AnnotationThe Growth of Hydroxyapatite from Solution. A New ConstantComposition Method

Z. AMJAD, P. KoUTSOUKOS, M. B. TomsON,* and G. H. NANCOLLAS

Department of Chemistry, State University of New Yor-k at Buffalo, Buffalo. New York14214. UISA

J Dent Res 57(9-10):909, September-October 1978

Hydroxyapatite (Ca5(P04),30H, HAP), is gen-erally considered to be the model compoundfor tooth and bone mineral. Calcification nu-cleation in vivo probably takes place on an or-ganic matrix which lowers the degree of super-saturation necessary for precipitation and directsthe regions of mineralization. This suggests thatseeded growth methods may be more appro-priate models for mineralization and some prog-ress toward the elucidation of the mechanismhas resulted from such studies at constant andat variable pH. Both natural and synthetic ma-terials have been used as seed to induce miner-alization in metastable calcium phosphate super-saturated solutions. The difficulties associatedwith these seeded growth studies are: (1) smallerrors in measured concentrations often precludedifferentiation between the various possible cal-cium phosphate phases; (2) as the calcium andphosphate concentrations in the growth mediadecrease, the marked changes in relative super-saturations probably modify the mechanism ofprecipitation; (3) it is generally not possible tomake quantitative physical or chemical measure-ments on the precipitated materials formed dur-ing the growth at various times because so littleactually precipitates.

These problems have been overcome in anew technique, reported here, wherein the rateof precipitation can be measured under condi-tions of constant solution composition for longperiods of time. The method enables macro-amounts of stoichiometric HAP to be formed bydirect precipitation at pH = 7.40 and 37 C.Both precipitation and dissolution may be stud-ied at constant Tca, Tp, pH, and ionic strengthsimilar to those in vivo. In a typical experiment,a metastable supersaturated solution is preparedby mixing solutions of calcium chloride and po-tassium dihydrogen phosphate at 37 C andbringing to the required pH by the careful ad-dition of dilute potassium hydroxide solution.Precipitation commences immediately upon theaddition of well-characterized seed material suchas tooth enamel, dentin or synthetic calciumphosphates. The release of protons (pH change0.003 or less) triggers the addition of titrants

Received for publication February 2, 1978.Accepted for publication April 18, 1978.This research was supported in part by the National

Institute of Dental Research, NIH, Research GrantDE-03223.

* Present Address: Rice University, EnvironmentalScience and Engineering Department, P. 0. Box 1892,Houston, Texas, 77001.

and aliquots of solution are removed periodi-cally for combined calcium and phosphate anal-yses In the earlier pH-stat approach the titrantconsisted of dilute base and the calcium andphosphate concentrations were allowed to de-crease during crystallization. In the presentmethod, additionis are made from mechanicallycoupled 1 0-ml burettes of calcium, phosphate.and base solutions at concentrations such thatthe composition of the supersaturated solutionremains constant during mineralization. It isalso very necessary to adjust the ionic strengthsof the solutions in order to compensate for ti-trant addition and to ensure constancy of activ-ity coefficients.

The table shows the results of a typical ex-periment, CCR42, at 37 C, pH = 7.40 with 80mg of HAP seed per liter. The initial volume

TABLE

CONSTANT CoMPosirION PRECIPITATION OF HAP(EXPT. CCR42)

Time TC. Tn Precipitation as %(min) (mM) (mM) of Original Seed

0 0.300 0.180 0120 0.300 0.182 23.3340 0.300 0.180 35.0425 0.300 0.183 43.1745 0.300 0.180 53.0

was 250 ml. It can be seen that the stoichiometryof the precipitated phase is constant, Tca/Tp =1.66 ± 0.01, for more than 12 hours, reflectingHAP precipitation. Confirmation of this phaseas HAP was given by the results of infrared andx-ray diffraction studies. It is significant thatmore than 50% of the original inoculating seedmaterial is grown directly as HAP. The resultsconfirm previous suggestions, based upon ap-proximate stoichiometry determinations usingconventional precipitation experiments, that atsufficiently low supersaturation, HAP can pre-cipitate without precursor formation both at thesurface of seed crystals and spontaneously fromsolution.

The method presented here makes it pos-sible, for the first time. to prepare macroscopicamounts of HAP by precipitation under condi-tions of temperature and electrolyte compositiontypical of those in vivo. Moreover, it enablesthe determination of the stoichiometries of theprecipitated phases with precision hitherto un-attainable.

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