/Luomanen/ Paunio/Frank
104
15
-
-
-
-
-
-
3.45 3.21 3.01 2.88 2.76 2.61 2.52
25 55 15 KM) 20 65 11
1.0.10 214 3(M) 2.0.10 218 220 2.1.10
Effects of Lasers on Hydroxyapatite
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Lased sample
pound present in accordance to table 1. However, the ab sence of strong (111) or (031) reflection of a-TCP at 7.31 Â as well as the (104) reflection at 6.49 Â suggest them to be rather HA than a-TCP or (f-TCP. Dissolution Kinetics o f Apatite after Lasing
Discussion It is most probable that during the lasing process the HA was heated over 1,100 °C and in this condition it was transformed to the a form of TCP because the temper ature range of 500-1,000 °C induces the formation of |5-TCP or whitlockite [Sakae, 1988]. It must be emphasized that we deliberately used a high energy level that caused melting in order to observe what happens to apatite crys tals under these extreme conditions. TCP is known to be more soluble than HA [Fowler and Kuroda, 1986]. Also solubility studies and estimation of the ionic activities of calcium phosphate salts after attain ing thermodynamic equilibrium have shown that HA is the less soluble calcium phosphate salt until pH 4.5, but for lower pH values CaHPO^ • 2H20 becomes less soluble [Nancollas, 1982], We have shown earlier that HA kinetic rates could be estimated fairly well in the 1- to 3-min time intervals in our HA dissolution studies [Thomann et al., 1990a, b, 1991]. This appeared to hold true also to the lased samples because the linearity was even better due to the very low and weak variation of bead surface with time. We have also previously shown that HA dissolution is a diffu sion controlled process through two adjacent layers, first a surface layer adherent to the solid interface, second the Ncrnst layer [Thomann et al., 1990a, b, 1991], It appears that the dissolution mechanism of the C 0 2Nd:YAG-lascd sample is comparable to that of HA if one assumes that the acting interface is saturated with respect to a-TCP and that in a first approach the dissolution is strongly self-inhibited. This self-inhibition was described previously [Thomann et al., 1990a, b, 1991] and is valid for
time (min.)
Fig. 9. Dissolution experiments performed at pH 3.7 under pHstat conditions. H* uptake and Ca2+ release kinetics versus time for (A) 10 mg of crude unlased HA and (B) approximately 1(H) mg of C 0 2Nd:YAG-lased material. Values are expressed in moles per square metre and they correspond to the amount of H + taken up or Ca2+ re leased by 1 m2 of material.
apaties of different origin. It is based mainly on the fact that the measured dissolution rates were 2 or 3 orders of magnitude below a pure diffusive process governed by ion transport through Nernst layer. Thus a model was pro posed in which kinetics are controlled by surface layer dif fusion and bulk diffusion [Thomann et al., 1990a, b, 1991]. Under such conditions proton fluxes J H+ could be ana lysed by: - V = R-Pc,.«l/(l + k)-(as-a)
in which R represents the number of protons necessary for the release of 1 Ca, PCa» is the mass transfer coefficient in the absence of chemical reaction, as is the calcium activity at the solid interface, a is the calcium activity in the bulk solution, k = Pca°/Pca defines the reduced permeability of the surface layer with respect to the permeability in the Nernst layer, and PCa is the mass transfer coefficient in the adherent layer. The existence of a positively charged membrane adher ent to the solid surface allows to explain the strong inhibi tion (high k value) observed experimentally. The presence of a positively charged interfacial layer containing strongly adsorbed calcium ions makes that layer to act as a perm selective membrane. In consequence, the transport of Ca2+ through the positively charged membrane is retarded and becomes the rate-limiting factor. The k values for C 0 2-Nd:YAG-lased HA were comparable to those for un-
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Beads caused by the 3,230-J -cm-2 irradiation with C 0 2Nd:YAG irradiation and powder received after respective lasing with Nd:YAG laser were used to compare the dis solution kinetics after lasing with that of unlased Bio-Rad apatite. The results showed that the Nd:YAG lasing alone had no effect on the Bio-Rad apatide. Proton uptake ki netics for C 0 2-Nd:YAG-lased sample and crude powder are shown in figure 9. The results indicate that the C 0 2Nd:YAG-lased material dissolved more rapidly than un lased HA samples.
lased HA. However, the higher solubility of a-TCP is caused by the (as-a) gradient of the pure Nernst layer. On the other hand, no difference was observed in the dissolu tion kinetics between unlascd crude HA and Nd:YAGlased material, which indicates that neither the specific surface area nor the chemical composition were altered by irradiation with only Nd:YAG laser. Since Stern and Sognnaes [1965] showed in their pio neering work that laser irradiation of enamel with a ruby laser increased its acid resistance, many attempts have been made to apply lasers in preventive dentistry. C 0 2 la ser irradiation, for example, has been suggested for sealing of enamel defects [Lenz et al., 1982], and Nd:YAG laser was found to be effective in inhibiting the formation of an in vitro incipient caries-like lesion [Yamamoto and Sato, 1980], The combination C 0 2-Nd:YAG laser that we used in the present study has not been studied with respect to its effect on dental tissues. We thought it necessary, however, first to obtain basic knowledge of the effects of this combi nation laser on apatite crystalline structure before further application. Our findings suggest that the HA-TCP trans formation observed was mainly due to the C 0 2 laser ir radiation since Nd:YAG alone did not cause any mea surable changes. Further, the study by Tagomori and Morioka [1989] has shown that the use of black stain on enamel greatly enhances Nd:YAG laser absorption in it. Because we used synthetic apatite no staining effort was made here. We have data in our laboratory showing that the combination of Nd:YAG irradiation to C 0 2 lasing strengthens its capacity to penetrate into enamel of ex tracted teeth even without staining (in preparation). Our findings are in agreement with those of Nelson et al. [1987], who studied surface alterations in human ena
mel after C 0 2 laser irradiation. At much lower energy lev els than what we used, they found melting and formation of tctracalcium diphosphate monoxide together with an apatite phase restricted to 10-20 urn in the surface enamel. In their study, however, the formation of artificial dissolu tion lesions was found to be inhibited after lasing, but only 50-J •cm-2 energy density was used in the laser device [Nel son et al., 1987] compared with the 500-3,230 J •cm-2 in that of the present study. It is evident that the transformation into TCP that we observed in synthetic HA after lasing is not beneficial if the same phenomenon takes place in dental enamel. Sub sequently, further studies are necessary. Because enamel is mainly composed of apatite crystallites, it may be better to evaluate in a first approach the effect of lower energy densities (also with different kinds of laser irradiations) on apatite before further surveys. At least the preservation of HA during lasing should be attempted with respect to the dissolving properties. Another interesting approach would be the incorporation of fluoride in the laser-ir radiated samples to increase their resistance against acid dissolution as was shown by Tagomori and Morioka [1989]. In this respect it would be interesting to attempt the for mation of fluoroapatites with reduced surface area by las ing.
Acknowledgements During this study the author J.H. Mcurman was a recipient of a research exchange grant from the Academy of Finland and Centre National de la Recherche Scientifique, France. Mme. J. Boissicr is thanked for her help with the X-ray diffraction analyses at the Ecole Européenne des Hautes Etudes des Industries Chimiques de Stras bourg, France.
Apfelbaum F, Mayer I, Feathcrstone JDB: The role of HPO; and CO) ions in the transformation of synthetic apatites to p-Ca3(P 0 4)2. J Inorg Biochem 1990;38:1-8. Borggreven JMPM, Van Dijk JWE, Driessens FCM: Effect of laser irradiation on the perme ability of bovine dental enamel. Arch Oral Biol 1980;25:831-832. Feathcrstone JDB, Nelson DGA: Laser effect on dental hard tissues. Adv Dent Res 1987; 1: 21-26. Ferreira JM, Palamara J. Phakcy PP. Rachinger WA. Orams HJ: Effects of continuous-wave COj laser on the ultrastructure of human den tal enamel. Arch Oral Biol 1989;34:551-562.
Fowler BO, Kuroda S: Changes in heated and in la ser irradiated human tooth enamel and their probable effects on solubility. Calcif Tissue Int 1986;38:197-208. Gramain P. Voegel JC. Gumpper M.Thomann JM: Surface properties and equilibrium kinetics of hydroxyapatite powder near the solubility equi librium. J Colloid Interface Sci 1987;118:148157. Hemmerle J: Etude ultrastructurale comparative de la biocompatibilité de différentes biocéra miques implantées dans le parodontc humain; DRS thesis, Strasbourg. 1990.
Joint Committee of Powdered Diffraction Society: International Centre for Diffraction Data. Swarthmore, 1989. Kuroda S. Fowler BO: Compositional, structural and phase changes in vitro laser irradiated hu man tooth enamel. Calcif Tissue Int 1984:36: 361-369. Lenz P, Gilde H. Walz R: Untersuchungen zur Schmelzversiegelung mit dem C 0 2-Laser. Dtsch Zahnärztl Z 1982;37:469-478. Nancollas GH: Phase transformation during pre cipitation of calcium salts; in Nancollas GH (ed): Biological Mineralization and Deminer alization. Dahlem Konferenzen. Berlin, Sprin ger, 1982, pp 79-99.
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References
Stern RH. Sognnaes RF: Laser effect on dental hard tissues. A preliminary report. J S Calcif Dent Assoc 1965:33:17-19. Stern RH, Sognnaes RF: Laser inhibition of dental caries suggested by first tests in vivo. J Am Dent Assoc 1972:85:1087-1090. Stem RH. Sognnaes RF. Goodman F: Iriser effect on in vitro enamel permeability and solubility. J Am Dent Assoc 1966:73:838-843. Stern RH. Vahl J, Sognnaes RF: Lased enamel: Ul trastructural observations of pulsed carbon dioxide laser effects. J Dent Res 1972:51:455460. Tagomori S, Morioka T: Combined effects of laser and fluoride on acid resistance of human den tal enamel. Caries Res 1989;23:225-231. Thomann JM. Gasser P. Bres EF. Voegel JC. Gramain P: Development of an automated experi mental set-up for the study of ionic exchange kinetics. Application to the ionic adsorption, equilibrium attainment and dissolution of apa tite compounds. Comp Meth Progr Biomed 1990a ;31:89-95.
Thomann JM. Voegel JC. Gramain P: Kinetics of dissolution of calcium hydroxyapatite powder. III. pH and sample conditioning effects. Calcif Tissue Int 1990b;46:121-129. Thomann JM. Voegel JC. Gramain P: Kinetics of dissolution of calcium hydroxyapatite powder. IV. intcrfacial calcium diffusion controlled process. Colloids Surface 1991;54:145-149. Voegel JC. Gramain P. Gumpper M. Thomann JM: Ionic adsorption properties and equilibration kinetics of biological enamel powder near ther modynamic equilibrium. J Cryst Growth 1987; 83:89-95. Yamamo’.o Y. Ooya K: Potential of yttrium-alu minium-garnet laser in caries prevention. J Oral Pathol 1974;3:7-15. Yamamoto Y, Sato K: Prevention of dental caries by Nd:YAG laser irradiation. J Dent Res 1980;59:2171-2177.
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Nelson DGA, Jongeblocd Wl.. Featherstone JDB: Laser irradiation of human dental enamel and dentine. NZ Dent J 1986a;82:74-77. Nelson DGA. Shariati M, Glena R. Featherstone JDB: Effect of pulsed low-energy infrared laser irradiation on artificial caries-like lesion for mation. Caries Res 1986b:20:289-299. Nelson DGA. Wefel JS, Jongeblocd WL. Feather stone JDB: Morphology, histology and crystal lography of human dental enamel treated with pulsed low-energy infrared laser radiation. Caries Res 1987:21:411—426. Oho T. Morioka T: A possible mechanism of ac quired acid resistance of human dental enamel by laser irradiation. Caries Res 1990:24:86-92. Sakae T: X-ray diffraction and thermal studies of crystals from the outer and inner layers of hu man dental enamel. Arch Oral Biol 1988:33: 707-713. Sato K: Relation between acid dissolution and his tological alteration of heated tooth enamel. Caries Res 1983:17:490-495.
J.H. Meumtanac J.C. Voegelc R. Rauhamaa-Makinena R Gasset* J.M. Thomannc J. Hemmerlec M. Luomanenh I. PaunioH R.M. Frankc
Effects of Carbon Dioxide, Nd:YAG and Carbon Dioxide-Nd:YAG Combination Lasers at High Energy Densities on Synthetic Hydroxyapatite
Departments of 3 Cariology and b Anatomy, University of Helsinki, Finland; c Centre de Recherches Odontologiques, U-157 INSERM, Université Louis-Pasteur. Strasbourg, France
Abstract The aim of this study was to determine the crystalline structure and chemical alterations of synthetic hydroxyapatite after irradiation with either C 0 2, Nd:YAG or C 0 2-Nd:YAG combination lasers at high energy densities of 5003,230 J-cm2. Further, dissolution kinetics of the lased material were analysed and compared with those of unlased apatite. Electron microscopy showed that the lased material consisted of two kinds of crystals. From the micrographs their diameters varied from 6(X) to 1,200 A and from 3,000 to 6,000 A, respec tively. The larger crystals showed 6.9-Angstrom periodic lattice fringes in the transmission electron microscope. a-Tricalcium phosphate (TCP) was identi fied by X-ray diffraction. Selective-area electron diffraction identified the large crystals to consist of tricalcium phosphate while the smaller crystals were probably hydroxyapatite. Assays of dissolution kinetics showed that at these high energy densities lased material dissolved more rapidly than unlased syn thetic hydroxyapatite due to the higher solubility of TCP.
Studies of the effects of laser irradiation on the dental hard tissues have shown that the solubility of the enamel surface can be affected by lasing. Many studies indicate that lasing improves the enamel surface and diminishes its liability to acid dissolution [Stern and Sognnaes, 1965, 1972; Stern et al., 1966, 1972; Yamamoto and Ooya, 1974; Yamamoto and Sato, 1980; Sato, 1983; Lenz et al., 1982; Nelson et al., 1986a, b, 1987; Featherstone and Nelson,
Received: February 12,1991 Accepted after revision: September 13. 1991
1987], but increased enamel permeability after lasing of bovine enamel has also been reported [Borggreven et al., 1980]. Lased enamel has been shown to be softer than un lased one, and the laser effect seems to depend, among other things, on the structure and orientation of enamel prisms [Ferreira et al., 1989], Recently, Oho and Morioka [1990] reported that lasing of enamel with an argon laser of 67 J-cm*2 resulted in microspaces which may entrap the
J.H. Mcurman Department of Cariology. University of Helsinki Mannerhcimintie 172 SF-00300 Helsinki (Finland)
© 1992S.Kargcr AG. Basel 0008-6568/92'0262-4X)77 $2.75/0
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Key Words Laser Hydroxyapatite Tricalcium phosphate
Materials and Methods Synthetic HA Synthetic HA (Biogcl HTP, Bio-Rad Laboratories, Richmond, USA) was used in this study. This crude powder employed contained 9.46 x lO-3 mol-g“1Ca and 5.89 x 10“3 mol-g“1PO] and had thus a Ca/P ratio of 1.61 instead of the theoretical value of 1.667 for an ideal apa tite structure and the value of about 1.65 generally found for sound human enamel [Voegel et al., 1987]. The amount of carbonate within the HA sample was approximately 0.23% [Gramain et al.. 1987]. An equilibration procedure of the powder close to saturation under Ar bubbling did not allow to reduce the amount even after 8 h. This be haviour shows that the carbonate ions are incorporated in the crystal line structure. However, the measured value is inferior to the 4.4% found for enamel powder [Voegel et al.. 1987]. The specific surface area 45 m2-g“' of the unlased sample was determined by N, ad
78
sorption (BET method). A very inprecise specific surface area of 0-0.1 n r • g“1was found with the aid of the same BET method for the lased apatite sample. However since this technique is not appropriate for small surface area determination, the lased sample was subse quently assimilated to spherical heeds. A rough geometrical estima tion led then to a value of 1.14 x 1()~2 m2-g“' for the lased sample. Laser Irradiation The laser device used in the experiments was a multiwavelength laser model 5050 Combo Nd:YAG/CO, laser (Lascrmatic Ltd., Hel sinki, Finland). The beam was focused in a 125-mm handpiece, and the diameter of the spot was 4.7 mm. Aliquots of 20 mg of HA were taken and lased for 20 s by using continuous wave mode on CO, wavelength, CO,-Nd:YAG irradiation wavelengths and plain Nd:YAG irradiation wavelength at the follow ing energy levels: 500,1.000 and 3,230 J-cm ':. The combination laser used 50% CO, irradiation with 50% Nd:YAG irradiation. The apatite powder samples were placed on specimen holders made either of steel or ceramic tile and all the experiments were made in triplicate. Material exposed to 3,230 J -cm“2 irradiation was used for the crystal lographic analyses and assessment of dissolution kinetics. Unlased apatite was used for reference. Scanning and Transmission Electron Microscopy SEM observation of lased and unlased samples was realized with a JEOL 820 scanning microscope operating at 6 kV. Samples were previously covered with a 300-Ângstrôm layer of Au-Pd in a Hummer Junior ion-sputtering device (Siemens AG, Karlsruhe, FRG). For TEM, apatite samples were embedded in Epon 812 and thin sections were prepared with a diamond knife. Sections were floated during sectioning on water (pH » 7.0) saturated with respect to HA (H ,0 equilibrated during at least 2 weeks with the crude HA powder in the presence of 1 mM NaN,). Grids were studied without staining in a JEOL 100B transmission electron microscope operating at 100 kV ac celerating voltage. Electron Diffraction Analysis Selective-area electron diffraction was performed on non-stained sections at 100 kV with a JEO L 100B electron microscope using an in termediary diaphragm of 10 or 20 pm and an anticontamination de vice. The diffracted area under these conditions had a surface of ap proximately 0.1-0.2 g nr. Lithium fluoride was used as control in the same sample holder. Representative crystal areas were studied and photographed. Measurements were then carried out on the photo graphs with the aid of a precision ruler (Siemens). Both unlased and lased materials were studied and the results were compared with the JCPDS reference files 9-432 for hydroxyapatite, 29-359 for a-TCP and 9-169 for |i-TCP [Joint Committee of Powdered Diffraction So ciety, 1989]. These crystalline forms were considered since apatite heating in the presence of COy. HPOy and/or P leads mainly to the formation of these compounds [Fowler and Kuroda, 1986: Apfelbaum et al., 1990], X-Ray Diffraction Analysis Lased apatite samples were pulverized in a Fritsch Pulverisette machine (FRG) and analysed with the aid of a computerized Siemens X-ray Diffractometer D-500 with a Diffrac-Socabim (France) pro gram. The diffraction diagrams were then compared with the JCPDS data bank files (see above). The crude unlased HA has been previ ously shown to possess HA structure [Hemmerle, 1990],
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ions released by acid dissolution. This would then explain why enamel lased at a low energy density may become more resistant to acid dissolution than unlased enamel. High-energy lasing (1,000-1(3,000 J-c n r2) causes melt ing of enamel apatite with subsequent formation of mod ified oxyhydroxyapatitc and a-tricalcium phosphate (aTCP), p-tricalcium phosphate (|TTCP) and tetracalcium phosphate [Kuroda and Fowler, 1984; Fowler and Kuroda, 1986], However, it is now widely accepted that heating or pyrolysis of synthetic hydroxyapatite (HA) or tooth enam el causes essentially the formation of a-TCP and [1-TCP [Fowler and Kuroda, 1986; Apfclbaum et al., 1990], These changes were shown either to decrease or increase the sol ubility depending on the Ca/P ratio in the formed miner als. Thus, the used energy density in the laser irradiation may modify the effects on enamel. Low-energy lasing, in particular, has been shown to increase the acid resistance of enamel while laser treatment with a high energy density may decrease it [Fowler and Kuroda, 1986], Lasing of enamel seems to cause an increase in its acid resistance also by enhancing the fluoride uptake in enamel [Tagomori and Morioka, 1989]. The aim of the present study was to analyse the effects of different irradiation processes with C 0 2, Nd:YAG or COr Nd:YAG combination lasers onto crystalline struc ture alteration of synthetic HA. Only high energy densities were used in order to maximize the anticipated crystalline transformation in the apatite. We chose the synthetic HA that is a well-defined compound particularly studied in our laboratory. This apatite was previously found to be a perfect model for describing the aspects of dissolution of synthetic and/or biological apatites such as dental enamel [Thomann et al., 1990a, b, 1991], The effect on apatite of the C 0 2-Nd:YAG combination laser has not been previ ously studied.
Dissolution Experiments The dissolution kinetics at a stirring speed of 1.860 rpm of the lased and unlased apatite samples were analysed by using an auto matic equipment where the proton consumption and calcium release kinetics were continuously followed at constant pH at 37±0.1°C ¡Thomann et al.. 1990a], The equipment comprises a double-walled thermostated reactor of 100 ml capacity (M ettler type 23517), an au toburette (M ettler type DV405), a complete stirring system (Mettler type 23830), a complete titration unit (Mettler type DV702), a ther mostat with ±1°C temperature regulation (Haakc type 000-9601), two titrator units (Mettler type DL21), a combined pH electrode (Mettler type DG111) and a calcium-selective electrode (Orion Research type 93-20). The whole set-up is interfaced with a microcomputer (Victor type V286). Amounts of about 1(H) mg of CO,-Nd:YAG-irradiated materials, 10 mg of plain YAG-irradiatcd material and 10 mg crude unlased HA powder were dissolved in 50 ml of solution. Due to the large specific surface area differences of the samples and in order to detect both proton uptake and calcium release kinetics against time using specific electrodes, 10 times more lased HA than unlased HA were used in the dissolution studies. The dissolution solution con tained initially 1 x HD1M C aC k 5 x 10"4M KH.PO, and 8 x ItU M KCI ensuring a constant ionic strength during the whole dissolution proc ess.
Results Morphological Alterations o f Apatite after Lasing
Fig. 1. Crude unlased HA showing plate let-shaped particles (SEM observation), x 260. Fig. 2. Higher magnification of a crude HA platelet observed by TEM. The particle is composed of the agglomeration of tiny crystallites having a mean diameter of 400 A. x 40.000.
tice planes of a-TCP with an equidistance of 7.31 or 6.85 A, respectively; or to (102) lattice planes of (1-TCP (table 1). This latter compound could be discarded, however, by Xray pattern analysis and selective-electron diffraction study (see below). X-Ray Diffraction a-TCP only was identified in the COr Nd:YAG lased ma terial by X-ray diffraction (table 1). The contribution of any other material, such as unalterated HA, was too small to have an effect on the X-ray diffraction pattern.
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Untransformed HA was composed by platelet-shaped particles of 2.6 ±0.9 pm thickness (fig. 1). At highermagnification (fig. 2) a platelet appeared as composed by many tiny crystallites having an average mean radius of about 400 A [Hemmerle, 1990]. In apatite samples lased with C 0 2 irradiation, melting was observed to begin at 500 J-c n r2 energy level among samples put onto the metallic specimen holder, while 1.000 J-cn r2 was needed for melting of the apatite on the ce ramic tile specimen holder. The effect of the combination COr Nd:YAG laser on the melting of apatite appeared to depend on the energy level of C 0 2 irradiation in the com bination used. In these samples the effect was the same as observed with C 0 2 irradiation alone. NdrYAG laser irra diation alone did not cause melting of the apatite in this study setting. Laser irradiation exceeding 500 J-cm-2 (with COr Nd:YAG laser) transformed the apatite powder into pearl-shaped formations at the focus site (fig. 3). Higher magnification revealed an irregular surface with deep pits and grooves (fig. 4). Thin sections of the lased material showed an arrangement of bigger crystals with a diameter of approximately 3,000-6,000 A adjacent to smaller 600- to 1,200-Angstrom crystals (fig. 5). High-resolution images of the big crystals revealed striations of approximately 6.9 A (fig. 6) corresponding most probably to (031) or (130) lat-
Electron Diffraction
Selective-area electron diffraction also revealed TCP at the bigger, approximately 3,000- to 6,000-Angstrom crystals (fig. 7) while the smaller, approximately 600- to 1,200-Angstrom crystals gave a diffraction pattern more close to HA (fig. 8). Although the smaller crystallites gave rise to Debye-Scherrer diagrams, they represent as de duced from TEM observations, only a few percent of the total amount of available crystals. Their identification was uncertain because the obtained reflections (not given) led to dhki values in the 1.5- to 3-Angstrom domain; this point together with the lower precision connected to DebyeScherrer rings did not allow us to define precisely the com-
Fig. 3. Low-magnification SEM image of a bead formed of syn thetic HA by CO:-Nd:YAG irradiation with 14 W +14 W for 20 s at 7()-mm focus, x 24. Fig. 4. Detail of the surface of the bead in figure 3. x 7,200. Fig. 5. TEM of CO;-Nd:YAG-lascd material showing the ar rangement of small crystals (SC) adjacent to a part of a large crystal (BC). x 150,000. Fig. 6. Periodic lattice fringes visualized at a high magnification by TEM. Equidistances shown are approximately 6.9 Â. x 1,500,000. Fig. 7. Electron diffraction pattern from the large crystal seen in figure 5. A reflection corresponds to (031) and B reflection to (261) of a-TCP. Fig. 8. Polycrystalline electron diffraction pattern obtained from small crystals in figure 5.
Table 1. Most important lattice equidistances and relative intensities with respect to (034) reflection deduced from X-ray diffraction pattern for a C 0 2-Nd:YAG-lased sample in comparison to theoretical values for HA file number 9-432, monoclinic form of a-TCP file number 29-359 and (1-TCP file number 9-169 of the Joint Committee of Powdered Diffraction Society [1989] Monoclinic a-TCP
d À
measured intensity
d A
theoretical intensity
hkl
—
_
7.35 6.33 5.86 5.19 4.00 3.90 3.88 3.69 3.01 2.94 2.91 2.86 2.63 2.60
39 23 19 17 46 52 52 52 26 21 KM) 46 83 58
12.28 7.31 6.31 5.84 5.18 4.01 3.90 3.88 3.69 3.01 2.94 2.91 2.86 2.62 2.60
001 031 201 132 131 201 162 132 261 361 191 034 335 290 400
-
-
-
7 39 15 11 14 24 41 48 33 17 20 1IM) 24 43 29 -
80
HA
-
d A
p-TCP theoretical intensity
8.17
11
-
-
hkl
KM) -
d A
theoretical intensity
8.15
11
-
-
hkl
102 -
-
-
-
6.49
-
-
-
-
-
-
-
-
-
-
-
-
4.07
9
200
-
-
-
5.21 4.06
20 15
110 204
3.88 3.44 3.17 3.08 2.81 2.78 2.72 2.63 2.26
9 KM) 11 17 KM) 60 60 25 30
Ill 211 102 210 211 112 300 202 310
Meurman/Voegcl/Rauhamaa-Makinen/ Gasser/Thomann/Hemmerle/Luomanen/ Paunio/Frank
104
15
-
-
-
-
-
-
3.45 3.21 3.01 2.88 2.76 2.61 2.52
25 55 15 KM) 20 65 11
1.0.10 214 3(M) 2.0.10 218 220 2.1.10
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Lased sample
pound present in accordance to table 1. However, the ab sence of strong (111) or (031) reflection of a-TCP at 7.31 Â as well as the (104) reflection at 6.49 Â suggest them to be rather HA than a-TCP or (f-TCP. Dissolution Kinetics o f Apatite after Lasing
Discussion It is most probable that during the lasing process the HA was heated over 1,100 °C and in this condition it was transformed to the a form of TCP because the temper ature range of 500-1,000 °C induces the formation of |5-TCP or whitlockite [Sakae, 1988]. It must be emphasized that we deliberately used a high energy level that caused melting in order to observe what happens to apatite crys tals under these extreme conditions. TCP is known to be more soluble than HA [Fowler and Kuroda, 1986]. Also solubility studies and estimation of the ionic activities of calcium phosphate salts after attain ing thermodynamic equilibrium have shown that HA is the less soluble calcium phosphate salt until pH 4.5, but for lower pH values CaHPO^ • 2H20 becomes less soluble [Nancollas, 1982], We have shown earlier that HA kinetic rates could be estimated fairly well in the 1- to 3-min time intervals in our HA dissolution studies [Thomann et al., 1990a, b, 1991]. This appeared to hold true also to the lased samples because the linearity was even better due to the very low and weak variation of bead surface with time. We have also previously shown that HA dissolution is a diffu sion controlled process through two adjacent layers, first a surface layer adherent to the solid interface, second the Ncrnst layer [Thomann et al., 1990a, b, 1991], It appears that the dissolution mechanism of the C 0 2Nd:YAG-lascd sample is comparable to that of HA if one assumes that the acting interface is saturated with respect to a-TCP and that in a first approach the dissolution is strongly self-inhibited. This self-inhibition was described previously [Thomann et al., 1990a, b, 1991] and is valid for
time (min.)
Fig. 9. Dissolution experiments performed at pH 3.7 under pHstat conditions. H* uptake and Ca2+ release kinetics versus time for (A) 10 mg of crude unlased HA and (B) approximately 1(H) mg of C 0 2Nd:YAG-lased material. Values are expressed in moles per square metre and they correspond to the amount of H + taken up or Ca2+ re leased by 1 m2 of material.
apaties of different origin. It is based mainly on the fact that the measured dissolution rates were 2 or 3 orders of magnitude below a pure diffusive process governed by ion transport through Nernst layer. Thus a model was pro posed in which kinetics are controlled by surface layer dif fusion and bulk diffusion [Thomann et al., 1990a, b, 1991]. Under such conditions proton fluxes J H+ could be ana lysed by: - V = R-Pc,.«l/(l + k)-(as-a)
in which R represents the number of protons necessary for the release of 1 Ca, PCa» is the mass transfer coefficient in the absence of chemical reaction, as is the calcium activity at the solid interface, a is the calcium activity in the bulk solution, k = Pca°/Pca defines the reduced permeability of the surface layer with respect to the permeability in the Nernst layer, and PCa is the mass transfer coefficient in the adherent layer. The existence of a positively charged membrane adher ent to the solid surface allows to explain the strong inhibi tion (high k value) observed experimentally. The presence of a positively charged interfacial layer containing strongly adsorbed calcium ions makes that layer to act as a perm selective membrane. In consequence, the transport of Ca2+ through the positively charged membrane is retarded and becomes the rate-limiting factor. The k values for C 0 2-Nd:YAG-lased HA were comparable to those for un-
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Beads caused by the 3,230-J -cm-2 irradiation with C 0 2Nd:YAG irradiation and powder received after respective lasing with Nd:YAG laser were used to compare the dis solution kinetics after lasing with that of unlased Bio-Rad apatite. The results showed that the Nd:YAG lasing alone had no effect on the Bio-Rad apatide. Proton uptake ki netics for C 0 2-Nd:YAG-lased sample and crude powder are shown in figure 9. The results indicate that the C 0 2Nd:YAG-lased material dissolved more rapidly than un lased HA samples.
lased HA. However, the higher solubility of a-TCP is caused by the (as-a) gradient of the pure Nernst layer. On the other hand, no difference was observed in the dissolu tion kinetics between unlascd crude HA and Nd:YAGlased material, which indicates that neither the specific surface area nor the chemical composition were altered by irradiation with only Nd:YAG laser. Since Stern and Sognnaes [1965] showed in their pio neering work that laser irradiation of enamel with a ruby laser increased its acid resistance, many attempts have been made to apply lasers in preventive dentistry. C 0 2 la ser irradiation, for example, has been suggested for sealing of enamel defects [Lenz et al., 1982], and Nd:YAG laser was found to be effective in inhibiting the formation of an in vitro incipient caries-like lesion [Yamamoto and Sato, 1980], The combination C 0 2-Nd:YAG laser that we used in the present study has not been studied with respect to its effect on dental tissues. We thought it necessary, however, first to obtain basic knowledge of the effects of this combi nation laser on apatite crystalline structure before further application. Our findings suggest that the HA-TCP trans formation observed was mainly due to the C 0 2 laser ir radiation since Nd:YAG alone did not cause any mea surable changes. Further, the study by Tagomori and Morioka [1989] has shown that the use of black stain on enamel greatly enhances Nd:YAG laser absorption in it. Because we used synthetic apatite no staining effort was made here. We have data in our laboratory showing that the combination of Nd:YAG irradiation to C 0 2 lasing strengthens its capacity to penetrate into enamel of ex tracted teeth even without staining (in preparation). Our findings are in agreement with those of Nelson et al. [1987], who studied surface alterations in human ena
mel after C 0 2 laser irradiation. At much lower energy lev els than what we used, they found melting and formation of tctracalcium diphosphate monoxide together with an apatite phase restricted to 10-20 urn in the surface enamel. In their study, however, the formation of artificial dissolu tion lesions was found to be inhibited after lasing, but only 50-J •cm-2 energy density was used in the laser device [Nel son et al., 1987] compared with the 500-3,230 J •cm-2 in that of the present study. It is evident that the transformation into TCP that we observed in synthetic HA after lasing is not beneficial if the same phenomenon takes place in dental enamel. Sub sequently, further studies are necessary. Because enamel is mainly composed of apatite crystallites, it may be better to evaluate in a first approach the effect of lower energy densities (also with different kinds of laser irradiations) on apatite before further surveys. At least the preservation of HA during lasing should be attempted with respect to the dissolving properties. Another interesting approach would be the incorporation of fluoride in the laser-ir radiated samples to increase their resistance against acid dissolution as was shown by Tagomori and Morioka [1989]. In this respect it would be interesting to attempt the for mation of fluoroapatites with reduced surface area by las ing.
Acknowledgements During this study the author J.H. Mcurman was a recipient of a research exchange grant from the Academy of Finland and Centre National de la Recherche Scientifique, France. Mme. J. Boissicr is thanked for her help with the X-ray diffraction analyses at the Ecole Européenne des Hautes Etudes des Industries Chimiques de Stras bourg, France.
Apfelbaum F, Mayer I, Feathcrstone JDB: The role of HPO; and CO) ions in the transformation of synthetic apatites to p-Ca3(P 0 4)2. J Inorg Biochem 1990;38:1-8. Borggreven JMPM, Van Dijk JWE, Driessens FCM: Effect of laser irradiation on the perme ability of bovine dental enamel. Arch Oral Biol 1980;25:831-832. Feathcrstone JDB, Nelson DGA: Laser effect on dental hard tissues. Adv Dent Res 1987; 1: 21-26. Ferreira JM, Palamara J. Phakcy PP. Rachinger WA. Orams HJ: Effects of continuous-wave COj laser on the ultrastructure of human den tal enamel. Arch Oral Biol 1989;34:551-562.
Fowler BO, Kuroda S: Changes in heated and in la ser irradiated human tooth enamel and their probable effects on solubility. Calcif Tissue Int 1986;38:197-208. Gramain P. Voegel JC. Gumpper M.Thomann JM: Surface properties and equilibrium kinetics of hydroxyapatite powder near the solubility equi librium. J Colloid Interface Sci 1987;118:148157. Hemmerle J: Etude ultrastructurale comparative de la biocompatibilité de différentes biocéra miques implantées dans le parodontc humain; DRS thesis, Strasbourg. 1990.
Joint Committee of Powdered Diffraction Society: International Centre for Diffraction Data. Swarthmore, 1989. Kuroda S. Fowler BO: Compositional, structural and phase changes in vitro laser irradiated hu man tooth enamel. Calcif Tissue Int 1984:36: 361-369. Lenz P, Gilde H. Walz R: Untersuchungen zur Schmelzversiegelung mit dem C 0 2-Laser. Dtsch Zahnärztl Z 1982;37:469-478. Nancollas GH: Phase transformation during pre cipitation of calcium salts; in Nancollas GH (ed): Biological Mineralization and Deminer alization. Dahlem Konferenzen. Berlin, Sprin ger, 1982, pp 79-99.
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