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The effect of fluoride treatment on bone mineral crystals in the rat
Bone, 13, 423429, (1992) Printed in the USA. All rights reserved.
Copyright
8756-3282192 $5.00 + .00 0 1992 Pergamon Press Ltd.
The Effect of Fluoride Treatment on Bone Mineral Crystals in the Rat M. D. GRYNPAS’
and C. REY*
’ Samuel Lunenfeld Research Institute of Mount Sinai Hospital and Department of Pathology, Mount Sinai Hospital and University of Toronto, Toronto, Canada ’ Luboratoire des Mate’riaux, Ecole Nationale SupCrieure de Chimie, Toulouse, France Address for correspondence and retxints: Dr. M. D. Grvnuas, Samuel Lunenfeld Research Institute, Mount Sinai Hospital. 600 University
Avenue,“Room 984, Toronto, On&o M5G 1X5 Canada. .
Abstract
dose of fluoride in their drinking water, mice accumulated six times as much fluoride in their bones as rats. Fluoride does not diffuse into already formed bones, but is incorporated during bone formation (Zipkin et al. 1952). This observation has been confirmed by x-ray microprobe analysis (Baud & Bang 1970; Bang 1978) and by density fractionation (Grynpas et al. 1986). However, earlier studies (Schraer et al. 1972) on growing rats exposed to different levels of fluoride for various lengths of time showed that changes in crystallinity did not correlate with the rate of fluoride accumulation in the bone mineral. Changes in crystallinity took place after the fluoride level had become constant. These data, as well as the data on the mineralization lag time, can be interpreted as a change in the kinetics of bone mineralization with increasing fluoride content in the systemic circulation, which means that it would take longer for fluoridated apatite bone crystals to get deposited and to mature than for the same to occur in apatite bone crystals. Fluoride can also affect putative precursor phases in the formation of bone mineral apatite. Eanes and Meyer (1978) have shown in vitro that in the case of octacalcium phosphate (OCP), which can occur as a transitional crystalline phase in apatite formation in vitro (Nancollas & Tomazic 1974), when the amount of fluoride approached 3% of the initial phosphate concentration, the presence of OCP could no longer be experimentally demonstrated. In fluoride-free preparations the apatite crystals appear as thin plates; in the high-fluoride preparations they appear as short and thin needles. The phase may be controlled in part by local conditions, particularly the amount of fluoride released by bone mineral dissolution. We appreciate that 60% of fluoride mobilized during bone resorption seems to be redeposited in the skeleton (Ming et al. 1988), and the redeposition of fluoride shows a positive correlation with bone mineral deposition. The aim of this paper is to analyse fine structural changes of bone mineral in order to better understand the mechanism of fluoride uptake and its influence on bone mineral behavior. Modifications of both long-range and short-range atomic arrangements of bone mineral have been analysed by using x-ray diffraction and infrared spectroscopy.
In order to investigate the effect of fluoride on bone mineral crystals, we gave groups of female rats 8 mM NaF/L water and distilled water to control groups. The rats were sacrificed at six weeks, three months, and six months. The fluor content of the bone was determined by neutron activation. X-ray diffraction showed no difference in bone crystal size/ strain with fluoride treatment. Fourier transform Infrared Spectroscopy (FTIR) showed an increased crystallinity in fluoride-fed animals, which seems to be associated with a decrease of labile phosphate environment. Three carbonate bands have been found in fluoridated and normal bone samples. The distribution of carbonate ions on type A and B sites is strongly affected by fluoride. Type A carbonate is always present in bone, but decreases with increasing bone fluoride content. A carbonate band found at 866 cm-’ may correspond to a fluoride interaction with type B carbonate ions. Lastly, phosphate bands have been found to be shifted towards high wave number, which is probably related to the change in unit cell size induced by the fluoride ion. All these changes induced by fluoride reduce the solubility of bone crystals by direct incorporation of fluoride ions in the apatite lattice and by decreasing the labile phosphate environments. Key Words: Bone minerals-Fluoride-Bone Crystals-Infrared-Phosphate-Carbonate.
crystals-
Introduction The amount of fluoride found in bone usually correlates with the amount of fluoride either in the water or in the diet (Legeros et al. 1982; Zipkin 1973). However, it is not clear when the fluoride is actually incorporated in the bone mineral itself. It has been shown that fluoride delays the initiation of mineralization of newly formed bone matrix and decreases the rate at which mineralization proceeds on previously calcified matrices (mineralization lag time) in animal and humans studies (Baylink et al. 1983; Harrison et al. 1984). Whether this delay in mineralization has a biological or a purely chemical origin has not been fully determined. This problem is complicated by the fact that incorporation of fluoride in bone mineral is dependent on age, as well as previous amount incorporated and ,bone type. The susceptibility to fluoride is also species-dependent, because for the same
Materials and Methods Female, weanling, Wistar-derived albino rats (25&300 g) were divided into control and experimental groups with four to six rats in each group. The experimental groups were given a fixed 423
M. D. Gynpas
424
% F IN CORTICAL BONE FROM RATS TREATED WITH FLUORIDE
07 1
./.FLUORIDATED
I
061
u CONTROL /4
-.i/../
3 I
P
7
x
I
I
-
2
0
---r-4
-7-
1
6
8
TIME (IN MONTHS)
Fig. 1. Bone fluoride content of control and F
treated rats
amount of supplemental NaF in their water: 8 mM NaF/L. The control groups were given distilled water. It was estimated that both groups received @4 p.g F/mg Ca inadvertently in the diet, which could not be completely eliminated. Both groups were fed
and C. Rey: Effect of fluoride treatment on bone mineral
a semisynthetic diet containing 0.5% Ca and 1% P, which has been described elsewhere (Harrison et al. 1984). The animals were maintained on this regimen with normal cage activity, one group for six weeks, one group for three months, and the last group for six months, at which times they were sacrificed. Care was taken that any noncortical bone elements were excluded to ensure that only the diaphyseal portions of the long bones were used with the metaphyses, epiphyses, periosteum, marrow elements, and trabecular bone discarded. Cortical bone fragments were then lyophilized for 24 hours to remove all water to prevent phase changes in the mineral. All specimens were stored in a desiccator. The specimens were then ground in a liquid N, bath to a powder form with a Spex Freezer Mill. For chemical analysis, small amounts of unfractionated bone powders were dissolved in hot 4:1 (V:V) HNO,/HClO, and analyzed by plasma emission spectroscopy for Ca and P. The fluoride content of the bone powders from the fluoridated group was determined using neutron activation analysis, as described elsewhere (Grynpas et al. 1987). X-ray diffraction of bone powder was performed on a Rigaku microdiffractometer using CuKa radiation at 160 MA, 60 KV. and using a highly crystalline mineral fluorapatite as a standard. The value of BOO2 and B130, the halfwidth of the hydroxyapatite [002] and [ 1301 reflections were measured in a step-scanning mode with 100 set preset time at each step of 0.02”. Because instrumental broadening was small compared to sample peak breadths, measured halfwidths were corrected for instrumental broadening by subtracting the square of BOO2 and B 130 respectively for the standard (fluorapatite) from the square of bone values, and taking the square root of the differences. D values related to the crystal size/strain in its long dimension (002) and in its cross section (130) were calculated from the corrected BOO2 and B 130 values by the Scherrer equation (Grynpas & Hunter 1988):
D=
I
550
880
cmFig. 2. Determination
bl
1
610
of the parameters
l
linked to different ionic locations and resolution factor: I-v4P04 2-&03
B1,2 case
where A is the x-ray wavelength, B,,, the breadth at half the height of the 002 and 130 peaks, and 8 the diffraction angle. K
“3
‘
K h radian
domain: Resolution
factor: * did3 domain: Relative intensities: ala
Type A CO?: h,bz
Labile Pod: 2
866 cm
-1
band:
s b,bz
860
I
M. D. Grynpas and C. Rey: Effect of fluoride treatment on bone mineral
425
Table I. Rat bone chemistry Sample 6 weeks rat fluor rat fluor 3 months rat fluor rat fluor 6 months rat fluor rat fluor
Ca%a
P%”
(+ ) (- )
25.6 + 1.6 26.4 2 0.9
12.9 2 0.6 12.8 2 0.5
.53 .59
65.1 65.5
(+ ) (- )
23.7 2 0.5 23.7 _f 0.5
12.4 + 0.2 12.4 _’ 0.2
.49 .47
61.7 61.8
(+ ) (- )
25.9 t 0.4 26.7 ? 0.2
12.6 t 0.1 13.1 + 0.1
.59 .58
64.6 66.7
CaiP Molar
(Ca + PO,)%=
Fluor (+ ) = fluoride treated; Fluor ( - ) = control. $ercent of fat free dry weight.
is a constant varying with crystal habit and chosen as 0.9 for the elongated crystallites of bone. Each sample was run in triplicate. Fourier Transform Infrared Spectroscopy (FTIR) The infrared spectra (IR) were recorded on an Analect Fourier Transform infrared spectrometer model FX60. The bone samples were grounded at liquid nitrogen temperature and incorporated in KBr pellets (2 mg sample/300 KBr). The Resolution Enhancement was obtained by using the self-deconvolution technique developed by Kauppinen et al. (1981). All spectra were deconvolved with the same computing parameters in order to allow their comparison. The estimations of resolution factor of vqP04 band, relative labile PO, and HPO, content were determined according to Fig. 2a; the relative amount of carbonate species were determined by reference to the type B carbonate band according to Fig. 2b.
ReSUlts Figure 1 shows that fluoride was incorporated in the bone in a linear fashion with time between six weeks and six months. On the other hand, fluoride had no effect on the Ca, P, WP molar ratio or on the mineral content of rat cortical bone (see Table I). Similarly, x-ray diffraction measurements (Table II) show no difference in crystal cross section (Doo2)or crystal breadth (D,,,) of the mineral crystals if we assume that the broadening of the x-ray diffraction lines are mainly due to the size of the apatite crystals. The IR spectra of fluoridated and control bone do not show any dramatic differences. However, the organic-mineral ratio, as determined by the intensity ratio of amino band to phosphate mineral band seems to be slightly lower after six weeks of fluoride diet than in control animals. This difference is not more perceptible in older animals. Besides, the mineral phase exhibits Table II. X-ray diffraction Sample F(+) 6 wk F(+) 3 mth F(+)6mth F(-)6wk F( -) 3 mth F( -) 6 mth
P 002 0.522 0.503 0.493 0.499 0.488 0.469
some changes concerning its crystallinity and the local environment of both phosphate and carbonate ions. The resolution of u4P04 bands is essentially linked to band broadening, and has frequently been proposed as a criterion of crystallinity of bone mineral (Termine & Posner 1966; TochonDangy 1978; Pellegrino & Biltz 1971; Rey et al. 1991a). All local irregularities in phosphate environment may affect the broadening of the IR bands and the resolution factor: distortions of the lattice occurring near the surface, or due to the presence of ionic substitutions, compositional heterogeneities, strains, etc. Thus, the resolution factor can be considered a global index of the crystallinity of apatite bone mineral. The crystallinity changes of bone mineral were evaluated by determining the u_JQ4 resolution factor; they are reported in Table III. The crystallinity of bone mineral from fluoride-fed animals always appears higher than that of controls. This result is consistent with the observation commonly made during fluoride diets or intoxication (Baud & Moghissi-Buchs 1965; Posner et al. 1963; Tochon-Danguy 1978; Very 1978). The improvement of crystallinity was specially sensitive for the youngest animals, but no significant variations were detected during prolonged diet. It may be noticed, as it is usually observed, that aging induced a clear increase of the crystallinity of normal rats’ bone mineral. Resolution-enhanced FTIR allows one to gather structural information on the local environments of phosphate and carbonate ions. The changes in phosphate environments are observable on v, and ~$0~ bands (Figs. 3 and 4). In the t+ domain, the very broad phosphate band could not be deconvolved as efficiently as in the u4 domain. Several faint maxima are observed on all spectra, but their attribution is still undetermined. The most significant feature in the ug domain is the band at 1125 cm-‘, which has been shown to correspond to a labile phosphate environment (Rey et al. 199 1c) . As already observed on chicken bones, the relative intensity of this band decreases slightly during aging, for normal animals, and the spectra in this domain becomes closer to that of a well defined apatite. The fluoride diet induces an important relative diminution of this phosphate location in the first six weeks of diet, which persists in older animals.
of rat cortical bone D,,(A) 156 162 166 164 167 174
F( + ) = treated; F( - ) = control.
* 2 2 + 2 2
P 130 2 1 3 3 1 6
1.38 1.38 1.33 1.41 1.42 1.36
Table III. Cxystallinityof bone mineral as measured by the resolution factor of resolution enhanced v,PO, bands
D&A) 61.5 61.2 63.6 60.2 59.7 62.3
* & ? -e * 5
1.6 0.9 1.3 1.2 1.7 1.6
Diet lasting time 6 weeks 3 months 6 months
F- fed (kO.01)
Controls (TO.01)
0.71 0.71 0.73
0.64 0.67 0.69
M. D. Grynpas and C. Rey: Effect of fluoride treatment
426
1130
970
1050
1130
cm’
1050
on bone mineral
970
l
Fig. 3. Resolution enhanced FUR spectra in the v,PO, domain: la---Controls
vx weeks; 1bF
fed six weeks; Za-Xontrols six months; 2b -F
fed six months
Some other changes in band intensities and location may be observed; however, the complexity of the spectra in this domain does not permit a complete description. The v,PO, domain shows a better resolution and the influence of the diet can be more easily described (Fig. 4). This domain contains three apatitic phosphate bands at about 560,575 and 600 cm-‘. On the high wave number side, a shoulder at 610-620 cm-’ may be noticed on the spectra of the youngest animals. It has been attributed to a labile phosphate location and has been shown to disappear gradually during aging (Rey et al. 1990). This evolution is not shown quite clearly in our samples; however, fluoride-fed animals always exhibited a labile phosphate band weaker than controls, which became nearly unobservable after several months of diet. On the low wave number side of the u,PO, domain, a broad absorption exists which has been assigned to HPO, ions (Rey et al. 1990). The intensity of
this absorption does not seem, however, to vary strongly because of the diet. Finally, a sensitive shift of the apatitic phosphate bands towards the high wave numbers may be noticed for all fluoride-rich minerals (Table IV). Important changes are also apparent in the $0, domain (Fig. 5). This domain contains two main absorptions due to carbonated ions in apatitic type A (CO, for OH) and type B (CO, for PO,) locations. A third location of carbonate ion is revealed by a shoulder at 866 cm- ’ In low fluoride-containing bone, this band has been assigned to an unstable environment of carbonate ion in a perturbed area of the crystal, probably near its surface (Rey et al. 1990). In fluoridated apatites, however, (Fig. 5) an intense band has been noticed in this domain (Legeros et al. 1968; Vignoles 1984). It has been attributed to an interaction of fluoride and carbonate ions tentatively described as a CO,,F association replacing a PO, ion (Vignoles et al. 1984). The up-
I
I
610
cmFig. 4. Resolution-enhanced fed six months.
610
550
FTIR spectra m the v,PO, domain:
la--Controls
550
l six weeks; IbF-
fed six weeks; 2a-Controls
six months; 2b-F
M. D. Grynpas
and C. Rey:
Effect of fluoride treatment on bone mineral
Table IV. Position of apatitic ~$0~ bands (in cm- ‘)
427
Table V. Relative intensities of carbonate and phosphate bands corresponding to local environments
V" to.4
u’
20.4 F- fed animals 6 weeks 3 months 6 months Controls 6 weeks 3 months 6 months
V"' 20.4
604.1 604.6 604.3
511.2 576.8 511.4
561.1 561.3 561.3
603.0 603.4 603.2
575.1 575.5 576.0
560.4 560.9 560.9
take of fluoride induces severe changes in the carbonate bands. The intensity ratio of type A/type B carbonate, especially, has been found to be invariable in all bone minerals and independent on the species or the age (Rey et al. 1989, 1991a). It decreases strongly, however, and constantly during the fluoride diet time (Table V). The relative intensity of the 866 cm- ’ band appears higher in the fluoride-fed group than in controls; however, in both groups no significant changes happened over time.
4 3
--
a80
860
cm -1
Fig. 5. Resolution-enhanced FHR spectra in the ~$0, Controls six weeks; l&Ffed six weeks; 2a-Controls 2&Ffed six months; 3--Synthetic, poorly crystalline
domain: lasix months; fluorapatite.
F- fed animals 6 weeks 3 months 6 months Controls 6 weeks 3 months 6 months
Labile PO,,
866 cm-’ CO, band
Type A CD3
0.42 0.35 0.41
0.68 0.70 0.71
0.62 0.59 0.55
0.50 0.43 0.48
0.63 0.62 0.65
0.73 0.73 0.71
Interpretation and Discussion The chemical measurements show that despite an increase in fluoride content of the bone up to 0.55% of bone dry weight, the bulk of the mineral chemistry remains unchanged (Ca, P, Ca/P, and Ca + PO,). We have not measured in this study the effect of fluoride on trace elements or carbonate in bone. However, it is known that fluoride can have a profound effect on these elements (Grynpas 1990). X-ray diffraction measurements also show no change in crystal size/strain, and therefore it is presumed that fluoride increased the packing of mineral crystals in bone (Grynpas 1990). The increase of crystallinity observed on the IR resolution factor cannot be linked to a change in crystal size/strain. It seems to be associated with a decrease of labile phosphate environment; however, such an environment has been shown not to be strictly related to crystallinity; in early dental enamel of better crystallinity than bone mineral, for instance, the relative amount of labile phosphate environment is higher (Rey 199 1b) . Although the decrease of labile phosphate content might not be the cause for the improvement of crystallinity, these events are temporally linked. Labile phosphate environment occurs essentially in recent biological or synthetic apatitic deposits (Rey et al. 1990); it has been shown to exhibit a preferential dissolution, and appears specially involved in the maturation process. The decrease of this species as well as the increase of crystallinity indicates that the maturity of fluoridated samples is higher than that of normal bone mineral of the same age. In vitro experiments have shown that fluoride ions possess the property of accelerating the maturation of apatitic calcium phosphates or the conversion of non-apatitic phosphates into apatites (Monte1 1958; Brown 1966; Chow & Brown 1973). This phenomenon, generally attributed to the formation of well organized, lowsolubility fluoridated apatite (Moreno et al. 1974; Legeros & Tung 1983), might occur in bone mineral as well. Three carbonate bands have been found in fluoridated and normal bone samples. The distribution of carbonate ions on type A and type B sites have been shown to be strongly affected by fluoride uptake. This phenomenon may be attributed to the preferential occupancy of the A site by fluoride ions. These ions have been shown to possess a strong affinity for A sites; they displace all other anions, including carbonate (Wallaeys 1952; Trombe 1972), and when the synthesis of apatite is made in fluoridecontaining solution, the type A carbonate is totally absent. In bones, however, type A carbonate is always present, suggesting that fluoride amounts at loci of precipitation are insufficient for a total occupancy of A sites. Besides, the decrease of type A carbonate content with increasing diet time is consistent with a progressive incorporation of fluoride into the mineral. The third carbonate band a 866 cm-’ might correspond to unstable carbonate and/or fluoride interaction with type B C0,2- ions. The
M. D. Grynpas and C. Rey: Effect of fluoride treatment on bone mineral
428
increase of this band in fluoridated bones might be assigned to fluoride-carbonate interaction. It is, however, actually impossible to differentiate these two carbonate environments. Fluoridecarbonate interactions have been attributed to the formation of CO,,F associations and it might be suggested that fluoride ions partially located on oxygen vacancies formed when CO, ions replace PO, ions (Vignoles 1984). Lastly, phosphate bands have been found to be shifted towards high wave numbers. Such band displacement is frequently observed in minerals and is generally attributed to the variations of unit-cell volume (Fowler 1974). This interpretation seems consistent with the well known effect of fluoride ions on apatitic lattice dimensions (Schaeken et al. 1975). It has been shown that fluoride diets have a marked effect on the short-range organization of the mineral. The main alterations of bone mineral apatite are a change in carbonate ion environments and a decrease of labile species content. It has been clearly shown that fluoride ions reduce the amount of type A carbonate environment and interact with type B carbonate ions of the structure, as in synthetic fluor-carbonate apatites (El F6ki et al. 1991). In addition, the new finding that fluoride uptake in bone decreases the amount of labile phosphate environment might explain some of the chemical properties of fluoridated bones, and especially their dissolution behavior. As these environments have been suggested to be involved in the ion reservoir function of bone mineral, it might be postulated that fluoride incorporation could modify the exchange process between bone mineral and the body fluids. Solubility studies have shown that bones from rats maintained on high-fluoride diets release much less mineral into buffered solutions than rats fed a regular diet (Ericsson & Ekberg 1985). The initial rate of dissolution of fluoridated bone was always significantly lower than the rate of dissolution of control bones (Grynpas & Cheng 1988). On another hand, the labile environments have been shown to be involved in the dissolution properties of the mineral (Rey et al. 1990). The decrease of the dissolution rate of fluoridated bone mineral might then be assigned to two different causes: (a) the direct, well documented effect of fluoride incorporation in apatite and (b) the decrease of labile phosphate environments. Beside the labile environments are clusters of atoms with a high energy, and they might be involved in other regulation processes of bone.
Acknowledgmenf:
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Gregory. T. M.; Chow, L. C. Effect of fluoride on enamel solu-
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M. D Fluonde effects on bone crysrals. J. Bone Min. Rer. 5(l):Sl6% 1990 M. D.: Cheng, P -T. Fluoride reduced the rate of dissolution of bone. Min. 5:1-9; 1988.
Grynpas. M. D.: Hunter. G. K. Bone mmeral and glycosaminoglycans in newborn and mature rabbits. J. Bone Min. Res. 3:15%164; 1988. Grynpas, M. D.: F’ritzker. K. P. H.: Hancock, R. G. V. Neutron activation analysis of bulk and selected trace elements in bones using a low flux slowpoke reactor. Bioiogicnl Trace Elements Research 13:333-344: 1987. Grynpas, M. D.: Simmons, E. D.; Pritzker. K. P. H.; Hancock, R. V.: Harrison. J E. Is fluoridated bone different from non-fluoridated bone? Yousuf, S. Ali, ed. Cell medrated calcification and matrix uesirles. New York: Elsevier. 1986, 40%414. Hamson. J. E : Hitchman, A. J. W ; Hasany. S A : Hitchman. A.. Tam, C S. The effect of diet calcium on fluoride toxicity in growing rats. Cnn. J. Physrol. Phormucol. 62:25‘%265; 1984. Kauppinen, I. K.; Moffat. D. J.; Mantsch, J. J., Cameron, D. I. (1981). Fourier self deconvolution: A method for resolving intrinsically overlapped bands. Appi Specr. 35~271-276 Legeros. R. Z.; Singer. L.: Ophaug, R. H.: Quirolgico, G.: Thein, A.: LeGeros. J P The effect of fluoride on the stability of synthetic and biological (bone mineral) apatites. Menczel, J.; Robin, G. C.; Makin, M.; Steinberg, R eds. Osteoporosis. New York: John Wiley & Sons: 1982; 327-341. Legeros. R. Z.; Trautz. 0. R.: Legeros, J. P.: Klein, E. Carbonate substitution u, the apatitic structure. Bull. Sot. Chim. Fr. 1712-1718; 1968. Legeros, R. Z.; Tung. M. S. Cheuxal stability of carbonare- and fluoridecontaining apatites. Caries Res. 17:419-l29; 1983. Mmg, K G ; Nopakun, J.: Messer, H M.; Ophaug, R.; Singer, L. Retention of skeletal fluoride during bone turmwer in rats. Washington. D.C.: American Institute of Nutrition; 1988; 362-366. Mantel. G. Contribution 2 I’ttude des m&hamsmes de synth&e de la fluorapatite.
Thtse d’Etat, Universit6 de Paris; 1958.
This research was
supported by grants from the Med-
ical Research Council of Canada and the National Institute of Health (U.S.A.). We also wish to thank the Arthritis Society of Canada for its support and Teresa Ng for typing the manuscript.
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Grynpas and C. Rey: Effect of fluoride treatment on bone mineral
vironments of the CO3 ion in ht emineral phase of enamel during its formation and maturation. C&if. Tissue hr. 49:25%268; l991b. Rey, C.; Shimizu, M.; Collins, B.; Glimchcr, M. J. Resolution-enhanced Fourier aansfonn infrared spectroscopy study of the environment of phosphate ion in the early deposits of a solid phase of calcium phosphate in bone and enamel and their evolution with age: 2. Investigation in the qPO, domain. C&if. Tissue ht. 49:38%388; 1991. Schaeken, H. G.; Verbeeck, R. M. H.; Driessens, F. C. M.; Thun, H. P. The variation of lattice parameters with composition in solid solutions of hydroxyapatite and fluorapatite. Bull. Sot. Chim. Be& 84:881-890; 1975. Schraer, H.; Posner, A. S.; Schraer, R.; Zipkin, I. Effect of fluoride on bone minerals. C&z. &hop. Rel. Res. 86:26&286; 1972. Tennine, J. D.; Power, A. S. Infrared determination of the percentage of crystallinity m apatitic calcium phosphates. Nature 211:268-270; 1966. Tochon-Danguy, H. I. Effect of fluorine on the crystallinity index of bone mineral substance. Fluoride and Bone Symp. CEMO 2nd. 1977, pp. 73-81. Trombe, J. C. Contribution g I’etude de la decomposition et de la reactivitt de
429 cenaines apatites hydroxylees ou fluodes alcalino-terreuses. Thtse d’Etat. Toulouse: Universit6 Paul Sabatier; 1972. Very, J. M. Effect of fluorine on the structure of bone crystalline mineral. Fluoride and Bone CEMO Symp.. 1978, pp. 68-72. Vignoles, M. Contribution a I’ttude des apatites carbonattes de type B. Thbse d’Etat. Institute National Plytechnique de Toulouse; 1984. Wallaeys, R. Contribution B I’tNde des apatites phosphocalciques. Ann. Chim. 7:801848; 1952. Zipkin, 1. Fluoride in the calcified structures. Zipkin, I., ed. Biological Mineralization. New York: Wiley-Interscience; 1973; 487.-505. Zipkin, I.; McClure, F. I. Deposition of fluorine in the bone and teeth of the growing rat. J. Nut. 4261 l-620; 1952.
Dale Received: January 29, 1992 Date Revised: June 9, 1992 Dare Accepted: June 15, 1992
Copyright
8756-3282192 $5.00 + .00 0 1992 Pergamon Press Ltd.
The Effect of Fluoride Treatment on Bone Mineral Crystals in the Rat M. D. GRYNPAS’
and C. REY*
’ Samuel Lunenfeld Research Institute of Mount Sinai Hospital and Department of Pathology, Mount Sinai Hospital and University of Toronto, Toronto, Canada ’ Luboratoire des Mate’riaux, Ecole Nationale SupCrieure de Chimie, Toulouse, France Address for correspondence and retxints: Dr. M. D. Grvnuas, Samuel Lunenfeld Research Institute, Mount Sinai Hospital. 600 University
Avenue,“Room 984, Toronto, On&o M5G 1X5 Canada. .
Abstract
dose of fluoride in their drinking water, mice accumulated six times as much fluoride in their bones as rats. Fluoride does not diffuse into already formed bones, but is incorporated during bone formation (Zipkin et al. 1952). This observation has been confirmed by x-ray microprobe analysis (Baud & Bang 1970; Bang 1978) and by density fractionation (Grynpas et al. 1986). However, earlier studies (Schraer et al. 1972) on growing rats exposed to different levels of fluoride for various lengths of time showed that changes in crystallinity did not correlate with the rate of fluoride accumulation in the bone mineral. Changes in crystallinity took place after the fluoride level had become constant. These data, as well as the data on the mineralization lag time, can be interpreted as a change in the kinetics of bone mineralization with increasing fluoride content in the systemic circulation, which means that it would take longer for fluoridated apatite bone crystals to get deposited and to mature than for the same to occur in apatite bone crystals. Fluoride can also affect putative precursor phases in the formation of bone mineral apatite. Eanes and Meyer (1978) have shown in vitro that in the case of octacalcium phosphate (OCP), which can occur as a transitional crystalline phase in apatite formation in vitro (Nancollas & Tomazic 1974), when the amount of fluoride approached 3% of the initial phosphate concentration, the presence of OCP could no longer be experimentally demonstrated. In fluoride-free preparations the apatite crystals appear as thin plates; in the high-fluoride preparations they appear as short and thin needles. The phase may be controlled in part by local conditions, particularly the amount of fluoride released by bone mineral dissolution. We appreciate that 60% of fluoride mobilized during bone resorption seems to be redeposited in the skeleton (Ming et al. 1988), and the redeposition of fluoride shows a positive correlation with bone mineral deposition. The aim of this paper is to analyse fine structural changes of bone mineral in order to better understand the mechanism of fluoride uptake and its influence on bone mineral behavior. Modifications of both long-range and short-range atomic arrangements of bone mineral have been analysed by using x-ray diffraction and infrared spectroscopy.
In order to investigate the effect of fluoride on bone mineral crystals, we gave groups of female rats 8 mM NaF/L water and distilled water to control groups. The rats were sacrificed at six weeks, three months, and six months. The fluor content of the bone was determined by neutron activation. X-ray diffraction showed no difference in bone crystal size/ strain with fluoride treatment. Fourier transform Infrared Spectroscopy (FTIR) showed an increased crystallinity in fluoride-fed animals, which seems to be associated with a decrease of labile phosphate environment. Three carbonate bands have been found in fluoridated and normal bone samples. The distribution of carbonate ions on type A and B sites is strongly affected by fluoride. Type A carbonate is always present in bone, but decreases with increasing bone fluoride content. A carbonate band found at 866 cm-’ may correspond to a fluoride interaction with type B carbonate ions. Lastly, phosphate bands have been found to be shifted towards high wave number, which is probably related to the change in unit cell size induced by the fluoride ion. All these changes induced by fluoride reduce the solubility of bone crystals by direct incorporation of fluoride ions in the apatite lattice and by decreasing the labile phosphate environments. Key Words: Bone minerals-Fluoride-Bone Crystals-Infrared-Phosphate-Carbonate.
crystals-
Introduction The amount of fluoride found in bone usually correlates with the amount of fluoride either in the water or in the diet (Legeros et al. 1982; Zipkin 1973). However, it is not clear when the fluoride is actually incorporated in the bone mineral itself. It has been shown that fluoride delays the initiation of mineralization of newly formed bone matrix and decreases the rate at which mineralization proceeds on previously calcified matrices (mineralization lag time) in animal and humans studies (Baylink et al. 1983; Harrison et al. 1984). Whether this delay in mineralization has a biological or a purely chemical origin has not been fully determined. This problem is complicated by the fact that incorporation of fluoride in bone mineral is dependent on age, as well as previous amount incorporated and ,bone type. The susceptibility to fluoride is also species-dependent, because for the same
Materials and Methods Female, weanling, Wistar-derived albino rats (25&300 g) were divided into control and experimental groups with four to six rats in each group. The experimental groups were given a fixed 423
M. D. Gynpas
424
% F IN CORTICAL BONE FROM RATS TREATED WITH FLUORIDE
07 1
./.FLUORIDATED
I
061
u CONTROL /4
-.i/../
3 I
P
7
x
I
I
-
2
0
---r-4
-7-
1
6
8
TIME (IN MONTHS)
Fig. 1. Bone fluoride content of control and F
treated rats
amount of supplemental NaF in their water: 8 mM NaF/L. The control groups were given distilled water. It was estimated that both groups received @4 p.g F/mg Ca inadvertently in the diet, which could not be completely eliminated. Both groups were fed
and C. Rey: Effect of fluoride treatment on bone mineral
a semisynthetic diet containing 0.5% Ca and 1% P, which has been described elsewhere (Harrison et al. 1984). The animals were maintained on this regimen with normal cage activity, one group for six weeks, one group for three months, and the last group for six months, at which times they were sacrificed. Care was taken that any noncortical bone elements were excluded to ensure that only the diaphyseal portions of the long bones were used with the metaphyses, epiphyses, periosteum, marrow elements, and trabecular bone discarded. Cortical bone fragments were then lyophilized for 24 hours to remove all water to prevent phase changes in the mineral. All specimens were stored in a desiccator. The specimens were then ground in a liquid N, bath to a powder form with a Spex Freezer Mill. For chemical analysis, small amounts of unfractionated bone powders were dissolved in hot 4:1 (V:V) HNO,/HClO, and analyzed by plasma emission spectroscopy for Ca and P. The fluoride content of the bone powders from the fluoridated group was determined using neutron activation analysis, as described elsewhere (Grynpas et al. 1987). X-ray diffraction of bone powder was performed on a Rigaku microdiffractometer using CuKa radiation at 160 MA, 60 KV. and using a highly crystalline mineral fluorapatite as a standard. The value of BOO2 and B130, the halfwidth of the hydroxyapatite [002] and [ 1301 reflections were measured in a step-scanning mode with 100 set preset time at each step of 0.02”. Because instrumental broadening was small compared to sample peak breadths, measured halfwidths were corrected for instrumental broadening by subtracting the square of BOO2 and B 130 respectively for the standard (fluorapatite) from the square of bone values, and taking the square root of the differences. D values related to the crystal size/strain in its long dimension (002) and in its cross section (130) were calculated from the corrected BOO2 and B 130 values by the Scherrer equation (Grynpas & Hunter 1988):
D=
I
550
880
cmFig. 2. Determination
bl
1
610
of the parameters
l
linked to different ionic locations and resolution factor: I-v4P04 2-&03
B1,2 case
where A is the x-ray wavelength, B,,, the breadth at half the height of the 002 and 130 peaks, and 8 the diffraction angle. K
“3
‘
K h radian
domain: Resolution
factor: * did3 domain: Relative intensities: ala
Type A CO?: h,bz
Labile Pod: 2
866 cm
-1
band:
s b,bz
860
I
M. D. Grynpas and C. Rey: Effect of fluoride treatment on bone mineral
425
Table I. Rat bone chemistry Sample 6 weeks rat fluor rat fluor 3 months rat fluor rat fluor 6 months rat fluor rat fluor
Ca%a
P%”
(+ ) (- )
25.6 + 1.6 26.4 2 0.9
12.9 2 0.6 12.8 2 0.5
.53 .59
65.1 65.5
(+ ) (- )
23.7 2 0.5 23.7 _f 0.5
12.4 + 0.2 12.4 _’ 0.2
.49 .47
61.7 61.8
(+ ) (- )
25.9 t 0.4 26.7 ? 0.2
12.6 t 0.1 13.1 + 0.1
.59 .58
64.6 66.7
CaiP Molar
(Ca + PO,)%=
Fluor (+ ) = fluoride treated; Fluor ( - ) = control. $ercent of fat free dry weight.
is a constant varying with crystal habit and chosen as 0.9 for the elongated crystallites of bone. Each sample was run in triplicate. Fourier Transform Infrared Spectroscopy (FTIR) The infrared spectra (IR) were recorded on an Analect Fourier Transform infrared spectrometer model FX60. The bone samples were grounded at liquid nitrogen temperature and incorporated in KBr pellets (2 mg sample/300 KBr). The Resolution Enhancement was obtained by using the self-deconvolution technique developed by Kauppinen et al. (1981). All spectra were deconvolved with the same computing parameters in order to allow their comparison. The estimations of resolution factor of vqP04 band, relative labile PO, and HPO, content were determined according to Fig. 2a; the relative amount of carbonate species were determined by reference to the type B carbonate band according to Fig. 2b.
ReSUlts Figure 1 shows that fluoride was incorporated in the bone in a linear fashion with time between six weeks and six months. On the other hand, fluoride had no effect on the Ca, P, WP molar ratio or on the mineral content of rat cortical bone (see Table I). Similarly, x-ray diffraction measurements (Table II) show no difference in crystal cross section (Doo2)or crystal breadth (D,,,) of the mineral crystals if we assume that the broadening of the x-ray diffraction lines are mainly due to the size of the apatite crystals. The IR spectra of fluoridated and control bone do not show any dramatic differences. However, the organic-mineral ratio, as determined by the intensity ratio of amino band to phosphate mineral band seems to be slightly lower after six weeks of fluoride diet than in control animals. This difference is not more perceptible in older animals. Besides, the mineral phase exhibits Table II. X-ray diffraction Sample F(+) 6 wk F(+) 3 mth F(+)6mth F(-)6wk F( -) 3 mth F( -) 6 mth
P 002 0.522 0.503 0.493 0.499 0.488 0.469
some changes concerning its crystallinity and the local environment of both phosphate and carbonate ions. The resolution of u4P04 bands is essentially linked to band broadening, and has frequently been proposed as a criterion of crystallinity of bone mineral (Termine & Posner 1966; TochonDangy 1978; Pellegrino & Biltz 1971; Rey et al. 1991a). All local irregularities in phosphate environment may affect the broadening of the IR bands and the resolution factor: distortions of the lattice occurring near the surface, or due to the presence of ionic substitutions, compositional heterogeneities, strains, etc. Thus, the resolution factor can be considered a global index of the crystallinity of apatite bone mineral. The crystallinity changes of bone mineral were evaluated by determining the u_JQ4 resolution factor; they are reported in Table III. The crystallinity of bone mineral from fluoride-fed animals always appears higher than that of controls. This result is consistent with the observation commonly made during fluoride diets or intoxication (Baud & Moghissi-Buchs 1965; Posner et al. 1963; Tochon-Danguy 1978; Very 1978). The improvement of crystallinity was specially sensitive for the youngest animals, but no significant variations were detected during prolonged diet. It may be noticed, as it is usually observed, that aging induced a clear increase of the crystallinity of normal rats’ bone mineral. Resolution-enhanced FTIR allows one to gather structural information on the local environments of phosphate and carbonate ions. The changes in phosphate environments are observable on v, and ~$0~ bands (Figs. 3 and 4). In the t+ domain, the very broad phosphate band could not be deconvolved as efficiently as in the u4 domain. Several faint maxima are observed on all spectra, but their attribution is still undetermined. The most significant feature in the ug domain is the band at 1125 cm-‘, which has been shown to correspond to a labile phosphate environment (Rey et al. 199 1c) . As already observed on chicken bones, the relative intensity of this band decreases slightly during aging, for normal animals, and the spectra in this domain becomes closer to that of a well defined apatite. The fluoride diet induces an important relative diminution of this phosphate location in the first six weeks of diet, which persists in older animals.
of rat cortical bone D,,(A) 156 162 166 164 167 174
F( + ) = treated; F( - ) = control.
* 2 2 + 2 2
P 130 2 1 3 3 1 6
1.38 1.38 1.33 1.41 1.42 1.36
Table III. Cxystallinityof bone mineral as measured by the resolution factor of resolution enhanced v,PO, bands
D&A) 61.5 61.2 63.6 60.2 59.7 62.3
* & ? -e * 5
1.6 0.9 1.3 1.2 1.7 1.6
Diet lasting time 6 weeks 3 months 6 months
F- fed (kO.01)
Controls (TO.01)
0.71 0.71 0.73
0.64 0.67 0.69
M. D. Grynpas and C. Rey: Effect of fluoride treatment
426
1130
970
1050
1130
cm’
1050
on bone mineral
970
l
Fig. 3. Resolution enhanced FUR spectra in the v,PO, domain: la---Controls
vx weeks; 1bF
fed six weeks; Za-Xontrols six months; 2b -F
fed six months
Some other changes in band intensities and location may be observed; however, the complexity of the spectra in this domain does not permit a complete description. The v,PO, domain shows a better resolution and the influence of the diet can be more easily described (Fig. 4). This domain contains three apatitic phosphate bands at about 560,575 and 600 cm-‘. On the high wave number side, a shoulder at 610-620 cm-’ may be noticed on the spectra of the youngest animals. It has been attributed to a labile phosphate location and has been shown to disappear gradually during aging (Rey et al. 1990). This evolution is not shown quite clearly in our samples; however, fluoride-fed animals always exhibited a labile phosphate band weaker than controls, which became nearly unobservable after several months of diet. On the low wave number side of the u,PO, domain, a broad absorption exists which has been assigned to HPO, ions (Rey et al. 1990). The intensity of
this absorption does not seem, however, to vary strongly because of the diet. Finally, a sensitive shift of the apatitic phosphate bands towards the high wave numbers may be noticed for all fluoride-rich minerals (Table IV). Important changes are also apparent in the $0, domain (Fig. 5). This domain contains two main absorptions due to carbonated ions in apatitic type A (CO, for OH) and type B (CO, for PO,) locations. A third location of carbonate ion is revealed by a shoulder at 866 cm- ’ In low fluoride-containing bone, this band has been assigned to an unstable environment of carbonate ion in a perturbed area of the crystal, probably near its surface (Rey et al. 1990). In fluoridated apatites, however, (Fig. 5) an intense band has been noticed in this domain (Legeros et al. 1968; Vignoles 1984). It has been attributed to an interaction of fluoride and carbonate ions tentatively described as a CO,,F association replacing a PO, ion (Vignoles et al. 1984). The up-
I
I
610
cmFig. 4. Resolution-enhanced fed six months.
610
550
FTIR spectra m the v,PO, domain:
la--Controls
550
l six weeks; IbF-
fed six weeks; 2a-Controls
six months; 2b-F
M. D. Grynpas
and C. Rey:
Effect of fluoride treatment on bone mineral
Table IV. Position of apatitic ~$0~ bands (in cm- ‘)
427
Table V. Relative intensities of carbonate and phosphate bands corresponding to local environments
V" to.4
u’
20.4 F- fed animals 6 weeks 3 months 6 months Controls 6 weeks 3 months 6 months
V"' 20.4
604.1 604.6 604.3
511.2 576.8 511.4
561.1 561.3 561.3
603.0 603.4 603.2
575.1 575.5 576.0
560.4 560.9 560.9
take of fluoride induces severe changes in the carbonate bands. The intensity ratio of type A/type B carbonate, especially, has been found to be invariable in all bone minerals and independent on the species or the age (Rey et al. 1989, 1991a). It decreases strongly, however, and constantly during the fluoride diet time (Table V). The relative intensity of the 866 cm- ’ band appears higher in the fluoride-fed group than in controls; however, in both groups no significant changes happened over time.
4 3
--
a80
860
cm -1
Fig. 5. Resolution-enhanced FHR spectra in the ~$0, Controls six weeks; l&Ffed six weeks; 2a-Controls 2&Ffed six months; 3--Synthetic, poorly crystalline
domain: lasix months; fluorapatite.
F- fed animals 6 weeks 3 months 6 months Controls 6 weeks 3 months 6 months
Labile PO,,
866 cm-’ CO, band
Type A CD3
0.42 0.35 0.41
0.68 0.70 0.71
0.62 0.59 0.55
0.50 0.43 0.48
0.63 0.62 0.65
0.73 0.73 0.71
Interpretation and Discussion The chemical measurements show that despite an increase in fluoride content of the bone up to 0.55% of bone dry weight, the bulk of the mineral chemistry remains unchanged (Ca, P, Ca/P, and Ca + PO,). We have not measured in this study the effect of fluoride on trace elements or carbonate in bone. However, it is known that fluoride can have a profound effect on these elements (Grynpas 1990). X-ray diffraction measurements also show no change in crystal size/strain, and therefore it is presumed that fluoride increased the packing of mineral crystals in bone (Grynpas 1990). The increase of crystallinity observed on the IR resolution factor cannot be linked to a change in crystal size/strain. It seems to be associated with a decrease of labile phosphate environment; however, such an environment has been shown not to be strictly related to crystallinity; in early dental enamel of better crystallinity than bone mineral, for instance, the relative amount of labile phosphate environment is higher (Rey 199 1b) . Although the decrease of labile phosphate content might not be the cause for the improvement of crystallinity, these events are temporally linked. Labile phosphate environment occurs essentially in recent biological or synthetic apatitic deposits (Rey et al. 1990); it has been shown to exhibit a preferential dissolution, and appears specially involved in the maturation process. The decrease of this species as well as the increase of crystallinity indicates that the maturity of fluoridated samples is higher than that of normal bone mineral of the same age. In vitro experiments have shown that fluoride ions possess the property of accelerating the maturation of apatitic calcium phosphates or the conversion of non-apatitic phosphates into apatites (Monte1 1958; Brown 1966; Chow & Brown 1973). This phenomenon, generally attributed to the formation of well organized, lowsolubility fluoridated apatite (Moreno et al. 1974; Legeros & Tung 1983), might occur in bone mineral as well. Three carbonate bands have been found in fluoridated and normal bone samples. The distribution of carbonate ions on type A and type B sites have been shown to be strongly affected by fluoride uptake. This phenomenon may be attributed to the preferential occupancy of the A site by fluoride ions. These ions have been shown to possess a strong affinity for A sites; they displace all other anions, including carbonate (Wallaeys 1952; Trombe 1972), and when the synthesis of apatite is made in fluoridecontaining solution, the type A carbonate is totally absent. In bones, however, type A carbonate is always present, suggesting that fluoride amounts at loci of precipitation are insufficient for a total occupancy of A sites. Besides, the decrease of type A carbonate content with increasing diet time is consistent with a progressive incorporation of fluoride into the mineral. The third carbonate band a 866 cm-’ might correspond to unstable carbonate and/or fluoride interaction with type B C0,2- ions. The
M. D. Grynpas and C. Rey: Effect of fluoride treatment on bone mineral
428
increase of this band in fluoridated bones might be assigned to fluoride-carbonate interaction. It is, however, actually impossible to differentiate these two carbonate environments. Fluoridecarbonate interactions have been attributed to the formation of CO,,F associations and it might be suggested that fluoride ions partially located on oxygen vacancies formed when CO, ions replace PO, ions (Vignoles 1984). Lastly, phosphate bands have been found to be shifted towards high wave numbers. Such band displacement is frequently observed in minerals and is generally attributed to the variations of unit-cell volume (Fowler 1974). This interpretation seems consistent with the well known effect of fluoride ions on apatitic lattice dimensions (Schaeken et al. 1975). It has been shown that fluoride diets have a marked effect on the short-range organization of the mineral. The main alterations of bone mineral apatite are a change in carbonate ion environments and a decrease of labile species content. It has been clearly shown that fluoride ions reduce the amount of type A carbonate environment and interact with type B carbonate ions of the structure, as in synthetic fluor-carbonate apatites (El F6ki et al. 1991). In addition, the new finding that fluoride uptake in bone decreases the amount of labile phosphate environment might explain some of the chemical properties of fluoridated bones, and especially their dissolution behavior. As these environments have been suggested to be involved in the ion reservoir function of bone mineral, it might be postulated that fluoride incorporation could modify the exchange process between bone mineral and the body fluids. Solubility studies have shown that bones from rats maintained on high-fluoride diets release much less mineral into buffered solutions than rats fed a regular diet (Ericsson & Ekberg 1985). The initial rate of dissolution of fluoridated bone was always significantly lower than the rate of dissolution of control bones (Grynpas & Cheng 1988). On another hand, the labile environments have been shown to be involved in the dissolution properties of the mineral (Rey et al. 1990). The decrease of the dissolution rate of fluoridated bone mineral might then be assigned to two different causes: (a) the direct, well documented effect of fluoride incorporation in apatite and (b) the decrease of labile phosphate environments. Beside the labile environments are clusters of atoms with a high energy, and they might be involved in other regulation processes of bone.
Acknowledgmenf:
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Gregory. T. M.; Chow, L. C. Effect of fluoride on enamel solu-
bdity and cariostasis. Caries Res. 11:118-141; 1977. Chow. L. C : Brown. W. E. Reaction of dicalcium phosphate dihydrate with fluonde. I Dent. Res. 52(6):122&1227; 1973. Eanes. E. D.; Meyer. J. L. The influence of fluoride on apatlte formation from unstable supersaturated solutions at pH 7.4. J. Denr. RCS. 57:617-624; 1978. El F&l. H.. Rey, C.; Vignoles. M. Carbonate ions in apatites: Infrared invesugatlons m the vj CO, domain. C&if Tissue fnr. 49:26%274: 1991. Encsson, Y.: Ekberg, 0. Dietetically provoked general and alveolar osteopenia m rat and Its prevention or cure by calcium and fluoride. J. Periodon. Res. 10:25&269: 1985. Fowler, B 0 Infrared studxs of apahtes II. Preparation of normal and isotopically substituted calcium, strontium and barium hydroxyapatites and spectrastructure-composition correlations. Inorg. Chem. 13(1):207-214; 1974. Fowler. B. 0.. Moreno, E. C.: Brown. W. E Infrared spectra of hydroxyapatite. octacalcium phosphate and pyrolysed octacalcium phosphate. Arch. Oral Eio[. 11~477-492: 1966. Grynpas. s175: Grynpas. Bone
M. D Fluonde effects on bone crysrals. J. Bone Min. Rer. 5(l):Sl6% 1990 M. D.: Cheng, P -T. Fluoride reduced the rate of dissolution of bone. Min. 5:1-9; 1988.
Grynpas. M. D.: Hunter. G. K. Bone mmeral and glycosaminoglycans in newborn and mature rabbits. J. Bone Min. Res. 3:15%164; 1988. Grynpas, M. D.: F’ritzker. K. P. H.: Hancock, R. G. V. Neutron activation analysis of bulk and selected trace elements in bones using a low flux slowpoke reactor. Bioiogicnl Trace Elements Research 13:333-344: 1987. Grynpas, M. D.: Simmons, E. D.; Pritzker. K. P. H.; Hancock, R. V.: Harrison. J E. Is fluoridated bone different from non-fluoridated bone? Yousuf, S. Ali, ed. Cell medrated calcification and matrix uesirles. New York: Elsevier. 1986, 40%414. Hamson. J. E : Hitchman, A. J. W ; Hasany. S A : Hitchman. A.. Tam, C S. The effect of diet calcium on fluoride toxicity in growing rats. Cnn. J. Physrol. Phormucol. 62:25‘%265; 1984. Kauppinen, I. K.; Moffat. D. J.; Mantsch, J. J., Cameron, D. I. (1981). Fourier self deconvolution: A method for resolving intrinsically overlapped bands. Appi Specr. 35~271-276 Legeros. R. Z.; Singer. L.: Ophaug, R. H.: Quirolgico, G.: Thein, A.: LeGeros. J P The effect of fluoride on the stability of synthetic and biological (bone mineral) apatites. Menczel, J.; Robin, G. C.; Makin, M.; Steinberg, R eds. Osteoporosis. New York: John Wiley & Sons: 1982; 327-341. Legeros. R. Z.; Trautz. 0. R.: Legeros, J. P.: Klein, E. Carbonate substitution u, the apatitic structure. Bull. Sot. Chim. Fr. 1712-1718; 1968. Legeros, R. Z.; Tung. M. S. Cheuxal stability of carbonare- and fluoridecontaining apatites. Caries Res. 17:419-l29; 1983. Mmg, K G ; Nopakun, J.: Messer, H M.; Ophaug, R.; Singer, L. Retention of skeletal fluoride during bone turmwer in rats. Washington. D.C.: American Institute of Nutrition; 1988; 362-366. Mantel. G. Contribution 2 I’ttude des m&hamsmes de synth&e de la fluorapatite.
Thtse d’Etat, Universit6 de Paris; 1958.
This research was
supported by grants from the Med-
ical Research Council of Canada and the National Institute of Health (U.S.A.). We also wish to thank the Arthritis Society of Canada for its support and Teresa Ng for typing the manuscript.
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Dale Received: January 29, 1992 Date Revised: June 9, 1992 Dare Accepted: June 15, 1992