Chapter VI Enamel Apatite Nucleation and Crystal Growth G. H. NANCOLLAS Chemistry Department, State University of New York at Buffalo, Buffalo, New York 14214, U.S.A. J Dent Res 58(B):861-869, March 1979
The course of enamel remineralization in aqueous solution is markedly dependent upon factors such as ionic medium supersaturation, pH, ionic strength, and the relative concentration of mineralizing surface. Although numerous attempts have been made to determine the mechanism of both the spontaneous and seeded precipitation reactions, it is only recently, through the use of a constant solution composition technique, that quantitative information can be obtained concerning the intermediate calcium phosphate phases which are formed during mineralization. Studies of surface phases in the presence of potential anti-calculus and anti-caries agents are facilitated using this
approach.
Introduction. The possibility of the repair, through remineralization, of enamel damaged bv caries attack is extremely attractive.1'2 Despite numerous attempts to achieve this goal, it has not been possible to reconstitute the enamel apatite crystals in a form identical with that in natural enamel. Of the few kinetic studies which have been made, most have been concerned with calcium phosphate mineralizing solutions considerably more supersaturated than those typical of in vivo conditions. Since the course of the mineralization reaction is markedly dependent upon the concentrations of lattice ions in the mineralizing medium,3-5 such studies may provide little insight into the mechanism of apatite remineralization at a tooth surface. In the oral environment, both mineralization and demineralization processes are involved in the post-eruptive maturation of dental enamel.6'7 The relatively high calcium and phosphate concentrations of saliva result in the deposition of mineral while demineralization occurs at the low pH regions produced by bacterial action. In an in vitro study of the remineralization of ground sections,8 it was shown that the initial rapid uptake of calcium phosphate slowed down after 48 hours and effectively stopped for three weeks. This result was interpreted in terms of the mineralization of a sound outer layer blocking access to the underlying white spot. Clinical evidence
also points to the remineralization of early carious lesions with the reversal of whitespot formation9,10,11 but the deposited mineral was not characterized chemically. More recently,12 a calcium phosphate buffer solution containing fluoride and solid dicalcium phosphate dihydrate (CaHPO4-2H20, DCPD) has been shown to be effective in the remineralization of sections of carious dentin in vitro, and x-ray diffraction measurements revealed the presence only of a relatively well-characterized apatite phase in the mineral deposited in the tissue.13 Much of the work on enamel remineralization has been done with bulk human enamel artificially demineralized in acid solutions, and the extent of reaction was followed either analytically14'15 or microscopically.16 The presence of fluoride in the remineralizing solution has been shown to enhance the formation of amorphous calcium phosphates in the upper surface of remineralizing enamel17, and calcium fluoride was observed when acidulated calcium fluoride-calcium phosphate solution was used. Recently,18 fluoride uptake by enamel surfaces exposed to mineralizing solutions was studied and it was shown that fluoride concentrations greater than 0.05 mM tended to form calcium fluoride deposits on the tooth surface while lower concentrations resulted mainly in fluoroapatite, FAP. In a pH- and pF-stat study of the kinetics of lesion remineralization15 it was shown that in the presence of 1 ppm fluoride, the material deposited was most likely fluoridated HAP; the overall rate of mineralization was about twice that in the absence of fluoride ion. In carious lesions formed in vivo, mineral deposits have been found with morphologies different from apatite19 and these have presumably been formed by the precipitation of calcium phosphate phases within the lesion. The chemical characterization of the deposits is difficult to achieve since, even if subsequent dissolution studies are made, small errors in measured concentrations can often preclude differentiation between the 861
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
862
J Dent Res Special Issue B, 1 9 79
NANCOLLAS
possible calcium phosphate phases. In the formation of plaque, aggregates of cells and intercellular binding proteins produce a porous structure at the tooth surface through which saliva may diffuse. In all plaques, the concentration of calcium, phosphate, and magnesium is markedly higher than in saliva and, during calculus formation, the organic material of the plaque becomes embedded in the mineralized deposit. The presence of magnesium ion appears to stabilize calcium phosphate precursors20, and a synergistic effect between magnesium and adenosine triphosphate in delaying the conversion of amorphous calcium phosphate to hydroxyapatite, HAP, has also recently been described21. The possibility of relating the mineralizing activity of dental plaque in vitro with dental calculus formation in vivo in the same individual22 makes mineralization kinetic experiments particularly attractive. Young plaque has been shown, by x-ray diffraction, to contain DCPD23, and the results of kinetic studies indicate that DCPD is a kinetically-favored phase in the mineralization of dental enamel at pH values below about 5.1.5 The importance of kinetic factors, coupled with a knowledge of thermodynamic precipitation driving forces, in determining the nature of the phases formed, is now well-established. As we shall see, however, the conventional in vitro mineralization experiments would require impossibly high precisions in the analytical data in order to distinguish between the calcium phosphate phases, particularly under the relatively low supersaturations typical of those in vivo where the amount of precipitated phase is small.
Mineralization of synthetic calcium phosphates. Hydroxyapatite, HAP, is generally considered to be the model compound for bone and tooth mineral and, although numerous studies have been made of HAP precipitation, there is still considerable uncertainty about the course of the reaction. The stoichiometry of the initially-formed solid expressed as the calcium/phosphate molar ratio (hereafter, Ca/P), is invariably less than the 1.67 required for HAP.24'25 Indeed, over a wide range of concentrations at physiologic pH, the value Ca/P is of the order
1.45 ± 0.05. The similarity of this stoichiometric ratio to the value, 1.50, for tricalcium phosphate, TCP,26 has prompted the proposal of TCP as the "amorphous" precursor to HAP formation in aqueous solutions. An autocatalytic conversion of TCP to HAP was suggested for both synthetic27 and natural28 apatite formation. The close crystallographic similarity of octacalcium phosphate, OCP, to HAP led Brown29 to propose this phase as an HAP precipitation precursor; the hydrolysis of OCP to HAP taking place with minimal lattice reorientation, one layer at a time. DCPD has also been invoked as the initially precipitating phase30 and, as the calcium phosphate of simplest stoichiometry, it is kinetically favored at 370C and in slightly acid solutions (pH values less than about
5.1).5
Numerous investigations have been directed in recent years at the growth of synthetic apatite seed crystals in metastable supersaturated solutions of calcium phosphate.3,4,3l-3S In these studies, the calcium- and phosphate-concentration changes, with time, following the addition of seed, were highly reproducible, provided that factors such as degree of supersaturation, temperature, ionic strength, and the presence of traces of foreign ions were carefully controlled. At physiological pH, the overall reaction involved not only the formation of different calcium phosphate phases but also the concomitant dissolution of a transient phase, stoichiometrically close to OCP, which is formed in the initial stages of the reaction.3 In Fig. 1 is shown a typical plot of calcium concentration against time following the addition of HAP seed crystals to a calcium phosphate solution supersaturated with respect to all phases, DCPD, OCP, TCP and HAP. The observed inflection in the growth curve probably reflects the superposition of a number of kinetic processes which take place at different rates and points to the involvement of more than one calcium phosphate phase in the precipitation. The results of a recent transmission electron microscopic investigation also support this multiphase explanation.36 The stoichiometry of the solids formed may be expressed in terms of the molar ratios of calcium to phosphate, L [Ca] /A(P04], which have precipitated at any instant. A [Ca] = Tc. (initial) - TCa (time t) and A [P04] =
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
Vol. 58(B)
863
Tp (initial) Tp (time t), where TCa and Tp -
total molar concentrations of calcium and phosphate, respectively, in the supersaturated solution. During the pH1.2 DCPD 00JSA statted seeded growth experiment at rela4' tively high supersaturation, such as that shown in Fig. 1, the value of A [Cal /A[P04 ] X ~~~~~~~~OCPso for the initially depositing phase was invariably greater than that of HAP, and, after TcP about 20 min. of reaction, decreased to 1.45 ± 0.05 which remained quite constant for MAP some 300 min. At longer times, the ratio z00 UhiA 400 goo gradually increased but the value of 1.67 required for HAP stoichiometry was not reached even after 3 days of reaction.3 A Fig. 1 Growth of calcium phosphate on [Ca] /'L P04 ] ratios consistently higher than 1.67 for the initial stages of the precipitation HAP seed crystals (200 mg 1-1). Curve A, plot of Taagainst time (initial TCa = 1.562 x 10-3M, process were also reported recently by T =0.973 x 10-3M, pH = 7.40, 250(Q). Curve B Moreno et al.37 from seed growth studies of SSA (m2g-1 right-hand ordinate) against at lower supersaturations (TCa = 0.3mM) pl'ot time at pH =7.40. Curve C, plot of SSA changes at in which the pH was allowed to decrease pH = 5.0. Approximate concentration levels correfrom an initial physiological value, 7.4, responding to saturation with respect to DCPD, to about 6.8 during the reaction. It was OCP, TCP and HAP are also shown. suggested that the high values of the ratio seen that there was a pronounced maximum were due either to the formation of an enriched calcium phase or to the initial at 120 mi. growth where the SSA was adsorption of calcium ions onto the HAP twice that for the seed material. The effecsurface. As the reaction proceeded, the A tive SSA of the growth product itself was [Cal /A[P041 value approached 1.67 as large (approx. 230 m2g-1) reflecting the microcrystallinity of the precursor phase more HAP precipitated,37 but at that stage, the data were not comparable with the formed on the surface of the HAP seed results of pH-stat work because the pH had crystals. The subsequent decrease in SSA already decreased markedly from the initial reflected the increased crystallinity of the value of 7.4. Evidence for OCP as a precur- solid as crystal growth proceeded. In the interpretation of the results of sor phase which is formed on HAP seed surfaces during the concentration in time mineralization experiments in vitro, the proflles such as that shown in Fig. 1, was degree of supersaturation with respect to obtained from dissolution kinetic studies.3 each of the calcium phosphate phases may Solid phases, withdrawn at known time be expressed in terms of the free energies intervals from the crystallization cell, were of formation. transferred to a sodium chloride solution of AG = RTln(IP/Kso) the same ionic strength as that of the growth medium and dissolution was followed at a pH where Kw is the thermodynamic solubility of 7.40, maintained by the pH-stat controlled product and the activity products, IP, of addition of dilute acid. The Ca/P ratio of the individual calcium phosphate phases the surface phase 1.34 + 0.02, which had are expressed, for dicalcium phosphate been formed in the crystallization experi- dihydrate, ments, was close to that for OCP. At longer IPDCPD = [Ca2+1 [HPO2-] dissolution times, however, this ratio was f24 considerably larger 1.57,3 indicating that for OCP, IPocp (lCa2+] f2 )4([PO3- ] OCP was no longer available for dissolution, f3)3 [HI] f, having already been dissolved or hydrolyzed for TCP, 'PTCP ([Ca2 f2 )3([PO4 in the earlier composite growth process. f3)2 The specific surface area, SSA, of the and for HAP, IPHAP =([Ca2 If2)5([PO34] product grown at various times in the pHf3)3Kw/[H+] f, at statted experiments high supersaturation is plotted against time in Fig. 1. It can be Kw is the thermodynamic activity product are the
I..
I
04
0
-
=
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
864
NANCOLLAS
of water at 3 70C. The activity coefficients be calculated from the extended form of the Debye-Huckel equation proposed by Davies.40 In the above expressions, the square brackets enclose the concentrations of the free ionic species. Neglect of activity coefficient corrections in the calculation of precipitation-driving forces results in errors amounting to several orders of magnitude in the ionic product terms for substances such as HAP. The epitaxial growth of one calcium phosphate phase upon another may play an important role in the remineralization of tooth components in vivo and studies have been under conditions closely simulating plaque and calculus production in the mouth with pH values of 4.5 - 5.1. Calcium phosphate solutions supersaturated with respect to HAP and DCPD were prepared by adjusting the pH of a mixture of calcium chloride and potassium dihydrogen phosphate solutions to 5.60 by the slow addition of potassium hydroxide solution. Following the addition of the HAP seed crystals, the rate curve in Fig. 2 shows an initial induction period, preceding the regular fall of concentration with time. It can be seen in Fig. 2 that the decrease in calcium and phosphate concentrations were the same to within experimental error (Ca/P = 1.02 ± 0.03) indicating the exclusive formation of DCPD, confirmed by x-ray diffraction, despite the fact that the solutions were also appreciably supersaturated with respect to the thermodynamically stable phase, HAP. The growth of DCPD seed crystals has been shown38 to follow a rate equation,1 where N is the number of moles of DCPD
J Dent Res Special Issue B, 19 79
may
-dN = KsN2 dt to be precipitated before equilibrium is reached, s is a function of the number of active growth sites, and K is the rate constant. The applicability of this equation in describing the crystallization following the delay period in Fig. 2 is illustrated in Fig. 3 in which the integrated form is plotted. All the calcium phosphate seed materials were effective nucleators for DCPD which, in the scanning electron microscope, was seen as large platelets growing from the surface of the seed crystals. HAP was a particularly effective surface for the nucleation and growth of DCPD. It can be seen that
Fig. 2 - Tp and TCa (°, left-hand ordinate) and relative rate of DCPD nucleation (v, righthand ordinate) plotted against time (initial TCa= 6.346 mM, Tp = 6.317 mM, pH = 5.59, 370C, 23 mgl-l HAP seed. -A G values (kcal mol-1) for HAP, 9.72: DCPD, 0.396: TCP, 0.486).
increasing the amount of HAP seed material produced an increase in the rate of DCPD growth (slopes of the linear portions of Fig. 3) with very little decrease in the duration of the initial induction effect. The latter was highly reproducible despite the fact that these initial stages reflected the nucleation of DCPD on the HAP seed substrate. The applicability of rate equation 1 for more than 70% of the growth reaction suggested that all the nucleation takes place during the initial induction period. The absence of additional secondary nucleation was also evidenced by the results of the scanning electron microscopic studies. It is interesting to note that no corrections were necessary for the approximately three-fold increase in surface area which occurred during the crystal growth. This strongly supports the suggestion that crystallization of DCPD took place on the growth sites formed during the induction period and that no new sites were produced during the subsequent crystal growth step. Similar observations have been made in the crystal growth of other sparingly-soluble electrolytes.39 Further evidence in support of
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
865
Vol. 58(B)
this suggestion was obtained from the results of specific surface area measurements of the seed material and of the solids extracted at various times, shown in Fig. 1. A modest decrease in total SSA from 39.7 m2g-1 initially, to 36.3 m2g-1 at the end of the induction period implied the production of an appreciable number of small DCPD nuclei. During the subsequent crystal growth steps of DCPD, the SSA decreased sharply to 29.7 m2g- 1, reflecting crystal growth on the already-formed DCPD nuclei. It is interesting to contrast these results with the SSA profile at physiological pH (Fig. 1) for which the marked increase in the early stages of crystal growth reflected the formation of microcrystalline precursors.
Fig. 3 - Plots of the integrated form of Eqn. 1. Initial TCa = 1.489 x 10-2, Tp = 1.504 x 10-2M, -AGDCPD = 0.406 kcal mol-1; -A GHAP = 7.13 kcal mol-1, o, 'A, v, replicated experiments with 228 mgl-l HAP seed, +, 114 mgl-l and A, 56 mgl-l HAP seed.
The formation of DCPD on the surface of the added HAP seed crystals involves a nucleation step and the subsequent crystal growth of the DCPD nuclei.41 The rate of heterogeneous nucleation is governed by the degree of lattice mismatch between DCPD and HAP and the supersaturation, sharply decreasing as the latter decreases. This is shown in Fig. 2 in which the relative nucleation rate was calculated from appropriate nucleation expressions.41 It can be
seen that the nucleation is essentially complete within the induction period, falling to zero at the commencement of the normal DCPD growth. The reproducibility of the time plots of calcium and phosphate concentration in these experiments is quite striking and the quadratic dependence of the rate of growth upon the supersaturation, following the induction period, is indicative of a surface-controlled process. This is further supported by the observed independence of the growth rate on the rate of stirring. It is significant that when the concentration of HAP seed crystals, added to the supersaturated solution, was increased to 226449 mgh-1, the course of the mineralization reaction was markedly different.41 In place of the ATCa/ATp value of unity at lower seed concentrations, the ratio at high seed, 1.52 ± 0.04, is indicative of more basic calcium phosphate phase. An even more sensitive indicator of calcium phosphate phase precipitated is the ratio of base added for constant pH to phosphate precipitated. The observed value, 1.88 + 0.06, also militates against DCPD as the growth phase. X-ray powder diffraction studies confirm the absence of peaks characteristic of DCPD or TCP, and indicate only an amorphous calcium phosphate phase. At the higher HAP seed concentrations, the rate of growth of the more basic phase is sufficiently fast to markedly reduce the effective DCPD nucleation rate in the initial periods so that this phase does not form. In marked contrast, for low HAP seed concentrations (Fig. 2), the rate of growth of the basic phase is near zero, thus maintaining TCa and Tp at sufficiently high levels to facilitate the nucleation of DCPD and its subsequent growth.
Mineralization of tooth components. A highly reproducible seeded growth technique has been used to investigate the mineralization of seed material prepared from human tooth enamel, dentin, and calculus.42,43 The kinetics of growth of calcium phosphate on human enamel has been studied at 25 and 370C and pH= 7.40 in mestastable supersaturation solutions of calcium phosphate (TCa = 1.50 X 10-3M, Tp = 0.87 x 10-3M). The grown material was characterized chemically, by specific surface area measurements, and by subsequent dissolution experiments. Upon the
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
866
J Dent Res Special Issue B, 19 79
NANCOLLAS
addition of seed material, growth started immediately as is shown in Fig. 4, which also indicates the calcium levels corresponding to saturation with DCPD, OCP and TCP. It can be seen that the solution became undersaturated with respect to DCPD after about 10 min., and to OCP after about 60 min. In the experiments in which the inorganic component of dentin was used as seed following the removal of the organic matrix, it can be seen (Fig. 4) that the more efficient hydrazine removal of the organic phase as compared with ethylenediamine, en, treatment, resulted in a more effective calcium phosphate nucleator. It is significant that increasing the seed concentration to 1.0 gl-t, and more markedly at 3.0 gl-l (Fig. 4), resulted in the appearance of an inflection in the growth curve. The initial rate of reaction normalized for seed concentration, --ATCa/
At'1Nd0.4,uMl-1min-lg-l,
decreased at 120
min. and increased again at 4 hours to -ATCa/t0.3 5 6,Ml- I min.- 1 g- 1 The inflections were also observed in the phosphate concentration, and in the potassium hydroxide required to maintain the pH at 7.4. The results are similar to those outlined above using synthetic seed material, and the evidence again points to the involvement of more than one phase in the seeded growth of dental enamel. In every experiment made at these higher supersaturations (TCa = 1.200mM, Tp = 0.719mM), the Ca/P ratio was 1.45 ± 0.05 during the first twentyfour hrs. of growth. A similar value was found in HAP seeded growth experiments and a gradual increase from 1.44 to 1.54 was observed after two days, but the 1.67 required for HAP stoichiometry was not reached even after three days of reaction. The difficulty of determining the stoichiometry of the precipitating phases from the small changes in calcium and phosphate concentrations in the supersaturated solution has already been discussed. In Fig. 4, the specific surface area, SSA, of the products grown at various time intervals is plotted as a function of time. It can be seen that there is a pronounced increase in the flrst 200 min of reaction until the composite SSA, rising to a broad maximum to about 300 min, is almost four times that of the seed material. This indicates that the value of the growth product itself is appreciable (X\'145 m2g9-). The concomitant change in morphology is apparent from scanning electron micro-
Fig. 4 - The growth of enamel seed at high supersaturation. TCa = 1.438 mM, Tp = 0.874 mM, 250C, A, 3.0 g seed 1-1, +, 1.0 g seed 1-1. v, TCa = 1.582 mM, T= 0.943 mM, 0.2 g seed 1-1 (hydrazine-treated), o, TCa = 1.492 mM, T = 0.876 mM, 0.2 g seed 1-1 (en treated). Plot oFSSA (@), TCa = 1.507 mM, T = 0.872 mM, 0.2 g seed 1-1) against time. flJIl represents saturation levels with respect to phase indicated.
graphs which show the formation of an intermediate phase consisting of hexagonal platelets on the surface of enamel seed. Studies of the kinetics of dissolution of this phase under pH-statted conditions, controlled to a value of 7.40 by the addition of 0.005M hydrochloric acid, indicated a chemical composition close to that of OCP for this intermediate phase when it dissolved into 0.006M potassium chloride solution.42 The use of sodium chloride resulted in a higher value, A TCa/ATp = 1.40, and for 0.002M magnesium chloride, the ratio was 1.61. These relatively higher values can be explained in terms of the known surface exchange of calcium for magnesium, and the tendency for sodium ion incorporation (up to 5%) in an apatite lattice. No evidence was found for potassium ion uptake. It has been shown42 that in lessconcentrated phosphate solutions (TC a 0.30 x 10-3M, Tp = 0.02 x 10-3M), supersaturated only with respect to TCP and HAP, the growth product has a stoichiometry close to the 1.67 required for HAP. It is significant that the scanning electron micrographs of the solid phases formed under these conditions indicated the complete absence of the intermediate hexagonal platelet-like phase observed at the higher concentrations. These results show that the nature of the calcium phosphate phase which is formed on the surface of the enamel seed material is markedly dependent
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
867
Vol. 58(B)
the degree of supersaturation with respect to calcium phosphate. The kinetics of growth of calcium phosphates on human enamel, whole human dentin and human calculus have been studied at 370C at pH values from 4.97 - 5.10 held constant by the pH-stat addition of base.43 The solutions were supersaturatedwith respect to both DCPD and HAP, and, as was found with synthetic HAP seed material, following an initial induction period, DCPD crystallization occurred on the surface of the solid phases in all cases. The typical plots of the intergraded rate equation1 in Fig. 5 show that, following well-defined induction periods the surfacecontrolled growth of DCPD closely followed this rate equation. The relative rate of mineralization is given by the slopes of these lines, and it can be seen that, as expected, synthetic HAP is the most effective nucleator of DCPD. In Fig. 5, the rate equation used takes into account both dilution, accompanying sampling of the supersaturated solution, and surface area changes of seed material during growth. f f(vb )dvb, where vb is the volume of solution added to maintain the pH, provides a more accurate assessment of the course of the mineralization than the ordinate (N-1-Ni-1) of Fig. 3. Scanning electron micrographs of the solid phases sampled during the induction periods clearly showed the nucleation of DCPD on the substrate surfaces in all cases, even though little change had taken place in the bulk solution calcium and phosphate concentrations. The rate of growth was also independent of stirring dynamics within the experimentally accessible range, strongly suggesting that nucleation was completed during the initial induction period and that the subsequent rate of growth of the DCPD was controlled by a surface reaction. It was found that the relative order of nucleating ability for DCPD is HAP>enameL>calculus>dentin. These differences may be explained by considering the number of active sites per unit surface area of substrate. HAP has the greatest nucleating activity, since enamel has 1-2%, calculus 10-30%, and dentin 20% of organic material which normally does not readily nucleate calcium phosphates. In addition, pyrophosphate and other substances thought to be present in enamel, calculus, and dentin, are known to act as inhibitors for DCPD crystallization. As expected, the rate upon
of the reaction is highly sensitive to pH changes since the degree of supersaturation with respect to both DCPD and HAP is also markedly dependent upon the pH. Similar situations occur in the mouth where bacterially-induced acidity is progressively neutralized by the buffering reaction of the saliva.
Fig. 5 Comparison of kinetics of growth of DCPD on HAP ( v ) enamel (+), calculus (A) and dentin (o), initial TCa = 1.52 x 10-2M, T = 1.506 x 10-2M, -AG values (kacl mol-1), ?63 for DCPD, 28.7 for HAP. Amount of seed used provides 0.83 m21 -1 in each case. -
Mineralization at constant solution composition. The pH-stat method was developed31 for the study of the kinetics of calcium phosphate-seeded crystal growth under conditions in which the pH was held constant by the controlled addition of base. Although the results were highly reproducible, the technique suffered from the disadvantage that the calcium and phosphate ionic concentrations decreased appreciably as the reaction proceeded toward solubility equilibrium. At each stage, therefore, the supersaturated solutions were metastable with respect to various calcium phosphate phases which could form and subsequently dissolve as the concentrations in the supersaturated solutions fell. In order to overcome the problems associated with these changing solution compositions during precipitation, and to model, more closely, in vivo conditions, a new method has been developed in which the chemical potentials of the solution species are kept constant during the reaction.44 Following the addition of
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
868
NANCOLLAS
well-characterized seed material to stable supersaturated solutions of calcium phosphate at the required pH, the concentrations of lattice ions were maintained constant by the simultaneous addition of reagent solutions containing calcium and phosphate ions, controlled by a glass electrode probe. The compositions of the reagent solutions were calculated from the results of exploratory measurements. The new technique represents a significant improvement in studies of the phases formed during tooth mineralization. The rate of reagent addition is a direct measure of the rate of crystal growth and the constancy of activity of the ion species in solution can be very precisely monitored. In a typical experiment (150 ml of supersaturated solution, initial TCa = 0.800 mM, Tp = 0.552 mM, 0.854 mM potassium hydroxide, 5.0 mg HAP seed), the use of calcium- and phosphate-containing reagent solutions with molar Ca/P = 1.45 resulted in crystal growth in which all solution parameters remained constant to within the experimental error in the analytical determination, ± 0.3%. In addition, it was found that the rate of reaction was directly proportional to the inoculating seed concentration, thus confirming that growth of the crystals occurred without interference from secondary nucleation. To have obtained a kinetic precipitation stoichiometry to this precision by techniques previously used would have required concentration analyses to at least ± 0.03%. In our preliminary experiments it was observed that, as the rate of precipitation exceeded a critical value of about 0.5 mg precipitate/min, the apparent Ca/P ratio of the solid phase became less than 1.45. Subsequent constant composition experiments at higher supersaturation (300 ml of supersaturated solution, initial TCa = 1.200 mM, Tp = 0.900 mM, 0.715 mM KOH, 24.27 mM kCl, 5.0 mg HAP seed) required reagent solutions with OCP stoichiometry, CA/P = 1.32 ± 0.01 (standard deviation) in order to maintain constant activities in the early stages of the reaction. Moreover, the formation of OCP was confirmed by xray diffraction. At longer time (15-20 min) hydrolysis to a more basic phase took place with a Ca/P of approximately 1.45, the value observed in so many previous calcium phosphate precipitation studies. It is highly significant that, using the constant composition method, more than twice the original
J Dent Res Special Issue B, 19 79
seed material could be grown as OCP in the early stages of the reaction. On the basis of our results, a model for calcium phosphate precipitation can be proposed in which OCP, formed as a precursor phase, hydrolyzes either partially or completely to HAP depending upon the rate of the precipitation reaction. The normally-observed higher Ca/P values can be accounted for by assuming that one in every three molecules of OCP transforms into HAP, leading to a Ca/P of 1.44, within 0.01 of the observed value. The hydrolysis probably takes place a layer at a time, as Brown et al. have proposed from a unit cell x-ray analysis of phases.45 The new constant composition technique has also been used to study the seeded growth of calcium phosphates at very low supersaturation (TCa = 0.200 mM, Tp = 0.120 mM) at physiological pH. The results confirm our previous suggestion3 that HAP will grow without the formation of a precursor phase. The HAP phase can be grown for 17 hours, representing some 21% of the original inoculating seed materials.46 These experiments have also been extended to higher pH (pH = 8.50, TCa = 0.300 mM, Tp = 0.180 mM) where HAP will also grow without the need to form a precursor phase. The newly-developed constant solution composition technique has thrown new light on the mechanism of calcium phosphate precipitation, both on synthetic seed and on tooth components. It is presently being used47 to study the mineralization of prepared enamel blocks (both whole and carious) under conditions close to those in vivo and in which the surface of the enamel can also be monitored microscopically during the highly reproducible kinetic experiments. Investigations of the influence of etching reagents used in preparing the substrate surface for remineralization and the effects of potential anti-calculus and anti-caries agents will be particularly interesting.47 In the latter case, the surface phases formed at sustained supersaturation even under "poisoned" conditions can be detetmined witha .precision hitherto unobtainable. Thus the results of recent studies of the kinetics of mineralization in the presence of traces of fluoride (0.1-5.0 p.p.m.)48 demonstrate the delicate balance between FAP and HAP formation on enamel and HAP substrates at physiological pH. At lower pH, fluoride and organic phos-
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
Vol. 58(B)
869
phonates can influence the nature of the calcium phosphate phase which grows on the seed substrate.43
Acknowledgment. This work was supported by the National Institute of Dental Research, NIH Research Grant Number DE 03223.
REFERENCES 1. VON DER FEHR, F. R.: In: Fluorine Research and Dental Caries Prevention (HARDWICK, J. L., HELD, R. R. and KONIG, KY G., eds.) pp. 83-98, Vol. 3, Pergamon Press, London,
1964. 2. KOULOURIDES, T.: Ann. N. Y. Acad. Sci. 153:84 (1968). 3. NANCOLLAS, G. H., TOMAZIC, B.: J. Phys. Chem. 78:2218 (1974). 4. TOMAZIC, D., NANCOLLAS, G. H.: J. Coll. Interface Sci 50:451 (1975). 5. BARONE, J. P., NANCOLLAS, G. H., TOMSON, M.: Calcif. Tiss. Res. 21:171 (1976). 6. BRINER, W. W., GRAY, J. A., FRANCIS, M. D.: J. Dent. Res. Suppl. (2) 53:239 (1974). 7. BRINER, W. W., ROSEN, S.: Calcif. Tiss. Res. 2:60 (1968). 8. JOHANSSON, B.: J. Dent. Res. 44:64 (1965). 9. VON DER FEHR,F. R., LOE, H., THEILADE, E.: Caries Res. 4:131 (1970). 10. BACKER DIRKS, O.: J. Dent. Res. 45: 503 (1966). 11. MILLER, W. A., MASELER, M.: Br. Dent. J. 112:187 (1962). 12. LEVINE, R. S.: Arch. Oral Biol. 17:1005
(1972). 13. LEVINE, R. Biol. 18:1351 14. FEAGIN, F. SONNE, B.
S., ROWLES, S. L.: Arch. Oral (1973). F., GONZALEZ, M., JEANG.: Calcif Tiss. Res. 10:113
(1972). 15. TEN CATE, J. M., ARENDS, J.: Caries Res. 11:277 (1977). 16. SILVERSTONE, L. M.: Caries Res. 11:134
(1977). 17. KRUTCHKOFF, D. J., ROWE, N. H.: J. Dent. Res. 50:1621 (1971). 18. PANTUMVANIT, P., FEAGIN, F. F., KOULOURIDES, T.: Caries Res. 11:52 (1977). 19. POOLE, D. F. G., SILVERSTONE, L. M.: In: Hard Tissue Growth, Repair and Remineralization; Ciba Foundation Symposium II Amsterdam: ASP(Elsevier, Excerpta Medica, North-Holland), 1973, pp. 35-36. 20. TOMAZIC, B., TOMSON, M., NANCOLLAS, G. H.: Arch. Oral Biol. 20:803 (1975). 21. BLUMENTHAL, N. C., BETTS, F., POSNER,
A. S.: Calcif. Tiss. Res. 23:245 (1977). 22. EILBERG, R. G., JUDY, K., IOVINO, E., KORNFELD, P., PHELAN, J., ELLISON, E.: J. Dent. Res. 52:45 (1973). 23. MANDEL, I. D.: J. Dent. Res. 53:246 (1974). 24. EANES, E. D., GILLESSEN, I. H., POSNER, A. S.: Nature 208:365 (1965). 25. WALTON, A. G., BODIN, W. J., FUREDI, H., SCHWARTZ, A.: Canad. J. Chem. 45: 2695 (1967). 26. DALLMAGNE, M. J., RICHELLE, L. J.: "Biological Mineralization" ZIPKIN, I., (ed), Wiley, New York, 1973, Ch. 2. 27. EANES, E. D., GILLESSEN, I. H., POSNER, A. S.: Proc. Int. Conf. Crystal Growth, Oxford, Pergamon Press, 1967, p 373. 28. TERMINE, J. D., POSNER, A. S.: Calcif. Tiss. Res. 1:8 (1967). 29. BROWN, W. E.: Clin. Orthop. 44:5318 (1966). 30. FRANCIS, M. D.: Ann. N. Y. Acad. Sci. 131:694 (1965). 31. NANCOLLAS, G. H., MOHAN, M. S.: Arch. Oral Biol. 15:731 (1970). 32. MEYER, J. L., NANCOLLAS, G. H.: Calcif. Tiss. Res. 13:295 (1973). 33. MEYER, J. L., EICK, J. D., NANCOLLAS, G. H., JOHNSON, L. N.: Calcif. Tiss. Res. 10:91 (1972). 34. MEYER, J. L., MCCALL, J. T., SMITH, L. H.: Calcif. Tiss. Res. 15:287 (1974). 35. EANES, E. D.: Calcif. Tiss. Res. 20:75 (1976). 36. EANES, E. D., MEYER, J. L.: Calcif. Tiss. Res. 23:259 (1977). 37. MORENO, E. C., ZAHRADNIK, R. T., GLAZMAN, A., HWU, R.: Calcif. Tiss. Res. 24: 47 (1977). 38. MARSHALL, R. W., NANCOLLAS, G. H.: J. Phys. Chem. 73:3 838 (1970). 39. LIU, S. T., NANCOLLAS, G. H.: J. Crystal Growth 6:281 (1970). 40. DAVIES, C. W.: Ion Association, London, Butterworths 1962. 41. BARONE, J. P., NANCOLLAS, G. H.: J. Coll. Interface Sci. 62:421 (1977). 42. TOMAZIC, B., TOMSON, M., NANCOLLAS, G. H.: Calcif. Tiss. Res. 19:263 (1976). 43. BARONE, J. P., NANCOLLAS, G. H.: J. Dent. Res., in press. 44. TOMSON, M., NANCOLLAS, G. H.: Science, submitted for publication. 45. BROWN, W. E., SMITH, J. P., LEHR, J. R., FRAZIER, W. A.: Nature 196:1050 (1962). 46. AMJAD, Z., KOUTSOUKOS, P., TOMSON, M., NANCOLLAS, G. H.: J. Dent. Res. submitted for publication. 47. AMJAD, Z., KUREK, B., NANCOLLAS, G. H.- unpublished results. 48. AMJAD, Z., NANCOLLAS, G. H.: unpublished results.
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
Session VI Discussion Dr. Termine: I have 2 questions. First, one problem we all have in coping with this sort of experiment is accounting for the competing ions that one finds in vivo. What information do you have on ions such as carbonate and magnesium in your new system? Second, in rat incisors, you can calculate that at optimum growth, the calcification front mineral doubles approximately every two minutes. What do you have to do to get crystals to replicate that fast? Dr. Nancollas: Once you exceed the critical limit, nucleation is extremely rapid and that is why one might question whether nucleation is the important factor in mineralization. Probably the mediation of crystal shape is more important subsequently than simply continued nucleation. The first question concerned the presence of other ions. We are now beginning to look at the effects of carbonate by this new method. One of the problems in using the older conventional techniques is that one could never see the concentration changes with sufficient precision, but now we can do so and carbonate seems to have a very appreciable effect. We can now look at carbonate incorporation into these apatites as well. Dr. Ingram: I noticed when you were doing DCP dihydrate precipitation you had a small plateau, an induction period before the thing got going, following the addition of the hydroxyapatite. Did you examine it at any earlier times than those that you
showed? Dr. Nancollas: We first sampled at 1/2 minute. Dr. Ingram: Did you notice anything unusual, like the actual calcium concentration in the solution going up, because what we may see is not the ordinary induction period, but the surface of the hydroxyapatite at that pH being transformed to DCP. I wonder if you used labelled apatites to start with, you might see in fact a small increase in calcium solubility, but the 45Ca could remain in the solution before it started to interact with the solid again. Dr. Nancollas: I didn't get a chance to
talk about it, but it is quite likely that DCP is the kinetically favored precursor. We can now do these experiments at a faster rate so that we can see pre-OCP phases. To answer your question, it is quite possible. We have conditioned our apatites at the pH of the experiment briefly before the experiment, but there is always initially some very rapid adjustment of concentration as the surface is put into the supersaturated solution. It is very difficult to get initial concentration changes which are meaningful. Dr. Ingram: So, if you were to look at the ion exchange between calcium and hydrated dicalcium phosphate you would probably see a few changes. Dr. Nancollas: That is right, very marked. Dr. Brown: The formation of platy crystals during the precipition of hydroxyapatite is an indication that octacalcium phosphate has been involved in their formation, because it is the salt with a platy habit. However, if platy crystals are formed from solutions that are undersaturated with respect to octacalcium phosphate, this could be a manifestation of monoclinic symmetry of the apatite. It should be kept in mind that at the nucleation stage, when the crystallite is about a unit cell thick, the distinction between hydroxyapatite and octacalcium phosphate becomes moot; the two structures are essentially identical when the ribbon is a single unit cell thick, since an octacalcium phosphate-like transition of the aqueous phase would probably be present in both cases. Dr. Arends: How do your results agree with the literature which shows that, at very short periods of reaction (as you can see from turbidity experiments for instance) the DCP is the very first precursor and that there may be a second precursor to make the picture even more complicated? Dr. Nancollas: That was what I was suggesting in answer to Dr. Ingram's comments. It is likely that DCP does not exist in solution in these specific experiments. Dr. Arends: So there may be two precursors? Dr. Nancollas: Yes, sequentially.
870 Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
The course of enamel remineralization in aqueous solution is markedly dependent upon factors such as ionic medium supersaturation, pH, ionic strength, and the relative concentration of mineralizing surface. Although numerous attempts have been made to determine the mechanism of both the spontaneous and seeded precipitation reactions, it is only recently, through the use of a constant solution composition technique, that quantitative information can be obtained concerning the intermediate calcium phosphate phases which are formed during mineralization. Studies of surface phases in the presence of potential anti-calculus and anti-caries agents are facilitated using this
approach.
Introduction. The possibility of the repair, through remineralization, of enamel damaged bv caries attack is extremely attractive.1'2 Despite numerous attempts to achieve this goal, it has not been possible to reconstitute the enamel apatite crystals in a form identical with that in natural enamel. Of the few kinetic studies which have been made, most have been concerned with calcium phosphate mineralizing solutions considerably more supersaturated than those typical of in vivo conditions. Since the course of the mineralization reaction is markedly dependent upon the concentrations of lattice ions in the mineralizing medium,3-5 such studies may provide little insight into the mechanism of apatite remineralization at a tooth surface. In the oral environment, both mineralization and demineralization processes are involved in the post-eruptive maturation of dental enamel.6'7 The relatively high calcium and phosphate concentrations of saliva result in the deposition of mineral while demineralization occurs at the low pH regions produced by bacterial action. In an in vitro study of the remineralization of ground sections,8 it was shown that the initial rapid uptake of calcium phosphate slowed down after 48 hours and effectively stopped for three weeks. This result was interpreted in terms of the mineralization of a sound outer layer blocking access to the underlying white spot. Clinical evidence
also points to the remineralization of early carious lesions with the reversal of whitespot formation9,10,11 but the deposited mineral was not characterized chemically. More recently,12 a calcium phosphate buffer solution containing fluoride and solid dicalcium phosphate dihydrate (CaHPO4-2H20, DCPD) has been shown to be effective in the remineralization of sections of carious dentin in vitro, and x-ray diffraction measurements revealed the presence only of a relatively well-characterized apatite phase in the mineral deposited in the tissue.13 Much of the work on enamel remineralization has been done with bulk human enamel artificially demineralized in acid solutions, and the extent of reaction was followed either analytically14'15 or microscopically.16 The presence of fluoride in the remineralizing solution has been shown to enhance the formation of amorphous calcium phosphates in the upper surface of remineralizing enamel17, and calcium fluoride was observed when acidulated calcium fluoride-calcium phosphate solution was used. Recently,18 fluoride uptake by enamel surfaces exposed to mineralizing solutions was studied and it was shown that fluoride concentrations greater than 0.05 mM tended to form calcium fluoride deposits on the tooth surface while lower concentrations resulted mainly in fluoroapatite, FAP. In a pH- and pF-stat study of the kinetics of lesion remineralization15 it was shown that in the presence of 1 ppm fluoride, the material deposited was most likely fluoridated HAP; the overall rate of mineralization was about twice that in the absence of fluoride ion. In carious lesions formed in vivo, mineral deposits have been found with morphologies different from apatite19 and these have presumably been formed by the precipitation of calcium phosphate phases within the lesion. The chemical characterization of the deposits is difficult to achieve since, even if subsequent dissolution studies are made, small errors in measured concentrations can often preclude differentiation between the 861
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
862
J Dent Res Special Issue B, 1 9 79
NANCOLLAS
possible calcium phosphate phases. In the formation of plaque, aggregates of cells and intercellular binding proteins produce a porous structure at the tooth surface through which saliva may diffuse. In all plaques, the concentration of calcium, phosphate, and magnesium is markedly higher than in saliva and, during calculus formation, the organic material of the plaque becomes embedded in the mineralized deposit. The presence of magnesium ion appears to stabilize calcium phosphate precursors20, and a synergistic effect between magnesium and adenosine triphosphate in delaying the conversion of amorphous calcium phosphate to hydroxyapatite, HAP, has also recently been described21. The possibility of relating the mineralizing activity of dental plaque in vitro with dental calculus formation in vivo in the same individual22 makes mineralization kinetic experiments particularly attractive. Young plaque has been shown, by x-ray diffraction, to contain DCPD23, and the results of kinetic studies indicate that DCPD is a kinetically-favored phase in the mineralization of dental enamel at pH values below about 5.1.5 The importance of kinetic factors, coupled with a knowledge of thermodynamic precipitation driving forces, in determining the nature of the phases formed, is now well-established. As we shall see, however, the conventional in vitro mineralization experiments would require impossibly high precisions in the analytical data in order to distinguish between the calcium phosphate phases, particularly under the relatively low supersaturations typical of those in vivo where the amount of precipitated phase is small.
Mineralization of synthetic calcium phosphates. Hydroxyapatite, HAP, is generally considered to be the model compound for bone and tooth mineral and, although numerous studies have been made of HAP precipitation, there is still considerable uncertainty about the course of the reaction. The stoichiometry of the initially-formed solid expressed as the calcium/phosphate molar ratio (hereafter, Ca/P), is invariably less than the 1.67 required for HAP.24'25 Indeed, over a wide range of concentrations at physiologic pH, the value Ca/P is of the order
1.45 ± 0.05. The similarity of this stoichiometric ratio to the value, 1.50, for tricalcium phosphate, TCP,26 has prompted the proposal of TCP as the "amorphous" precursor to HAP formation in aqueous solutions. An autocatalytic conversion of TCP to HAP was suggested for both synthetic27 and natural28 apatite formation. The close crystallographic similarity of octacalcium phosphate, OCP, to HAP led Brown29 to propose this phase as an HAP precipitation precursor; the hydrolysis of OCP to HAP taking place with minimal lattice reorientation, one layer at a time. DCPD has also been invoked as the initially precipitating phase30 and, as the calcium phosphate of simplest stoichiometry, it is kinetically favored at 370C and in slightly acid solutions (pH values less than about
5.1).5
Numerous investigations have been directed in recent years at the growth of synthetic apatite seed crystals in metastable supersaturated solutions of calcium phosphate.3,4,3l-3S In these studies, the calcium- and phosphate-concentration changes, with time, following the addition of seed, were highly reproducible, provided that factors such as degree of supersaturation, temperature, ionic strength, and the presence of traces of foreign ions were carefully controlled. At physiological pH, the overall reaction involved not only the formation of different calcium phosphate phases but also the concomitant dissolution of a transient phase, stoichiometrically close to OCP, which is formed in the initial stages of the reaction.3 In Fig. 1 is shown a typical plot of calcium concentration against time following the addition of HAP seed crystals to a calcium phosphate solution supersaturated with respect to all phases, DCPD, OCP, TCP and HAP. The observed inflection in the growth curve probably reflects the superposition of a number of kinetic processes which take place at different rates and points to the involvement of more than one calcium phosphate phase in the precipitation. The results of a recent transmission electron microscopic investigation also support this multiphase explanation.36 The stoichiometry of the solids formed may be expressed in terms of the molar ratios of calcium to phosphate, L [Ca] /A(P04], which have precipitated at any instant. A [Ca] = Tc. (initial) - TCa (time t) and A [P04] =
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
Vol. 58(B)
863
Tp (initial) Tp (time t), where TCa and Tp -
total molar concentrations of calcium and phosphate, respectively, in the supersaturated solution. During the pH1.2 DCPD 00JSA statted seeded growth experiment at rela4' tively high supersaturation, such as that shown in Fig. 1, the value of A [Cal /A[P04 ] X ~~~~~~~~OCPso for the initially depositing phase was invariably greater than that of HAP, and, after TcP about 20 min. of reaction, decreased to 1.45 ± 0.05 which remained quite constant for MAP some 300 min. At longer times, the ratio z00 UhiA 400 goo gradually increased but the value of 1.67 required for HAP stoichiometry was not reached even after 3 days of reaction.3 A Fig. 1 Growth of calcium phosphate on [Ca] /'L P04 ] ratios consistently higher than 1.67 for the initial stages of the precipitation HAP seed crystals (200 mg 1-1). Curve A, plot of Taagainst time (initial TCa = 1.562 x 10-3M, process were also reported recently by T =0.973 x 10-3M, pH = 7.40, 250(Q). Curve B Moreno et al.37 from seed growth studies of SSA (m2g-1 right-hand ordinate) against at lower supersaturations (TCa = 0.3mM) pl'ot time at pH =7.40. Curve C, plot of SSA changes at in which the pH was allowed to decrease pH = 5.0. Approximate concentration levels correfrom an initial physiological value, 7.4, responding to saturation with respect to DCPD, to about 6.8 during the reaction. It was OCP, TCP and HAP are also shown. suggested that the high values of the ratio seen that there was a pronounced maximum were due either to the formation of an enriched calcium phase or to the initial at 120 mi. growth where the SSA was adsorption of calcium ions onto the HAP twice that for the seed material. The effecsurface. As the reaction proceeded, the A tive SSA of the growth product itself was [Cal /A[P041 value approached 1.67 as large (approx. 230 m2g-1) reflecting the microcrystallinity of the precursor phase more HAP precipitated,37 but at that stage, the data were not comparable with the formed on the surface of the HAP seed results of pH-stat work because the pH had crystals. The subsequent decrease in SSA already decreased markedly from the initial reflected the increased crystallinity of the value of 7.4. Evidence for OCP as a precur- solid as crystal growth proceeded. In the interpretation of the results of sor phase which is formed on HAP seed surfaces during the concentration in time mineralization experiments in vitro, the proflles such as that shown in Fig. 1, was degree of supersaturation with respect to obtained from dissolution kinetic studies.3 each of the calcium phosphate phases may Solid phases, withdrawn at known time be expressed in terms of the free energies intervals from the crystallization cell, were of formation. transferred to a sodium chloride solution of AG = RTln(IP/Kso) the same ionic strength as that of the growth medium and dissolution was followed at a pH where Kw is the thermodynamic solubility of 7.40, maintained by the pH-stat controlled product and the activity products, IP, of addition of dilute acid. The Ca/P ratio of the individual calcium phosphate phases the surface phase 1.34 + 0.02, which had are expressed, for dicalcium phosphate been formed in the crystallization experi- dihydrate, ments, was close to that for OCP. At longer IPDCPD = [Ca2+1 [HPO2-] dissolution times, however, this ratio was f24 considerably larger 1.57,3 indicating that for OCP, IPocp (lCa2+] f2 )4([PO3- ] OCP was no longer available for dissolution, f3)3 [HI] f, having already been dissolved or hydrolyzed for TCP, 'PTCP ([Ca2 f2 )3([PO4 in the earlier composite growth process. f3)2 The specific surface area, SSA, of the and for HAP, IPHAP =([Ca2 If2)5([PO34] product grown at various times in the pHf3)3Kw/[H+] f, at statted experiments high supersaturation is plotted against time in Fig. 1. It can be Kw is the thermodynamic activity product are the
I..
I
04
0
-
=
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
864
NANCOLLAS
of water at 3 70C. The activity coefficients be calculated from the extended form of the Debye-Huckel equation proposed by Davies.40 In the above expressions, the square brackets enclose the concentrations of the free ionic species. Neglect of activity coefficient corrections in the calculation of precipitation-driving forces results in errors amounting to several orders of magnitude in the ionic product terms for substances such as HAP. The epitaxial growth of one calcium phosphate phase upon another may play an important role in the remineralization of tooth components in vivo and studies have been under conditions closely simulating plaque and calculus production in the mouth with pH values of 4.5 - 5.1. Calcium phosphate solutions supersaturated with respect to HAP and DCPD were prepared by adjusting the pH of a mixture of calcium chloride and potassium dihydrogen phosphate solutions to 5.60 by the slow addition of potassium hydroxide solution. Following the addition of the HAP seed crystals, the rate curve in Fig. 2 shows an initial induction period, preceding the regular fall of concentration with time. It can be seen in Fig. 2 that the decrease in calcium and phosphate concentrations were the same to within experimental error (Ca/P = 1.02 ± 0.03) indicating the exclusive formation of DCPD, confirmed by x-ray diffraction, despite the fact that the solutions were also appreciably supersaturated with respect to the thermodynamically stable phase, HAP. The growth of DCPD seed crystals has been shown38 to follow a rate equation,1 where N is the number of moles of DCPD
J Dent Res Special Issue B, 19 79
may
-dN = KsN2 dt to be precipitated before equilibrium is reached, s is a function of the number of active growth sites, and K is the rate constant. The applicability of this equation in describing the crystallization following the delay period in Fig. 2 is illustrated in Fig. 3 in which the integrated form is plotted. All the calcium phosphate seed materials were effective nucleators for DCPD which, in the scanning electron microscope, was seen as large platelets growing from the surface of the seed crystals. HAP was a particularly effective surface for the nucleation and growth of DCPD. It can be seen that
Fig. 2 - Tp and TCa (°, left-hand ordinate) and relative rate of DCPD nucleation (v, righthand ordinate) plotted against time (initial TCa= 6.346 mM, Tp = 6.317 mM, pH = 5.59, 370C, 23 mgl-l HAP seed. -A G values (kcal mol-1) for HAP, 9.72: DCPD, 0.396: TCP, 0.486).
increasing the amount of HAP seed material produced an increase in the rate of DCPD growth (slopes of the linear portions of Fig. 3) with very little decrease in the duration of the initial induction effect. The latter was highly reproducible despite the fact that these initial stages reflected the nucleation of DCPD on the HAP seed substrate. The applicability of rate equation 1 for more than 70% of the growth reaction suggested that all the nucleation takes place during the initial induction period. The absence of additional secondary nucleation was also evidenced by the results of the scanning electron microscopic studies. It is interesting to note that no corrections were necessary for the approximately three-fold increase in surface area which occurred during the crystal growth. This strongly supports the suggestion that crystallization of DCPD took place on the growth sites formed during the induction period and that no new sites were produced during the subsequent crystal growth step. Similar observations have been made in the crystal growth of other sparingly-soluble electrolytes.39 Further evidence in support of
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
865
Vol. 58(B)
this suggestion was obtained from the results of specific surface area measurements of the seed material and of the solids extracted at various times, shown in Fig. 1. A modest decrease in total SSA from 39.7 m2g-1 initially, to 36.3 m2g-1 at the end of the induction period implied the production of an appreciable number of small DCPD nuclei. During the subsequent crystal growth steps of DCPD, the SSA decreased sharply to 29.7 m2g- 1, reflecting crystal growth on the already-formed DCPD nuclei. It is interesting to contrast these results with the SSA profile at physiological pH (Fig. 1) for which the marked increase in the early stages of crystal growth reflected the formation of microcrystalline precursors.
Fig. 3 - Plots of the integrated form of Eqn. 1. Initial TCa = 1.489 x 10-2, Tp = 1.504 x 10-2M, -AGDCPD = 0.406 kcal mol-1; -A GHAP = 7.13 kcal mol-1, o, 'A, v, replicated experiments with 228 mgl-l HAP seed, +, 114 mgl-l and A, 56 mgl-l HAP seed.
The formation of DCPD on the surface of the added HAP seed crystals involves a nucleation step and the subsequent crystal growth of the DCPD nuclei.41 The rate of heterogeneous nucleation is governed by the degree of lattice mismatch between DCPD and HAP and the supersaturation, sharply decreasing as the latter decreases. This is shown in Fig. 2 in which the relative nucleation rate was calculated from appropriate nucleation expressions.41 It can be
seen that the nucleation is essentially complete within the induction period, falling to zero at the commencement of the normal DCPD growth. The reproducibility of the time plots of calcium and phosphate concentration in these experiments is quite striking and the quadratic dependence of the rate of growth upon the supersaturation, following the induction period, is indicative of a surface-controlled process. This is further supported by the observed independence of the growth rate on the rate of stirring. It is significant that when the concentration of HAP seed crystals, added to the supersaturated solution, was increased to 226449 mgh-1, the course of the mineralization reaction was markedly different.41 In place of the ATCa/ATp value of unity at lower seed concentrations, the ratio at high seed, 1.52 ± 0.04, is indicative of more basic calcium phosphate phase. An even more sensitive indicator of calcium phosphate phase precipitated is the ratio of base added for constant pH to phosphate precipitated. The observed value, 1.88 + 0.06, also militates against DCPD as the growth phase. X-ray powder diffraction studies confirm the absence of peaks characteristic of DCPD or TCP, and indicate only an amorphous calcium phosphate phase. At the higher HAP seed concentrations, the rate of growth of the more basic phase is sufficiently fast to markedly reduce the effective DCPD nucleation rate in the initial periods so that this phase does not form. In marked contrast, for low HAP seed concentrations (Fig. 2), the rate of growth of the basic phase is near zero, thus maintaining TCa and Tp at sufficiently high levels to facilitate the nucleation of DCPD and its subsequent growth.
Mineralization of tooth components. A highly reproducible seeded growth technique has been used to investigate the mineralization of seed material prepared from human tooth enamel, dentin, and calculus.42,43 The kinetics of growth of calcium phosphate on human enamel has been studied at 25 and 370C and pH= 7.40 in mestastable supersaturation solutions of calcium phosphate (TCa = 1.50 X 10-3M, Tp = 0.87 x 10-3M). The grown material was characterized chemically, by specific surface area measurements, and by subsequent dissolution experiments. Upon the
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
866
J Dent Res Special Issue B, 19 79
NANCOLLAS
addition of seed material, growth started immediately as is shown in Fig. 4, which also indicates the calcium levels corresponding to saturation with DCPD, OCP and TCP. It can be seen that the solution became undersaturated with respect to DCPD after about 10 min., and to OCP after about 60 min. In the experiments in which the inorganic component of dentin was used as seed following the removal of the organic matrix, it can be seen (Fig. 4) that the more efficient hydrazine removal of the organic phase as compared with ethylenediamine, en, treatment, resulted in a more effective calcium phosphate nucleator. It is significant that increasing the seed concentration to 1.0 gl-t, and more markedly at 3.0 gl-l (Fig. 4), resulted in the appearance of an inflection in the growth curve. The initial rate of reaction normalized for seed concentration, --ATCa/
At'1Nd0.4,uMl-1min-lg-l,
decreased at 120
min. and increased again at 4 hours to -ATCa/t0.3 5 6,Ml- I min.- 1 g- 1 The inflections were also observed in the phosphate concentration, and in the potassium hydroxide required to maintain the pH at 7.4. The results are similar to those outlined above using synthetic seed material, and the evidence again points to the involvement of more than one phase in the seeded growth of dental enamel. In every experiment made at these higher supersaturations (TCa = 1.200mM, Tp = 0.719mM), the Ca/P ratio was 1.45 ± 0.05 during the first twentyfour hrs. of growth. A similar value was found in HAP seeded growth experiments and a gradual increase from 1.44 to 1.54 was observed after two days, but the 1.67 required for HAP stoichiometry was not reached even after three days of reaction. The difficulty of determining the stoichiometry of the precipitating phases from the small changes in calcium and phosphate concentrations in the supersaturated solution has already been discussed. In Fig. 4, the specific surface area, SSA, of the products grown at various time intervals is plotted as a function of time. It can be seen that there is a pronounced increase in the flrst 200 min of reaction until the composite SSA, rising to a broad maximum to about 300 min, is almost four times that of the seed material. This indicates that the value of the growth product itself is appreciable (X\'145 m2g9-). The concomitant change in morphology is apparent from scanning electron micro-
Fig. 4 - The growth of enamel seed at high supersaturation. TCa = 1.438 mM, Tp = 0.874 mM, 250C, A, 3.0 g seed 1-1, +, 1.0 g seed 1-1. v, TCa = 1.582 mM, T= 0.943 mM, 0.2 g seed 1-1 (hydrazine-treated), o, TCa = 1.492 mM, T = 0.876 mM, 0.2 g seed 1-1 (en treated). Plot oFSSA (@), TCa = 1.507 mM, T = 0.872 mM, 0.2 g seed 1-1) against time. flJIl represents saturation levels with respect to phase indicated.
graphs which show the formation of an intermediate phase consisting of hexagonal platelets on the surface of enamel seed. Studies of the kinetics of dissolution of this phase under pH-statted conditions, controlled to a value of 7.40 by the addition of 0.005M hydrochloric acid, indicated a chemical composition close to that of OCP for this intermediate phase when it dissolved into 0.006M potassium chloride solution.42 The use of sodium chloride resulted in a higher value, A TCa/ATp = 1.40, and for 0.002M magnesium chloride, the ratio was 1.61. These relatively higher values can be explained in terms of the known surface exchange of calcium for magnesium, and the tendency for sodium ion incorporation (up to 5%) in an apatite lattice. No evidence was found for potassium ion uptake. It has been shown42 that in lessconcentrated phosphate solutions (TC a 0.30 x 10-3M, Tp = 0.02 x 10-3M), supersaturated only with respect to TCP and HAP, the growth product has a stoichiometry close to the 1.67 required for HAP. It is significant that the scanning electron micrographs of the solid phases formed under these conditions indicated the complete absence of the intermediate hexagonal platelet-like phase observed at the higher concentrations. These results show that the nature of the calcium phosphate phase which is formed on the surface of the enamel seed material is markedly dependent
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
867
Vol. 58(B)
the degree of supersaturation with respect to calcium phosphate. The kinetics of growth of calcium phosphates on human enamel, whole human dentin and human calculus have been studied at 370C at pH values from 4.97 - 5.10 held constant by the pH-stat addition of base.43 The solutions were supersaturatedwith respect to both DCPD and HAP, and, as was found with synthetic HAP seed material, following an initial induction period, DCPD crystallization occurred on the surface of the solid phases in all cases. The typical plots of the intergraded rate equation1 in Fig. 5 show that, following well-defined induction periods the surfacecontrolled growth of DCPD closely followed this rate equation. The relative rate of mineralization is given by the slopes of these lines, and it can be seen that, as expected, synthetic HAP is the most effective nucleator of DCPD. In Fig. 5, the rate equation used takes into account both dilution, accompanying sampling of the supersaturated solution, and surface area changes of seed material during growth. f f(vb )dvb, where vb is the volume of solution added to maintain the pH, provides a more accurate assessment of the course of the mineralization than the ordinate (N-1-Ni-1) of Fig. 3. Scanning electron micrographs of the solid phases sampled during the induction periods clearly showed the nucleation of DCPD on the substrate surfaces in all cases, even though little change had taken place in the bulk solution calcium and phosphate concentrations. The rate of growth was also independent of stirring dynamics within the experimentally accessible range, strongly suggesting that nucleation was completed during the initial induction period and that the subsequent rate of growth of the DCPD was controlled by a surface reaction. It was found that the relative order of nucleating ability for DCPD is HAP>enameL>calculus>dentin. These differences may be explained by considering the number of active sites per unit surface area of substrate. HAP has the greatest nucleating activity, since enamel has 1-2%, calculus 10-30%, and dentin 20% of organic material which normally does not readily nucleate calcium phosphates. In addition, pyrophosphate and other substances thought to be present in enamel, calculus, and dentin, are known to act as inhibitors for DCPD crystallization. As expected, the rate upon
of the reaction is highly sensitive to pH changes since the degree of supersaturation with respect to both DCPD and HAP is also markedly dependent upon the pH. Similar situations occur in the mouth where bacterially-induced acidity is progressively neutralized by the buffering reaction of the saliva.
Fig. 5 Comparison of kinetics of growth of DCPD on HAP ( v ) enamel (+), calculus (A) and dentin (o), initial TCa = 1.52 x 10-2M, T = 1.506 x 10-2M, -AG values (kacl mol-1), ?63 for DCPD, 28.7 for HAP. Amount of seed used provides 0.83 m21 -1 in each case. -
Mineralization at constant solution composition. The pH-stat method was developed31 for the study of the kinetics of calcium phosphate-seeded crystal growth under conditions in which the pH was held constant by the controlled addition of base. Although the results were highly reproducible, the technique suffered from the disadvantage that the calcium and phosphate ionic concentrations decreased appreciably as the reaction proceeded toward solubility equilibrium. At each stage, therefore, the supersaturated solutions were metastable with respect to various calcium phosphate phases which could form and subsequently dissolve as the concentrations in the supersaturated solutions fell. In order to overcome the problems associated with these changing solution compositions during precipitation, and to model, more closely, in vivo conditions, a new method has been developed in which the chemical potentials of the solution species are kept constant during the reaction.44 Following the addition of
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
868
NANCOLLAS
well-characterized seed material to stable supersaturated solutions of calcium phosphate at the required pH, the concentrations of lattice ions were maintained constant by the simultaneous addition of reagent solutions containing calcium and phosphate ions, controlled by a glass electrode probe. The compositions of the reagent solutions were calculated from the results of exploratory measurements. The new technique represents a significant improvement in studies of the phases formed during tooth mineralization. The rate of reagent addition is a direct measure of the rate of crystal growth and the constancy of activity of the ion species in solution can be very precisely monitored. In a typical experiment (150 ml of supersaturated solution, initial TCa = 0.800 mM, Tp = 0.552 mM, 0.854 mM potassium hydroxide, 5.0 mg HAP seed), the use of calcium- and phosphate-containing reagent solutions with molar Ca/P = 1.45 resulted in crystal growth in which all solution parameters remained constant to within the experimental error in the analytical determination, ± 0.3%. In addition, it was found that the rate of reaction was directly proportional to the inoculating seed concentration, thus confirming that growth of the crystals occurred without interference from secondary nucleation. To have obtained a kinetic precipitation stoichiometry to this precision by techniques previously used would have required concentration analyses to at least ± 0.03%. In our preliminary experiments it was observed that, as the rate of precipitation exceeded a critical value of about 0.5 mg precipitate/min, the apparent Ca/P ratio of the solid phase became less than 1.45. Subsequent constant composition experiments at higher supersaturation (300 ml of supersaturated solution, initial TCa = 1.200 mM, Tp = 0.900 mM, 0.715 mM KOH, 24.27 mM kCl, 5.0 mg HAP seed) required reagent solutions with OCP stoichiometry, CA/P = 1.32 ± 0.01 (standard deviation) in order to maintain constant activities in the early stages of the reaction. Moreover, the formation of OCP was confirmed by xray diffraction. At longer time (15-20 min) hydrolysis to a more basic phase took place with a Ca/P of approximately 1.45, the value observed in so many previous calcium phosphate precipitation studies. It is highly significant that, using the constant composition method, more than twice the original
J Dent Res Special Issue B, 19 79
seed material could be grown as OCP in the early stages of the reaction. On the basis of our results, a model for calcium phosphate precipitation can be proposed in which OCP, formed as a precursor phase, hydrolyzes either partially or completely to HAP depending upon the rate of the precipitation reaction. The normally-observed higher Ca/P values can be accounted for by assuming that one in every three molecules of OCP transforms into HAP, leading to a Ca/P of 1.44, within 0.01 of the observed value. The hydrolysis probably takes place a layer at a time, as Brown et al. have proposed from a unit cell x-ray analysis of phases.45 The new constant composition technique has also been used to study the seeded growth of calcium phosphates at very low supersaturation (TCa = 0.200 mM, Tp = 0.120 mM) at physiological pH. The results confirm our previous suggestion3 that HAP will grow without the formation of a precursor phase. The HAP phase can be grown for 17 hours, representing some 21% of the original inoculating seed materials.46 These experiments have also been extended to higher pH (pH = 8.50, TCa = 0.300 mM, Tp = 0.180 mM) where HAP will also grow without the need to form a precursor phase. The newly-developed constant solution composition technique has thrown new light on the mechanism of calcium phosphate precipitation, both on synthetic seed and on tooth components. It is presently being used47 to study the mineralization of prepared enamel blocks (both whole and carious) under conditions close to those in vivo and in which the surface of the enamel can also be monitored microscopically during the highly reproducible kinetic experiments. Investigations of the influence of etching reagents used in preparing the substrate surface for remineralization and the effects of potential anti-calculus and anti-caries agents will be particularly interesting.47 In the latter case, the surface phases formed at sustained supersaturation even under "poisoned" conditions can be detetmined witha .precision hitherto unobtainable. Thus the results of recent studies of the kinetics of mineralization in the presence of traces of fluoride (0.1-5.0 p.p.m.)48 demonstrate the delicate balance between FAP and HAP formation on enamel and HAP substrates at physiological pH. At lower pH, fluoride and organic phos-
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
Vol. 58(B)
869
phonates can influence the nature of the calcium phosphate phase which grows on the seed substrate.43
Acknowledgment. This work was supported by the National Institute of Dental Research, NIH Research Grant Number DE 03223.
REFERENCES 1. VON DER FEHR, F. R.: In: Fluorine Research and Dental Caries Prevention (HARDWICK, J. L., HELD, R. R. and KONIG, KY G., eds.) pp. 83-98, Vol. 3, Pergamon Press, London,
1964. 2. KOULOURIDES, T.: Ann. N. Y. Acad. Sci. 153:84 (1968). 3. NANCOLLAS, G. H., TOMAZIC, B.: J. Phys. Chem. 78:2218 (1974). 4. TOMAZIC, D., NANCOLLAS, G. H.: J. Coll. Interface Sci 50:451 (1975). 5. BARONE, J. P., NANCOLLAS, G. H., TOMSON, M.: Calcif. Tiss. Res. 21:171 (1976). 6. BRINER, W. W., GRAY, J. A., FRANCIS, M. D.: J. Dent. Res. Suppl. (2) 53:239 (1974). 7. BRINER, W. W., ROSEN, S.: Calcif. Tiss. Res. 2:60 (1968). 8. JOHANSSON, B.: J. Dent. Res. 44:64 (1965). 9. VON DER FEHR,F. R., LOE, H., THEILADE, E.: Caries Res. 4:131 (1970). 10. BACKER DIRKS, O.: J. Dent. Res. 45: 503 (1966). 11. MILLER, W. A., MASELER, M.: Br. Dent. J. 112:187 (1962). 12. LEVINE, R. S.: Arch. Oral Biol. 17:1005
(1972). 13. LEVINE, R. Biol. 18:1351 14. FEAGIN, F. SONNE, B.
S., ROWLES, S. L.: Arch. Oral (1973). F., GONZALEZ, M., JEANG.: Calcif Tiss. Res. 10:113
(1972). 15. TEN CATE, J. M., ARENDS, J.: Caries Res. 11:277 (1977). 16. SILVERSTONE, L. M.: Caries Res. 11:134
(1977). 17. KRUTCHKOFF, D. J., ROWE, N. H.: J. Dent. Res. 50:1621 (1971). 18. PANTUMVANIT, P., FEAGIN, F. F., KOULOURIDES, T.: Caries Res. 11:52 (1977). 19. POOLE, D. F. G., SILVERSTONE, L. M.: In: Hard Tissue Growth, Repair and Remineralization; Ciba Foundation Symposium II Amsterdam: ASP(Elsevier, Excerpta Medica, North-Holland), 1973, pp. 35-36. 20. TOMAZIC, B., TOMSON, M., NANCOLLAS, G. H.: Arch. Oral Biol. 20:803 (1975). 21. BLUMENTHAL, N. C., BETTS, F., POSNER,
A. S.: Calcif. Tiss. Res. 23:245 (1977). 22. EILBERG, R. G., JUDY, K., IOVINO, E., KORNFELD, P., PHELAN, J., ELLISON, E.: J. Dent. Res. 52:45 (1973). 23. MANDEL, I. D.: J. Dent. Res. 53:246 (1974). 24. EANES, E. D., GILLESSEN, I. H., POSNER, A. S.: Nature 208:365 (1965). 25. WALTON, A. G., BODIN, W. J., FUREDI, H., SCHWARTZ, A.: Canad. J. Chem. 45: 2695 (1967). 26. DALLMAGNE, M. J., RICHELLE, L. J.: "Biological Mineralization" ZIPKIN, I., (ed), Wiley, New York, 1973, Ch. 2. 27. EANES, E. D., GILLESSEN, I. H., POSNER, A. S.: Proc. Int. Conf. Crystal Growth, Oxford, Pergamon Press, 1967, p 373. 28. TERMINE, J. D., POSNER, A. S.: Calcif. Tiss. Res. 1:8 (1967). 29. BROWN, W. E.: Clin. Orthop. 44:5318 (1966). 30. FRANCIS, M. D.: Ann. N. Y. Acad. Sci. 131:694 (1965). 31. NANCOLLAS, G. H., MOHAN, M. S.: Arch. Oral Biol. 15:731 (1970). 32. MEYER, J. L., NANCOLLAS, G. H.: Calcif. Tiss. Res. 13:295 (1973). 33. MEYER, J. L., EICK, J. D., NANCOLLAS, G. H., JOHNSON, L. N.: Calcif. Tiss. Res. 10:91 (1972). 34. MEYER, J. L., MCCALL, J. T., SMITH, L. H.: Calcif. Tiss. Res. 15:287 (1974). 35. EANES, E. D.: Calcif. Tiss. Res. 20:75 (1976). 36. EANES, E. D., MEYER, J. L.: Calcif. Tiss. Res. 23:259 (1977). 37. MORENO, E. C., ZAHRADNIK, R. T., GLAZMAN, A., HWU, R.: Calcif. Tiss. Res. 24: 47 (1977). 38. MARSHALL, R. W., NANCOLLAS, G. H.: J. Phys. Chem. 73:3 838 (1970). 39. LIU, S. T., NANCOLLAS, G. H.: J. Crystal Growth 6:281 (1970). 40. DAVIES, C. W.: Ion Association, London, Butterworths 1962. 41. BARONE, J. P., NANCOLLAS, G. H.: J. Coll. Interface Sci. 62:421 (1977). 42. TOMAZIC, B., TOMSON, M., NANCOLLAS, G. H.: Calcif. Tiss. Res. 19:263 (1976). 43. BARONE, J. P., NANCOLLAS, G. H.: J. Dent. Res., in press. 44. TOMSON, M., NANCOLLAS, G. H.: Science, submitted for publication. 45. BROWN, W. E., SMITH, J. P., LEHR, J. R., FRAZIER, W. A.: Nature 196:1050 (1962). 46. AMJAD, Z., KOUTSOUKOS, P., TOMSON, M., NANCOLLAS, G. H.: J. Dent. Res. submitted for publication. 47. AMJAD, Z., KUREK, B., NANCOLLAS, G. H.- unpublished results. 48. AMJAD, Z., NANCOLLAS, G. H.: unpublished results.
Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
Session VI Discussion Dr. Termine: I have 2 questions. First, one problem we all have in coping with this sort of experiment is accounting for the competing ions that one finds in vivo. What information do you have on ions such as carbonate and magnesium in your new system? Second, in rat incisors, you can calculate that at optimum growth, the calcification front mineral doubles approximately every two minutes. What do you have to do to get crystals to replicate that fast? Dr. Nancollas: Once you exceed the critical limit, nucleation is extremely rapid and that is why one might question whether nucleation is the important factor in mineralization. Probably the mediation of crystal shape is more important subsequently than simply continued nucleation. The first question concerned the presence of other ions. We are now beginning to look at the effects of carbonate by this new method. One of the problems in using the older conventional techniques is that one could never see the concentration changes with sufficient precision, but now we can do so and carbonate seems to have a very appreciable effect. We can now look at carbonate incorporation into these apatites as well. Dr. Ingram: I noticed when you were doing DCP dihydrate precipitation you had a small plateau, an induction period before the thing got going, following the addition of the hydroxyapatite. Did you examine it at any earlier times than those that you
showed? Dr. Nancollas: We first sampled at 1/2 minute. Dr. Ingram: Did you notice anything unusual, like the actual calcium concentration in the solution going up, because what we may see is not the ordinary induction period, but the surface of the hydroxyapatite at that pH being transformed to DCP. I wonder if you used labelled apatites to start with, you might see in fact a small increase in calcium solubility, but the 45Ca could remain in the solution before it started to interact with the solid again. Dr. Nancollas: I didn't get a chance to
talk about it, but it is quite likely that DCP is the kinetically favored precursor. We can now do these experiments at a faster rate so that we can see pre-OCP phases. To answer your question, it is quite possible. We have conditioned our apatites at the pH of the experiment briefly before the experiment, but there is always initially some very rapid adjustment of concentration as the surface is put into the supersaturated solution. It is very difficult to get initial concentration changes which are meaningful. Dr. Ingram: So, if you were to look at the ion exchange between calcium and hydrated dicalcium phosphate you would probably see a few changes. Dr. Nancollas: That is right, very marked. Dr. Brown: The formation of platy crystals during the precipition of hydroxyapatite is an indication that octacalcium phosphate has been involved in their formation, because it is the salt with a platy habit. However, if platy crystals are formed from solutions that are undersaturated with respect to octacalcium phosphate, this could be a manifestation of monoclinic symmetry of the apatite. It should be kept in mind that at the nucleation stage, when the crystallite is about a unit cell thick, the distinction between hydroxyapatite and octacalcium phosphate becomes moot; the two structures are essentially identical when the ribbon is a single unit cell thick, since an octacalcium phosphate-like transition of the aqueous phase would probably be present in both cases. Dr. Arends: How do your results agree with the literature which shows that, at very short periods of reaction (as you can see from turbidity experiments for instance) the DCP is the very first precursor and that there may be a second precursor to make the picture even more complicated? Dr. Nancollas: That was what I was suggesting in answer to Dr. Ingram's comments. It is likely that DCP does not exist in solution in these specific experiments. Dr. Arends: So there may be two precursors? Dr. Nancollas: Yes, sequentially.
870 Downloaded from jdr.sagepub.com at UNIV OF PITTSBURGH on March 10, 2015 For personal use only. No other uses without permission.
