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A XRD and TEM study on the transformation of amorphous calcium phosphate in the presence of magnesium
,. . . . . . . .
C R Y S T A L G R O W T H
ELSEVIER
Journal of Crystal Growth 165 (1996) 98-105
A XRD and TEM study on the transformation of amorphous calcium phosphate in the presence of magnesium Francesco Abbona a,,, Alain Baronnet b a Dipartimento di Scienze della Terra, Unicersith della Calabria, 1-80736 Arcacacata di Rende (CS), ltaly 6 CRMCC, CNRS, Campus de Luminy, Case 913, F-13218 Marseille Cedex 9, France
Received 15 December 1995; accepted 5 February 1996
Abstract The evolution of the amorphous calcium phosphate (ACP), precipitated at 25°C from solutions of concentration [Ca] = [P] = 5 mM at pH = 7.94, was followed at fixed time intervals by combined X-ray, electron diffraction and TEM imaging. The same runs were carried out in the presence of magnesium ions (C = 1 raM). Only one kind of amorphous compound was observed (ACPI), which seems to change directly into apatite (HAP) without crystalline intermediate. Edge dislocations and grain boundaries were found by HRTEM analysis in HAP. The effects of magnesium on conversion of ACP into HAP and on the crystal structure and habit of HAP are interpreted in terms of incorporation and surface adsorption.
1. Introduction When calcium phosphates are precipitated from aqueous solutions, an amorphous phase is easily obtained, which is commonly denoted as ACP (amorphous calcium phosphate) and usually evolves into an hydroxyapatite-like phosphate (HAP). Intensive researches have been carried out mostly under physiological conditions. In spite of unquestionable progresses, some uncertainties remain about the conversion mechanism. ACP is said to convert directly into H A P by some authors [1-4], while others state that the transformation takes place through an intermediate like OCP [5-8] or by ACP dissolution and subsequent H A P crystallization [9]. Recently Christoffersen et al. [5] were able to show the occurrence of two amorphous calcium phosphates, A C P I and ACP2, having the same composition, but differ-
Corresponding author.
ing in morphology and solubility. Also the role of Mg, a cation common in the natural milieu where calcium phosphates form, is far from being fully understood. Recent results indicate that Mg does not affect the formation of ACP1, but only delay its conversion to ACP2 [6] and that it first decreases, then increases the H A P crystallinity during maturation [ 10]. The present work has been undertaken in order to study the conversion of ACP out of physiological conditions through a combined X-ray and TEM investigation and to examine the effect of Mg on the transformation, with special regard to the two new amorphous phases and their role in the process.
2. Experimental procedure All experiments were carried out at 25 + 0.2°C with solutions of the same concentration: [Ca] = [P]
0022-0248/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved PII S 0 0 2 2 - 0 2 4 8 ( 9 6 ) 0 0 1 5 6 - X
F. Abbona, A. Baronnet/Journal (~f Co'sml Growth 165 (1996) 98 105
= 5 mM, as used and tested previously [11]. These solutions were prepared by rapidly mixing equal volumes of a solution of CaCIo • 6 H 2 0 10 mM (A) and a solution of (NH4)2HPO 4 10 mM (B). Several
99
solutions were prepared and left at rest for fixed time intervals: t = 0, 2, 4, 8, 16, 32 min; 2, 5, 12, 24, 48 h; 6 months. At each of these times the precipitates were filtered by pressure suction through a Millipore
f
4. ~"-A
. . . .
Fig. 1. Analyses of precipitates from Mg-free solutions: t = 0: (a) electron-micrograph and -diffraction of the spherical amorphous ACP! particles: (b) two grain boundaries showing lattice fringes (d = 8.3 ,~) due to (1010) planes of HAP; t = 24 h: (c) thin flakes of HAP with few blades; (d) two grains differently oriented. White spots: the OH channels, surrounded by Ca ions, parallel to the c axis. Top right: a polygonized crystallite occurring after 48 h; t = 6 months: (e) elongated laminar crystals of HAP (the bar = 1500 A); (f) HAP blades showing fringes.
100
F. Abbona, A. Baronnet / Journal of Cr)'stal Growth 165 (1996) 98-105
0.22 p,m filter; they were treated with absolute ethanol to stop any reaction and then dried with acetone. The samples were submitted to X-ray and electron diffraction, to S E M and T E M studies and to X-ray energy dispersive ( E D S ) microanalysis. For T E M observations, a suspension o f the run product
in acetone was allowed to settle d o w n onto a carbon-coated Cu grid. L o w magnification brightfield images and selected area diffraction patterns of representative groups o f particles were systematically recorded with a J E O L 2000 F X electron microscope. High resolution T E M i m a g i n g was p e r f o r m e d
Fig. 2. Analyses of precipitates from Mg-doped solutions, t = 0: (a) the most common ACP1 spherical particles; (b) a second form of ACP similar to ACP2, observed in one sample; t = 32 min: (c) aggregates of curved, crystallizing flakes (the bar = 1000 ,~); (d) lattice fringes at d = 8.3 and 3.3 ,~, corresponding to HAP; t = 6 months: (e) HAP short blades with irregular external shape; (f) HAP crystallite showing two-dimensional order, damaged by the electron beam.
F. Abbona, A. Baronnet / Journal of Crystal Growth 165 (1996) 98-105
in multiple-beam bright field mode with the objective aperture allowed to cut space frequencies beyond the resolution of the instrument (2.8 A ~). C a / P and M g / C a ratios were checked with a X-ray energy dispersive Si(Li) detector (TRACOR Northern) attached to the microscope and also by atomic absorption spectrophotometry. Another series of experiments was carried out under the same conditions in the presence of Mg, which was added as MgClz.6H20 to the solution A. The final concentrations were: [Ca] = [P] = 5 mM; [Mg] = 1 mM. This is the maximum concentration of Mg allowing the conversion of ACP into HAP; at higher concentrations whitlockite is formed [11]. To check reproducibility, experiments were duplicated.
3. Results 3.1. From pure solutions
The first phase which precipitates immediately after the mixing of solutions is amorphous. It consists of spherical or disc-shaped particles loosely aogglomerated with a diameter in the range 400-1000 A and mean value of 700 A (Fig. la). In one sample collected in the very first moment (t = 0 rain) sharp fringes were observed with spacings of about 8.3 A (Fig. lb). This distance corresponds to the (10~0) planes of HAP (8.17 A). Edge dislocations are observed at the boundaries where the (1010) planes of two flakes meet under small tilt angles (Fig. lb). The bulk of precipitate remains amorphous for at least 16 min; it starts to crystallize within 30'. The X-ray and electron diffraction patterns taken after 30 min correspond to very poorly crystalline HAP. Crystallization is accompanied by a morphological change of the particles, which decompose into very thin roundish flakes sometimes folded and wrapped. Rare fringes distant 8.3 and 3.3 A, corresponding to the (0002) planes, are observed. The ordered ~gions are very small (from 130 X 33 to 250 X 125 A2) and irregular in shape. After 24 h the flakes have changed into short strips of greater size (about 900 X 120 ~2) (Fig. lc). Most flakes show HRTEM fringes at 8.3 and few at 9.1 A. An hexagonal array of white spots bear evidence of the channels in the HAP structure seen along [0001] (Fig. ld). With aging the crystal-
101
lites tend to become better polygonized (Fig. l d). After 6 months all the precipitate is formed by rather regular, thin laths having a mean size of 1400 x 250 ,~2 (Fig. le). Few crystallites are needle-like and seem to be hollow. New fringes are found at 5.2 and 4.1 A, corresponding to the (1011) and (1120) planes respectively (Fig. lf). The occurrence of OCP was never observed. 3.2. From solutions doped with Mg
Also from these solutions the phase which precipitates first is amorphous and consists of spherical particles of smaller size (from 300 to 1100 A, with a mean value of about 500 ,~) (Fig. 2a). In one sample (Fig. 2b) thin fibers or flakes interlaced giving the cluster a soft and gel-like aspect reveal a weak crystallinity by electron diffraction, attributable to HAP. However two hours are necessary for crystallization to start and at least five hours for HAP crystallinity to clearly disclose. The flakes are numerous, interwoven and curved (Fig. 4c) and exhibit fringes having distances of 8.3 and 3.3 ,~ (Fig. 4d). After six months the crystallites consist of irregular blades showing fringes at 8.3 A (Figs. 2e, 2f), with maximum size of 1000 X 250 ~2. They are easily damaged by the electron beam (Fig. 2f). The atomic ratio Mg/Ca, measured in the solid phase, is progressively decreasing with time: from 0.04 it drops to 0.009 in the precipitate aged for 48 h (Fig. 3). The same trend was already noticed [5].
Mg/Ca ) solid 0.04.
0.03-
0.02-
0.01-
0 0
I
1
12
24
I
36
I
b
48 hours
Fig. 3. Change of the M g / C a atom ratio in the solid phases with time.
F. Abbona, A. Baronnet/Journal of Co'stal Growth 165 (1996) 98 105
102
aS(Ca2+)aS(HPO~ )
4. Discussion [~HAP =
4.1. Supersaturation The solutions we used in our experiments were highly supersaturated. The degree of supersaturation was calculated by a computer programme already applied [12]. For HAP it is given by the ratio
Mg 0 mM 1 mM
1 0.025 0.027
pH 7.943 7.928
ACP 30.8 24.7
B 16.5 15.3
M 32.9 30.5
(where I = ionic strength; B = brnshite; M = monetite; OCP = octacalcium phosphate; W = whitlockite; S = struvite). The value for ACP, which is not a true phase, is only indicative of its tendency to precipitate; this happens whenever [3ACp > 1. The presence of Mg implies a low supersaturation with respect to struvite, MgNH4PO4.6H20 , but does not alter in a significant way the supersaturation with respect to the calcium phosphates and the other physico-cbemical properties of the solutions. As 13ACe >> 1, ACP nucleates as first phase instead of HAP (Ostwald's rule). Its immediate precipitation decreases supersaturation with respect to the other phases, so that their nucleation is hindered.
4.2. Morphology The most frequent morphology of ACP is spherical and corresponds to ACP1, as found by Christoffersen et al. [5]. The second type of ACP, which was denoted ACP2 by the same authors and represents the evolution of ACPI, was observed in two samples only, aged for 4 min and 24 h in the presence of Mg. They were recognized on the basis of their morphological resemblance with ACP2, but, unlike the latter, they already showed some degree of crystallinity attributable to HAP (Fig. 2b). The spherical particles of our ACP evolve into clusters of thin flakes which seem to be morphologically different from ACP2, as quoted in literature [5,6,13], and are already crystalline, even if very poorly. The difference is possibly due to the solution composition, especially the pH which controls the ion speciation. Christoffersen
a4(H ÷ ) K~p(HAP)
where a is the ion activity, K,p(HAP) is the thermodynamic solubility product at 25°C ( 1 0 - 7 " 5 0 ) . The values of 13 for the most frequent phases are the following ones:
OCP 9.0 X l07 6.7 X l 0 7
W 5.8 X l 0 6 4.9 X 10 6
HAP 7.0 X 10 17 4.9 X 1017
S --
3
et al. [5] used a little more concentrated ([Ca] X [P] = 6 0 mM 2) and less basic solution (pH i = 7.4). These conditions are favourable to the nucleation of brushite and/or OCP after ACP and also of ACP2, which is found to be in pseudo-equilibrium with a solution at pH = 5.69 [13]. The same ACP2 is considered necessary for the development of OCP [5]. Our solutions, being more basic (pH i = 7.94), do not allow the formation of either brushite or OCP as distinct phases and are likely incompatible with ACP2; they directly lead to poorly crystallized HAP through only one amorphous phase.
4.3. Nucleation Due to high supersaturation, nucleation of ACP1 is very likely homogeneous. Direct nucleation of HAP through a homogeneous mechanism in pure calcium solutions is also possible, as shown by the fringes found just at the beginning; this event, which was observed only once, is likely casual due to the turbulent regime of mixing. As a matter of fact, the first HAP nuclei appear about half an hour after the precipitation of ACP and are associated with it. This relationship and thermodynamic considerations have suggested an heterogeneous nucleation of HAP on ACP as support [8]. However, in our case the HAP nuclei do not emerge from the surface of ACP particles, but appear to be homogeneously distributed within the amorphous particles. A perhaps more plausible mechanism is the formation of HAP nuclei by internal rearrangement of ACP through a solution mediated process, as already proposed by Heughe-
103
F. Abbona, A. Baronnet / Journal of Crystal Growth 165 (1996) 98-105
baert [14] and confirmed by Lazic for basic solutions [91. From a structural point of view, the conversion of ACP into HAP may be interpreted by considering that each C a 2 + in ACP has the same three innermost shells as in HAP out to a distance of 3.1 ,~ [4]. In the HAP structure the calcium polyhedra form columns running along the c-axis; these are surrounded by P O 4 tetrahedra and have great density in the (1010) layers. To extend order to longer distance and form HAP embryos, the Ca polyhedra occurring in ACP have to align themselves along the c-axis and properly arrange in the (10~0) layers with the P O 4 tetrahedra; the rearrangement is accompanied by an exchange of ions with solution. Once the embryos have reached the critical size, they can grow and cause the dissolution of ACP. This process will end by about 24 h; from then a continuous, slow increase of HAP crystallinity will take place.
I
'
/~
,
i
', i : ,
•
A progressive change in the crystal habit of HAP from the initial roundish flakes to the final dominant blades has been observed. The platy habit is ascribed to the presence of chloride in the crystallizing medium [15] or to the previous occurrence of OCP [16]. In our case chloride is very likely the responsible of flatness. The crystallites aged for 6 months in pure solutions exhibit shape and structure (Fig. If) comparable to those found in the human tooth enamel [17] and human alveolar bone apatite crystals [18]. The clearer axial zone, seen on some tiny needle-like crystals, may be indicative of screw dislocation and would suggest a slow recrystallization from superficial solution [ 19].
4.5. Effects of magnesium Magnesium exerts strong effects on morphology, crystal perfection, nucleation and growth kinetics. It reduces the size of ACP particles and makes the flakes more flexible and equant. It controls also the structure and crystal habit throughout the whole crystallization process. The effect is reflected in the final HAP crystallites: they are smaller, shorter, more irregular in shape and more rich in defects than those grown in pure milieu. Their crystallinity degree is
:
I
,
[
"~,~¢%~
:.i ~ ¸
i,.,
i
~
r~.
i~
:
I
,
i
i i' 20 °
28
30'
Fig. 4. X-ray diffraction pattern (Cu K) of hydroxyapatite after 6 months of aging in Mg-free (a) and Mg-doped solutions (b).
continuously increasing, whereas by FTIR first a decrease, then an increase in the crystallinity index was observed [10]. Anyway, it is lower than that of pure HAP, as shown by the peaks at dhk I = 2.82, 2.78, 2.72, 2.63 A, which overlap in the X-ray pattern (Fig. 4). Magnesium does not affect the formation of ACP1, but delays the HAP nucleation for at least two hours (whereas in pure solutions it occurs within 30 min). As the transformation of ACP into HAP and the subsequent HAP maturation are accompanied by a decrease of the M g / C a ratio in the solid phases, it is plausible to admit incorporation of magnesium by ACP [4,20]. Let us accept the alternative cluster model of Betts and Posner for ACP, in which fragments of HAP of composition Cag(PO4) 6 would be already present [21]. We can reasonably admit that Mg substitutes for Ca in these clusters. The replacement does not affect the immediate environment of calcium [20], but will cause local deformation with eventual shrinking of the columns of calcium and distortion of PO 4 polyhedra, as magnesium has a lower ionic radius than calcium (0.66 versus 0.99 ,~) and a shorter bond length with oxygen ( M g - O is about 2.1 A, whereas C a - O in HAP is in the range 2.443-2.555 ,~). Due to mechanical strains induced by Mg ions, nucleation of HAP is hindered and the HAP nuclei start to form when Mg ions will leave the structure. At the end only crystallites survive, which contain magnesium at a level which do not prejudice the structural stability; the others will dissolve. The slow release of Mg from o
4.4. Crystal habit
':
o
104
F. Abbona, A. Baronnet / Journal of Crystal Growth 165 (1996) 98-105
both ACP and HAP is one additional factor of the delayed HAP nucleation and growth. Magnesium can be also adsorbed at the surface of the HAP crystallites in the kink sites instead of calcium, as proved by the irregular shape and shortness of crystallites. Adsorption is very likely preferential on the end facets and by slowing down their growth rate increase their importance and will cause shortness. 4.6. E v o l u t i o n o f A C P
As concerns ACP and its evolution, our results are similar to those found by some authors (e.g., Refs. [1,7,9,22]) and at some variance with others (e.g., Refs. [5,6,13,23]) The differences can be attributed to the solution concentration and composition, mainly pH. At very low concentration (C < 2 mM) and neutral pH HAP may directly nucleate from solution [24]. At higher concentration and neutral or acidic pH ACP precipitates first and later evolves into brushite a n d / o r OCP and successively into HAP. (Just under these conditions ACP1 and ACP2 were found.) In basic solutions the amorphous phosphate is replaced by HAP without apparent intermediates. The latter case does not exclude the possibility that during conversion interlayering of OCP and HAP may occur, causing a higher d~0~0 spacing of HAP [25]. As a matter of fact higher values of dj0T0 were observed (d = 9.1 A) in some of our samples, but no interlayered mixtures were detected by HRTEM. Lamellar mixed crystals of OCP and HAP were observed by other authors at HRTEM, but only in the solutions where OCP may occur, i.e. at pH = 6.5, and in the presence of F [26]. This confirms once more the determinant role of pH and composition.
5. Conclusion By precipitation of calcium phosphates from basic solutions of low concentration at room temperature, an amorphous phase was obtained which corresponds to the form ACPI recently discovered. The other amorphous form denoted ACP2 was not clearly detected. From TEM studies it follows that ACP1 changes its morphology and evolves into plate-like
or ribbon-like clusters revealing an embryonic HAP structure. The only fringes observed at the high resolution of TEM in precipitates with and without magnesium correspond to HAP. No other crystalline phase was found. The conversion of ACP into HAP appears to be a solution mediated process. This is not the only mechanism, even if it is the usual one, as the TEM study showed that HAP may nucleate directly from solution at the same time as ACP. The crystallites from pure solutions have the shape of long, rectangular and thin blades, similar to those occurring in the human tooth enamel. The presence of magnesium in the solution prolongs by at least four times the induction period of HAP and make worse the crystal shape and the crystallinity, and smaller their size. During the aging of the precipitates, the increase of crystallinity is accompanied by the decrease of magnesium in the solid phases. The influence of Mg is attributed to its incorporation in the ACP clusters and HAP pre-nuclei and also to its adsorption onto the surfaces of ACP and of HAP crystallites.
Acknowledgements The financial supports by CNR (PB 88.01700.05) and MURST (60%) are gratefully acknowledged.
References [1] E.D. Eanes, Coll. Int. CNRS No. 23 (1975) 295. [2] J.C. Heughebaert and G. Montel, Calcif. Tiss. Int. 34 (1982) 103. [3] W. Kibalczyc and K. Bondarczuk, J. Crystal Growth 7l (1985) 751. [4] J.E. Harries, D.W.L. Hukins, C. Holt and S.S. Hasnain, J. Crystal Growth 84 (1987) 563. [5] J. Christoffersen, M.R. Christoffersen, W. Kibalczyc and A. Andersen, L Crystal Growth 94 (1989) 767. [6] W. Kibalczyc, J. Christoffersen, M.R. Christoffersen, A. Zielenkiewicz and W. Zielenkiewicz, J. Crystal Growth 106 (1990) 355. [7] E.D. Eanes and J.L. Meyer, Calcif. Tiss. Res. 23 (1977) 259. [8] J.L. Meyer and E.D. Eanes, Calcif. Tiss. Res. 25 (1978) 59. [9] S. Lazic, J. Crystal Growth 147 (1995) 147. [10] F. Apfelbaum, I. Mayer, C. Rey and A. Lebugle, J. Crystal Growth 144 (1994) 304. [11] F. Abbona and M. Franchini-Angela, J. Crystal Growth 104 (1990) 661.
F. Abbona, A. Baronnet/Journal of Co'sml Growth 165 (1996) 98 105 [12] H.E. Lundager Madsen, N~phrologie 5 (1984) 151. [13] M.R, Christoffersen, J. Christoffersen and W. Kibalczyk, J. Crystal Growth 106 (1990) 349. [14] J.C. Heughebaert, Thesis, Toulouse (1977). [15] P.L. Koutsoukos and G.H. Nancollas, J. Crystal Growth 55 (1981) 369. [16] W.E. Brown, J.P. Smith, J.R. Lehr and A.W. Frazier. Nature 196 (1962) 1050. [17] R.M. Frank and J.C. Voegel, Coll. Int. CNRS No. 230 (1975) 369. [18] F. Cuisinier, E.F. Bres. J. Hemmerle, J.-C. Voegel and R.M. Frank, Calcif. Tiss. Int. 40 (1987) 332. [19] J. Puech, J.-C. Heughebaert and G. Montel, J. Crystal Growth 56 (1982) 20.
105
[20] C. Holt, M.J.J.M. van Kemenade, J.E. Harries, L.S. Nelson, R.T. Bailey, D.W.L. Hukins, S.S. Hasnain and P.L. De Bruyn, J. Crystal Growth 92 (1988) 239. [21] F. Betts and A.S. Posner, Trans. Am. Cryst. Assoc. 10 (1974) 73. [22] E.D. Eanes, J.D. Termine and M.U. Nylen, Calcif. Tiss. Res. 12 (1973) 143. [23] L. Brecevic and H. Furedi-Milhofer, Calcif. Tiss. Res. 10 (1972) 82. [24] A.L. Boskey and A.S. Posner, J. Phys. Chem. 80 (1976) 40. [25] W.E. Brown, L.W. Schroeder and J.S. Ferris, J. Phys. Chem. 83 (1979) 1385. [26] M. Iijima, H. Tohda and Y. Moriwaki, J. Crystal Growth 116 (1992) 319.
C R Y S T A L G R O W T H
ELSEVIER
Journal of Crystal Growth 165 (1996) 98-105
A XRD and TEM study on the transformation of amorphous calcium phosphate in the presence of magnesium Francesco Abbona a,,, Alain Baronnet b a Dipartimento di Scienze della Terra, Unicersith della Calabria, 1-80736 Arcacacata di Rende (CS), ltaly 6 CRMCC, CNRS, Campus de Luminy, Case 913, F-13218 Marseille Cedex 9, France
Received 15 December 1995; accepted 5 February 1996
Abstract The evolution of the amorphous calcium phosphate (ACP), precipitated at 25°C from solutions of concentration [Ca] = [P] = 5 mM at pH = 7.94, was followed at fixed time intervals by combined X-ray, electron diffraction and TEM imaging. The same runs were carried out in the presence of magnesium ions (C = 1 raM). Only one kind of amorphous compound was observed (ACPI), which seems to change directly into apatite (HAP) without crystalline intermediate. Edge dislocations and grain boundaries were found by HRTEM analysis in HAP. The effects of magnesium on conversion of ACP into HAP and on the crystal structure and habit of HAP are interpreted in terms of incorporation and surface adsorption.
1. Introduction When calcium phosphates are precipitated from aqueous solutions, an amorphous phase is easily obtained, which is commonly denoted as ACP (amorphous calcium phosphate) and usually evolves into an hydroxyapatite-like phosphate (HAP). Intensive researches have been carried out mostly under physiological conditions. In spite of unquestionable progresses, some uncertainties remain about the conversion mechanism. ACP is said to convert directly into H A P by some authors [1-4], while others state that the transformation takes place through an intermediate like OCP [5-8] or by ACP dissolution and subsequent H A P crystallization [9]. Recently Christoffersen et al. [5] were able to show the occurrence of two amorphous calcium phosphates, A C P I and ACP2, having the same composition, but differ-
Corresponding author.
ing in morphology and solubility. Also the role of Mg, a cation common in the natural milieu where calcium phosphates form, is far from being fully understood. Recent results indicate that Mg does not affect the formation of ACP1, but only delay its conversion to ACP2 [6] and that it first decreases, then increases the H A P crystallinity during maturation [ 10]. The present work has been undertaken in order to study the conversion of ACP out of physiological conditions through a combined X-ray and TEM investigation and to examine the effect of Mg on the transformation, with special regard to the two new amorphous phases and their role in the process.
2. Experimental procedure All experiments were carried out at 25 + 0.2°C with solutions of the same concentration: [Ca] = [P]
0022-0248/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved PII S 0 0 2 2 - 0 2 4 8 ( 9 6 ) 0 0 1 5 6 - X
F. Abbona, A. Baronnet/Journal (~f Co'sml Growth 165 (1996) 98 105
= 5 mM, as used and tested previously [11]. These solutions were prepared by rapidly mixing equal volumes of a solution of CaCIo • 6 H 2 0 10 mM (A) and a solution of (NH4)2HPO 4 10 mM (B). Several
99
solutions were prepared and left at rest for fixed time intervals: t = 0, 2, 4, 8, 16, 32 min; 2, 5, 12, 24, 48 h; 6 months. At each of these times the precipitates were filtered by pressure suction through a Millipore
f
4. ~"-A
. . . .
Fig. 1. Analyses of precipitates from Mg-free solutions: t = 0: (a) electron-micrograph and -diffraction of the spherical amorphous ACP! particles: (b) two grain boundaries showing lattice fringes (d = 8.3 ,~) due to (1010) planes of HAP; t = 24 h: (c) thin flakes of HAP with few blades; (d) two grains differently oriented. White spots: the OH channels, surrounded by Ca ions, parallel to the c axis. Top right: a polygonized crystallite occurring after 48 h; t = 6 months: (e) elongated laminar crystals of HAP (the bar = 1500 A); (f) HAP blades showing fringes.
100
F. Abbona, A. Baronnet / Journal of Cr)'stal Growth 165 (1996) 98-105
0.22 p,m filter; they were treated with absolute ethanol to stop any reaction and then dried with acetone. The samples were submitted to X-ray and electron diffraction, to S E M and T E M studies and to X-ray energy dispersive ( E D S ) microanalysis. For T E M observations, a suspension o f the run product
in acetone was allowed to settle d o w n onto a carbon-coated Cu grid. L o w magnification brightfield images and selected area diffraction patterns of representative groups o f particles were systematically recorded with a J E O L 2000 F X electron microscope. High resolution T E M i m a g i n g was p e r f o r m e d
Fig. 2. Analyses of precipitates from Mg-doped solutions, t = 0: (a) the most common ACP1 spherical particles; (b) a second form of ACP similar to ACP2, observed in one sample; t = 32 min: (c) aggregates of curved, crystallizing flakes (the bar = 1000 ,~); (d) lattice fringes at d = 8.3 and 3.3 ,~, corresponding to HAP; t = 6 months: (e) HAP short blades with irregular external shape; (f) HAP crystallite showing two-dimensional order, damaged by the electron beam.
F. Abbona, A. Baronnet / Journal of Crystal Growth 165 (1996) 98-105
in multiple-beam bright field mode with the objective aperture allowed to cut space frequencies beyond the resolution of the instrument (2.8 A ~). C a / P and M g / C a ratios were checked with a X-ray energy dispersive Si(Li) detector (TRACOR Northern) attached to the microscope and also by atomic absorption spectrophotometry. Another series of experiments was carried out under the same conditions in the presence of Mg, which was added as MgClz.6H20 to the solution A. The final concentrations were: [Ca] = [P] = 5 mM; [Mg] = 1 mM. This is the maximum concentration of Mg allowing the conversion of ACP into HAP; at higher concentrations whitlockite is formed [11]. To check reproducibility, experiments were duplicated.
3. Results 3.1. From pure solutions
The first phase which precipitates immediately after the mixing of solutions is amorphous. It consists of spherical or disc-shaped particles loosely aogglomerated with a diameter in the range 400-1000 A and mean value of 700 A (Fig. la). In one sample collected in the very first moment (t = 0 rain) sharp fringes were observed with spacings of about 8.3 A (Fig. lb). This distance corresponds to the (10~0) planes of HAP (8.17 A). Edge dislocations are observed at the boundaries where the (1010) planes of two flakes meet under small tilt angles (Fig. lb). The bulk of precipitate remains amorphous for at least 16 min; it starts to crystallize within 30'. The X-ray and electron diffraction patterns taken after 30 min correspond to very poorly crystalline HAP. Crystallization is accompanied by a morphological change of the particles, which decompose into very thin roundish flakes sometimes folded and wrapped. Rare fringes distant 8.3 and 3.3 A, corresponding to the (0002) planes, are observed. The ordered ~gions are very small (from 130 X 33 to 250 X 125 A2) and irregular in shape. After 24 h the flakes have changed into short strips of greater size (about 900 X 120 ~2) (Fig. lc). Most flakes show HRTEM fringes at 8.3 and few at 9.1 A. An hexagonal array of white spots bear evidence of the channels in the HAP structure seen along [0001] (Fig. ld). With aging the crystal-
101
lites tend to become better polygonized (Fig. l d). After 6 months all the precipitate is formed by rather regular, thin laths having a mean size of 1400 x 250 ,~2 (Fig. le). Few crystallites are needle-like and seem to be hollow. New fringes are found at 5.2 and 4.1 A, corresponding to the (1011) and (1120) planes respectively (Fig. lf). The occurrence of OCP was never observed. 3.2. From solutions doped with Mg
Also from these solutions the phase which precipitates first is amorphous and consists of spherical particles of smaller size (from 300 to 1100 A, with a mean value of about 500 ,~) (Fig. 2a). In one sample (Fig. 2b) thin fibers or flakes interlaced giving the cluster a soft and gel-like aspect reveal a weak crystallinity by electron diffraction, attributable to HAP. However two hours are necessary for crystallization to start and at least five hours for HAP crystallinity to clearly disclose. The flakes are numerous, interwoven and curved (Fig. 4c) and exhibit fringes having distances of 8.3 and 3.3 ,~ (Fig. 4d). After six months the crystallites consist of irregular blades showing fringes at 8.3 A (Figs. 2e, 2f), with maximum size of 1000 X 250 ~2. They are easily damaged by the electron beam (Fig. 2f). The atomic ratio Mg/Ca, measured in the solid phase, is progressively decreasing with time: from 0.04 it drops to 0.009 in the precipitate aged for 48 h (Fig. 3). The same trend was already noticed [5].
Mg/Ca ) solid 0.04.
0.03-
0.02-
0.01-
0 0
I
1
12
24
I
36
I
b
48 hours
Fig. 3. Change of the M g / C a atom ratio in the solid phases with time.
F. Abbona, A. Baronnet/Journal of Co'stal Growth 165 (1996) 98 105
102
aS(Ca2+)aS(HPO~ )
4. Discussion [~HAP =
4.1. Supersaturation The solutions we used in our experiments were highly supersaturated. The degree of supersaturation was calculated by a computer programme already applied [12]. For HAP it is given by the ratio
Mg 0 mM 1 mM
1 0.025 0.027
pH 7.943 7.928
ACP 30.8 24.7
B 16.5 15.3
M 32.9 30.5
(where I = ionic strength; B = brnshite; M = monetite; OCP = octacalcium phosphate; W = whitlockite; S = struvite). The value for ACP, which is not a true phase, is only indicative of its tendency to precipitate; this happens whenever [3ACp > 1. The presence of Mg implies a low supersaturation with respect to struvite, MgNH4PO4.6H20 , but does not alter in a significant way the supersaturation with respect to the calcium phosphates and the other physico-cbemical properties of the solutions. As 13ACe >> 1, ACP nucleates as first phase instead of HAP (Ostwald's rule). Its immediate precipitation decreases supersaturation with respect to the other phases, so that their nucleation is hindered.
4.2. Morphology The most frequent morphology of ACP is spherical and corresponds to ACP1, as found by Christoffersen et al. [5]. The second type of ACP, which was denoted ACP2 by the same authors and represents the evolution of ACPI, was observed in two samples only, aged for 4 min and 24 h in the presence of Mg. They were recognized on the basis of their morphological resemblance with ACP2, but, unlike the latter, they already showed some degree of crystallinity attributable to HAP (Fig. 2b). The spherical particles of our ACP evolve into clusters of thin flakes which seem to be morphologically different from ACP2, as quoted in literature [5,6,13], and are already crystalline, even if very poorly. The difference is possibly due to the solution composition, especially the pH which controls the ion speciation. Christoffersen
a4(H ÷ ) K~p(HAP)
where a is the ion activity, K,p(HAP) is the thermodynamic solubility product at 25°C ( 1 0 - 7 " 5 0 ) . The values of 13 for the most frequent phases are the following ones:
OCP 9.0 X l07 6.7 X l 0 7
W 5.8 X l 0 6 4.9 X 10 6
HAP 7.0 X 10 17 4.9 X 1017
S --
3
et al. [5] used a little more concentrated ([Ca] X [P] = 6 0 mM 2) and less basic solution (pH i = 7.4). These conditions are favourable to the nucleation of brushite and/or OCP after ACP and also of ACP2, which is found to be in pseudo-equilibrium with a solution at pH = 5.69 [13]. The same ACP2 is considered necessary for the development of OCP [5]. Our solutions, being more basic (pH i = 7.94), do not allow the formation of either brushite or OCP as distinct phases and are likely incompatible with ACP2; they directly lead to poorly crystallized HAP through only one amorphous phase.
4.3. Nucleation Due to high supersaturation, nucleation of ACP1 is very likely homogeneous. Direct nucleation of HAP through a homogeneous mechanism in pure calcium solutions is also possible, as shown by the fringes found just at the beginning; this event, which was observed only once, is likely casual due to the turbulent regime of mixing. As a matter of fact, the first HAP nuclei appear about half an hour after the precipitation of ACP and are associated with it. This relationship and thermodynamic considerations have suggested an heterogeneous nucleation of HAP on ACP as support [8]. However, in our case the HAP nuclei do not emerge from the surface of ACP particles, but appear to be homogeneously distributed within the amorphous particles. A perhaps more plausible mechanism is the formation of HAP nuclei by internal rearrangement of ACP through a solution mediated process, as already proposed by Heughe-
103
F. Abbona, A. Baronnet / Journal of Crystal Growth 165 (1996) 98-105
baert [14] and confirmed by Lazic for basic solutions [91. From a structural point of view, the conversion of ACP into HAP may be interpreted by considering that each C a 2 + in ACP has the same three innermost shells as in HAP out to a distance of 3.1 ,~ [4]. In the HAP structure the calcium polyhedra form columns running along the c-axis; these are surrounded by P O 4 tetrahedra and have great density in the (1010) layers. To extend order to longer distance and form HAP embryos, the Ca polyhedra occurring in ACP have to align themselves along the c-axis and properly arrange in the (10~0) layers with the P O 4 tetrahedra; the rearrangement is accompanied by an exchange of ions with solution. Once the embryos have reached the critical size, they can grow and cause the dissolution of ACP. This process will end by about 24 h; from then a continuous, slow increase of HAP crystallinity will take place.
I
'
/~
,
i
', i : ,
•
A progressive change in the crystal habit of HAP from the initial roundish flakes to the final dominant blades has been observed. The platy habit is ascribed to the presence of chloride in the crystallizing medium [15] or to the previous occurrence of OCP [16]. In our case chloride is very likely the responsible of flatness. The crystallites aged for 6 months in pure solutions exhibit shape and structure (Fig. If) comparable to those found in the human tooth enamel [17] and human alveolar bone apatite crystals [18]. The clearer axial zone, seen on some tiny needle-like crystals, may be indicative of screw dislocation and would suggest a slow recrystallization from superficial solution [ 19].
4.5. Effects of magnesium Magnesium exerts strong effects on morphology, crystal perfection, nucleation and growth kinetics. It reduces the size of ACP particles and makes the flakes more flexible and equant. It controls also the structure and crystal habit throughout the whole crystallization process. The effect is reflected in the final HAP crystallites: they are smaller, shorter, more irregular in shape and more rich in defects than those grown in pure milieu. Their crystallinity degree is
:
I
,
[
"~,~¢%~
:.i ~ ¸
i,.,
i
~
r~.
i~
:
I
,
i
i i' 20 °
28
30'
Fig. 4. X-ray diffraction pattern (Cu K) of hydroxyapatite after 6 months of aging in Mg-free (a) and Mg-doped solutions (b).
continuously increasing, whereas by FTIR first a decrease, then an increase in the crystallinity index was observed [10]. Anyway, it is lower than that of pure HAP, as shown by the peaks at dhk I = 2.82, 2.78, 2.72, 2.63 A, which overlap in the X-ray pattern (Fig. 4). Magnesium does not affect the formation of ACP1, but delays the HAP nucleation for at least two hours (whereas in pure solutions it occurs within 30 min). As the transformation of ACP into HAP and the subsequent HAP maturation are accompanied by a decrease of the M g / C a ratio in the solid phases, it is plausible to admit incorporation of magnesium by ACP [4,20]. Let us accept the alternative cluster model of Betts and Posner for ACP, in which fragments of HAP of composition Cag(PO4) 6 would be already present [21]. We can reasonably admit that Mg substitutes for Ca in these clusters. The replacement does not affect the immediate environment of calcium [20], but will cause local deformation with eventual shrinking of the columns of calcium and distortion of PO 4 polyhedra, as magnesium has a lower ionic radius than calcium (0.66 versus 0.99 ,~) and a shorter bond length with oxygen ( M g - O is about 2.1 A, whereas C a - O in HAP is in the range 2.443-2.555 ,~). Due to mechanical strains induced by Mg ions, nucleation of HAP is hindered and the HAP nuclei start to form when Mg ions will leave the structure. At the end only crystallites survive, which contain magnesium at a level which do not prejudice the structural stability; the others will dissolve. The slow release of Mg from o
4.4. Crystal habit
':
o
104
F. Abbona, A. Baronnet / Journal of Crystal Growth 165 (1996) 98-105
both ACP and HAP is one additional factor of the delayed HAP nucleation and growth. Magnesium can be also adsorbed at the surface of the HAP crystallites in the kink sites instead of calcium, as proved by the irregular shape and shortness of crystallites. Adsorption is very likely preferential on the end facets and by slowing down their growth rate increase their importance and will cause shortness. 4.6. E v o l u t i o n o f A C P
As concerns ACP and its evolution, our results are similar to those found by some authors (e.g., Refs. [1,7,9,22]) and at some variance with others (e.g., Refs. [5,6,13,23]) The differences can be attributed to the solution concentration and composition, mainly pH. At very low concentration (C < 2 mM) and neutral pH HAP may directly nucleate from solution [24]. At higher concentration and neutral or acidic pH ACP precipitates first and later evolves into brushite a n d / o r OCP and successively into HAP. (Just under these conditions ACP1 and ACP2 were found.) In basic solutions the amorphous phosphate is replaced by HAP without apparent intermediates. The latter case does not exclude the possibility that during conversion interlayering of OCP and HAP may occur, causing a higher d~0~0 spacing of HAP [25]. As a matter of fact higher values of dj0T0 were observed (d = 9.1 A) in some of our samples, but no interlayered mixtures were detected by HRTEM. Lamellar mixed crystals of OCP and HAP were observed by other authors at HRTEM, but only in the solutions where OCP may occur, i.e. at pH = 6.5, and in the presence of F [26]. This confirms once more the determinant role of pH and composition.
5. Conclusion By precipitation of calcium phosphates from basic solutions of low concentration at room temperature, an amorphous phase was obtained which corresponds to the form ACPI recently discovered. The other amorphous form denoted ACP2 was not clearly detected. From TEM studies it follows that ACP1 changes its morphology and evolves into plate-like
or ribbon-like clusters revealing an embryonic HAP structure. The only fringes observed at the high resolution of TEM in precipitates with and without magnesium correspond to HAP. No other crystalline phase was found. The conversion of ACP into HAP appears to be a solution mediated process. This is not the only mechanism, even if it is the usual one, as the TEM study showed that HAP may nucleate directly from solution at the same time as ACP. The crystallites from pure solutions have the shape of long, rectangular and thin blades, similar to those occurring in the human tooth enamel. The presence of magnesium in the solution prolongs by at least four times the induction period of HAP and make worse the crystal shape and the crystallinity, and smaller their size. During the aging of the precipitates, the increase of crystallinity is accompanied by the decrease of magnesium in the solid phases. The influence of Mg is attributed to its incorporation in the ACP clusters and HAP pre-nuclei and also to its adsorption onto the surfaces of ACP and of HAP crystallites.
Acknowledgements The financial supports by CNR (PB 88.01700.05) and MURST (60%) are gratefully acknowledged.
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