Langmuir 1988,4 , 907-910
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Crystallization of Calcite on Collagen Type I Evangelos Dalas and Petros G. Koutsoukos* Department of Chemistry and the Research Institute of Chemical Engineering and High Temperature Chemical Processes, P.O. Box 1239, University Campus, GR-261 10 Patras, Greece Received October 14, 1987. In Final Form: February 23, 1988 Bovine Achilles tendon collagen type I was found to be a substrate favoring the deposition of calcite crystalsfrom stable supersaturated solutions at pH 8.50 and at 25 "C. The induction periods varied markedly with supersaturation, and kinetics results yielded values of 58 mJ m-2 for the surface energy of the calcite overgrowth. The crystallization was studied at constant solution supersaturation, thus making it possible for relatively large amounts of the overgrowth to be formed and be identified exclusively as calcite. The apparent order found from kinetics data was that typically found for the crystallization of calcite on synthetic calcite crystals, thus suggesting a similar, surface-controlled mechanism.
Introduction The formation of calcium carbonate polymorphs, mainly calcite and aragonite, has been reported in a number of cases ranging from pancreatic stones in both humans and cattle1i2 to animal phyla, algae and in mollusk^,^ and gallstone^.^ It is believed that an organic matrix is associated with the calcium carbonate mineraL5 It is important therefore to clarify the nature of the calcium carbonate polymorph forming by heterogeneous nucleation. It has been found that supersaturation is critical in determining the calcium carbonate polymorph precipitating. Thus, a t high degrees of supersaturation, where spontaneous precipitation occurs vaterite forms predominantly even a t 25 O C , 6 while at low supersaturations, calcite forms directly in stable supersaturated solutions seeded with ~ a l c i t e .The ~ organic matrix is considered to play a principal role in the deposition of calcium carbonate serving as a substrate which facilitates nucleation of crystals by a process similar to e p i t a ~ y .Moreover, ~~~ it should be noted that the amount of free Ca2+ions in the biological fluids is very low due to extensive complexation with proteins and other biological macromolecules. It is likely therefore that the solution composition is such that spontaneous precipitation of calcium carbonate takes place. In the present work we have selected collagen type I produced from tendon collagen, which is a well-characterized macromolecule possessing crystallinity, in order to evaluate its capability of inducing calcium carbonate formation. The study was done in stable supersaturated solutions, and the constant solution composition was u ~ e d . ~This J ~ method is particularly suited for studying the formation of a new phase on a substrate, since the (1) Verine, H. J. Bull Comp. Pathol. 1973,3, 5. (2)Moore, E.W.; Verine, H. J. J. Am. Phys. SOC.1987,G707. (3)Crenshaw, M. A. In Biological Mineralization and Demineralization; Nancollas, G . H., Ed.; Dahlem Konf., Springer Verlag: Berlin, Heidelberg, New York, 1982;pp 243-257. (4)Saito, K.;Omori, H.; Kanno, S.; Hirata, Y.; Okado, T.; Mori, S.; Nakadate, K. Gastroenterol. Jpn. 1986,21,162. (5)Watabe, N.In Progress in Crystal Growth and Characterization; Pamplin, B., Ed.; Pergamon: Oxford, 1981;Vol. 4. (6) Xyla, A.; Koutaoukos, P. G. 31st International Congress of Pure and Applied Chemistry; July 13-18, Sofia, Bulgaria, 1987;Vol. 3,pp 7, 158. (7) Giannimaras, E.; Koutaoukos, P. G. 31st International Congress of Pure and Applied Chemistry; July 13-18,Sofia, Bulgaria, 1987;Vol. 3, p 41. (8) Wilbur, K. M. In Physiology of Molluscs; Wilbur, K. M., Yonge, C. M., Eds.; Academic: New York, 1964;(Washington, DC)Vol. 1, pp 243-282. (9)Tomson, M. B.; Nancollas, G. H. Science 1977,200, 1059. (IO)Kazmierczak, T. F.; Tomson, M. B.; Nancollas, G. H. J . Phys. Chem. 1982,86,103.
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initial conditions of precipitation are maintained. Initial rates and induction periods may be precisely measured even a t very low degrees of supersaturation, where the extent and the rate of the precipitate formation are very low. Based on the measured rates, nucleation parameters for the heterogeneous nucleation of calcium carbonate on collagen substrates are estimated.
Experimental Section The experiments described herein were done at 25 A 0.1 "C in a 0.250-dm3, double-walled Pyrex vessel thermostated by circulating water. Triply distilled, C02-freewater was used for the solution preparations. The supersaturatedsolutions, volume totaling 0.200 dm3, were prepared in the reaction vessel from calcium nitrate and sodium bicarbonate solutions as described in detail elsewhere." The pH of the solution was subsequently adjusted to 8.50 by the addition of standard potassium hydroxide solution. The initial conditions of the working solutions were such that the supersaturatedsolutions were stable for at least 4 days. The solution pH was measured by a glass/saturatedcalomel pair of electrodes (Radiometer G202C and K402, respectively), standardized before and after each experiment by NBS buffer solutions at pH 6.865 (0.025 m KH2P04+ 0.025 m Na2HP04)and at pH 9.18 (0.01 m Borax). The solutions were stirred by a magnetic stirrer with a Teflon-coated stirring bar at ca. 350 rpm. Following the pH adjustment, 100 mg of dry collagen type I (Sigma) were introduced in to the solution. The solid material formed a good suspension within 2 min. No change in pH was observed upon collagen addition. The collagen used was crystalline in X-ray and had a BET specific surface area (Perkin Elmer Sorptometer Model 212D) of 6.25 m2g-l and a MW of 50O00. The precipitation started followingwell-defied induction periods that were shorter the higher the supersaturation. Following the onset of precipitation, the concomitant proton release triggered the addition of titrants from two mechanically coupled glass burets of a modified pH stat (Metrohn,Model 614);pH changes as little as 0.005 units were sufficient to trigger the titrant additions. The two burets contained calcium nitrate and sodium carbonate at the molar stoichiometry dictated by the precipitating calcite. Provision was made in the titrants for avoiding dilution in the working solution and for the amount of potassium hydroxide required for pH adjustment of the working solution. More explicitly, the concentration of the titrant in the two burets was calculated as follows: buret 1: (Ca(N03)z)(Cx + 2x1 buret 2: Cx(NazC03)+ 2x(NaHC03)+ 2y(KOH) where x is the molar concentration of calcium nitrate or sodium bicarbonate in the working solution and y the amount of potassium hydroxide required for the pH adjustment in the working solution. (11)Koutsoukos, P. G.; Kontoyannis, C. G. J. Chem. Soc., Farad. Trans. 1 1984,80,1181.
0 1988 American Chemical Society
908 Langmuir, Vol. 4, No. 4, 1988
Dalas and Koutsoukos
Table I. Crystal Growth of Calcite on Insoluble Collagen Type I at Sustained Supersaturationa ion 7, Cat, strength, AGdcitm AGar AGvat, AGniono, R, exDeriment min lo3 mol-dm" lo2 mol~dm-~ kJ-mol-l kJ.mc?' kJ.mol-l kJ.mol-' lo7 rnol.min-'.m2 -2.68 -1.46 3.00 7.2 -3.08 -1.40 3.90 1 300 2.50 6.0 -2.72 -2.31 2 400 -1.10 -1.04 2.60 5.4 -2.51 4 550 2.25 -2.10 -0.89 -0.83 2.09 2.00 4.8 -2.27 -1.86 5 700 -0.65 4.60 1.79 6 780 7.75 4.2 -2.00 -1.59 -0.33 1.17 -0.38 7 1000 1.50 3.6 -1.69 -1.28 -0.07 -0.02 0.61 9
~~~
"Conditons: 25 "C, pH 8.50, 0.5 mg of collagen/mL. Total calcium = Cat, total carbonate = Ct. For maintenance of the ionic strength constant an amount 2C of inert electrolyte (potassium nitrate) was added in the working solution where C is a constant (expressing how many times the titrants are more concentrated than the working solution). In our experiments, C was chosen as 10. The choice of the best value for C requires preliminary experiments. During the course of the reaction, samples were withdrawn and fiitered through membrane filters (0.2 fim, Millipore), and the filtrates were analyzed for calcium by atomic absorption spectroscopy (Varian 1200) and by a spectrophotometric titration with EDTA using murexide as indicator. The analyses confirmed the maintenance of supersaturation in the solution (which was better than 2% in all experiments). The time lapsed between the addition of collagen in solution and the beginning of the titrant additions was taken as the induction period for the nucleation of calcium carbonate on collagen. From the slope of the recorder traces (titrant addition with time) the rates were calculated. Initial rates were taken for the kinetics treatment. The solid phases formed during the course of precipitation were examined by infrared spectroscopy (Perkin-Elmer 467), powder X-ray diffraction (Phillips, 1300/00),Cu K a radiation, optical (olympus) and scanning electron microscopy (Amray), thermogravimetric analysis, and a differential scanning calorimetry (Du Pont 910) system coupled with a 990 programmer recorder.
Results and Discussion The solution speciation in all experiments was computed from the pH, the total calcium mass balance, and the electroneutrality conditions assuming a system in which the partial pressure of COz is kept constant. Since the solution pH is high and the volume of the working solution is such that the air over the solution is minimal, this assumption is valid.1° The driving force for the formation of the various calcium carbonate polymorphs, which is the change in Gibbs free energy for the transition from the supersaturated solution to equilibrium, AGp, was calculated from eq 1:
In eq 1R is the gas constant, Tis the absolute temperature, parentheses denote the activities of the ions, and K L is the thermodynamicsolubility of the polymorph considered. A value of 1.279 X low8was taken for the thermodynamic solubility product of calcium carbonate monohydrate, a polymorph favored in the presence of organic molecules.12J3 The solubility product obtained from ref 14 was corrected for all ion pairs possible including Ca, Mg carbonates, and hydrolysis products. In all cases, an induction period preceded the onset of calcium carbonate precipitation. Spectroscopic examination by X-ray diffraction and IR techniques confirmed the exclusive formation of calcite. The absence of the mono(12) Kitano, Y. Bull. Chem. SOC.Jpn. 1962,35, 1973. (13) Giannimaras, E.; Koutaoukos, P. G . Langmuir, in press. (14) Hull, H.; Turnbull, A. G. Geocheim. Cosmochim. Acta 1973,37, 685.
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Figure 1. Plots of the volume of titrants added as a function of time for the growth of calcite on collagen type I a t constant solution composition. Experiment numbers correspond to Table
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hydrate polymorph was also ruled out by thermogravimetric and differential scanning calorimetry using as standards for comparison calcite samples on which the monohydrate salt had been deposited. The induction period, 7,observed varied inversely proportional with supersaturation. The initial conditions of the experiments are summarized in Table I. The reproducibility of the reported rates was f4% and of the induction periods &15%. Plots of the volume of titrants added as a function of time are shown in Figure 1. The experimental conditions in the present work were chosen so that the supersaturated solutions were stable,15yet the overall free energy for the formation of the critical nucleus, AG,, will be lower than that corresponding to homogeneous nucleation, AGN, by a factor +, which is
