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Application of the turbidity measurements to study in situ crystallization of calcium phosphates

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Kinetics of the initial crystallization stages of calcium phosphates from supersaturated aqueous solutions containing NaCl, NaN3, and Tris–buffer was followed by turbidity measurements during 10 h. The crystallization was studied inside a silica cell
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  Colloids and SurfacesA: Physicochemical and Engineering Aspects 203 (2002) 237–244 Application of the turbidity measurements to study in situcrystallization of calcium phosphates Elena I. Dorozhkina, Sergey V. Dorozhkin * Research Institute of Fertilizers ,  Kudrinskaja sq .  1 - 155  ,  123242   Moscow D - 242  ,  Russia Received 5 January 2001; accepted 24 October 2001 Abstract Kinetics of the initial crystallization stages of calcium phosphates from supersaturated aqueous solutions containingNaCl, NaN 3 , and Tris–buffer was followed by turbidity measurements during 10 h. The crystallization was studiedinside a silica cell at 25 °C and solution pH 7.4. The crystals obtained were found to consist of a mixture of poorcrystallized hydroxyapatite (HA) and octacalcium phosphate (OCP). The threshold concentration (when crystalliza-tion started) measured by the turbidity technique appeared to be 5.5 mM. Presence of poly- L -glutamic acid previouslyadsorbed onto the silica seemed to promote the crystallization, while similar layer of poly- L -aspartic acid was foundto have no influence. When dissolved, both polypeptides were found to inhibit the crystallization. © 2002 ElsevierScience B.V. All rights reserved. Keywords :   Calcium phosphates; Crystallization kinetics; Turbidity measurements; Silica cell; Influence of polypeptides; Promotionand inhibitionwww.elsevier.com / locate / colsurfa 1. Introduction Several types of glasses and glass-ceramics areknown to possess the bioactive properties [1]. Thelatter means that having been implanted inside thehuman body in the places of bone defects, artifi-cial bone substitutes made of those glasses pro-mote in vivo formation of a newly formed bone.Bone is known to be a complicated compositeconsisting of approximately 70% of biological ap-atite (i.e. non-stoichiometric, poor crystallized,sodium- and magnesium-containing carbonateap-atite [2]), 20% of organic matrix (mainly, colla-gen), and 10% of water [3,4]. Therefore, from thechemical point of view, the process of bone for-mation is just in vivo crystallization of calciumphosphates onto (or simultaneously with) a poly-meric matrix of collagen. Taking into consider-ation the bioactive properties of several glasses,investigations on calcium phosphates crystalliza-tion on the surface of silica-contained materials(especially in the presence of biological com-pounds) appear to have a medical application[5,6]. * Corresponding author. Present address: Solid State Chem-istry, Department of Chemistry, Ruhr-University of Bochum,D-44780 Bochum, Germany. Tel.:  + 49-234-32-26635; fax: + 49-234-32-14558. E  - mail address :   sergey.dorozhkin@ruhr-uni-bochum.de(S.V. Dorozhkin).0927-7757 / 02 / $ - see front matter © 2002 Elsevier Science B.V. All rights reserved.PII: S0927-7757(01)01108-6  E  . I  .  Dorozhkina ,  S  . V  .  Dorozhkin  /   Colloids and Surfaces A :   Physicochem .  Eng  .  Aspects  203 (2002) 237  –  244  238 Due to the medical importance (dental caries,bone substitutes, biomineralization, biomimetics),in vitro dissolution and crystallization of calciumphosphates have been studied a lot ([1,2,7  –  10],and references therein). Usually both processesare studied just from aqueous undersaturated orsupersaturated solutions, respectively. In fl uence of solution concentration, temperature, and hydro-dynamics are investigated. Constant compositiondevice,  fi rst introduced by Nancollas et al. [11,12],is currently the most advanced experimental tech-nique. It allows keeping the ionic concentrationsconstant, which provides a good simulation of thein vivo crystallization conditions. The crystalliza-tion kinetics is followed by measuring amounts of the stock solutions of acid / base, calcium, andphosphate added [11,12]. However, for slightlysupersaturated solutions investigations on the ini-tial crystallization stages are complicated becauseamounts of the stock solutions added are small.Moreover, investigations on the in fl uence of or-ganic and biological compounds on crystallizationkinetics by the constant composition device arenot easy, because these compounds are dangerousfor Ca-selective electrode.However, inside the human body, dissolution / precipitation processes occur in the presence of hundreds of organic, biological, and polymericcompounds. Part of these compounds is in solu-tion, another part is in the solid state, but most of them have an in fl uence on calcium phosphatecrystallization [13,14]. Usually dissolved com-pounds are crystal growth inhibitors, while thesame molecules immobilized on the surface act ascrystal nucleators [15  –  18]. For example, bonesialoproteins [19,20] and poly- L -glutamic acid[19  –  21] were found to have such double in fl uenceon calcium phosphate crystallization. For crystal-lization, the above dualism of biological com-pounds might be due to their in fl uence on thenucleation process. Homogeneous nucleation(from a solution) of calcium phosphates is inhib-ited by the Ca-chelating properties of biologicalmolecules (this decreases solution supersatura-tion), while heterogeneous nucleation (on the sur-face) might be promoted by the adsorbedbiological molecules those either change the sur-face charge on silica or create necessary nucle-ation sites on silica surface by the speci fi cconformation. In the case of dissolution, poly- L -glutamic and poly- L -aspartic acids when dissolvedwere found to act as a driving force (due to theCa-chelating properties), but when adsorbed theyinhibited dissolution of hydroxyapatite (HA,Ca 10 (PO 4 ) 6 (OH) 2 ) [22].The goal of this paper is to study the in fl uenceof the above polypeptides on calcium phosphatescrystallization. We have chosen turbidity mea-surements because this technique is suitable forinvestigations of even rather concentrated suspen-sions [23] and it is easy to perform. Moreover,when supersaturated solutions are continuouslypumped through the silica cell, the turbidity mea-surements provide in situ following the initialstages of crystallization kinetics. 2. Materials and methods Sodium salts of poly- L -glutamic (P-4886, 51300Da) and poly- L -aspartic (P-6762, 28800 Da) acidswere purchased from Sigma Chemical Co. Highpurity calcium chloride and potassium hy-drophosphate (both from Reachim, Russia) wereused as the initial chemicals. Buffered solutionscontaining ions of calcium (solution A) and phos-phate (solution B) of 7  –  18 mM each were pre-pared by dissolving of the above salts in a buffercontained 150 mM of NaCl, 0.01% of NaN 3  and10 mM of Tris (tris  –  hydroxymethy-laminomethane). Solution pH 7.40  0.01 was ad- justed by HCl. Solutions of polypeptides wereprepared in a similar way: 60 mg of each weredissolved in 100 ml of either the same buffer, orbuffered solutions of A and B. All the solutionswere  fi ltrated through the Millipore 0.22   m  fi ltersbefore using.The saturated buffer solution (SBS) of calciumphosphate was also prepared. To do so, equalamounts of solutions A and B (both with andwithout polypeptides) with concentration of 15mM each were mixed and kept stirring in a closedvessel at 25  ° C during 1 week followed by  fi ltra-tion. Chemical analysis of the SBS gave values of 2.5  0.3 mM for calcium and 3.7  0.2 mM forphosphate (Ca:P  0.67) and solution pH 7.05   E  . I  .  Dorozhkina ,  S  . V  .  Dorozhkin  /   Colloids and Surfaces A :   Physicochem .  Eng  .  Aspects  203 (2002) 237  –  244   239 0.02. Similar analysis of the precipitates obtainedduring the SBS preparation gave Ca:P = 1.52  0.04. X-ray diffraction (XRD) patterns (Fig. 1,pattern 1) and FTIR (Fourier transform infra red)spectra (Fig. 2, spectrum 1) of the precipitatesrevealed a mixture of poor crystallized non-stoi-chiometric HA and octacalcium phosphate (OCP,Ca 8 (HPO 4 ) 2 (PO 4 ) 4 · 5H 2 O). Presence of thepolypeptides during SBS preparation was foundto have an in fl uence to the properties of neitherSBS nor a mixture of HA and OCP precipitated(solution pH, Ca:P ratio, crystal sizes, spectra of XRD and FTIR were similar to those obtainedwithout the polypeptides).Turbidity experiments were performed usingFEK-2M spectrophotometer (LOMO, Russia) at  = 650 nm (red light  fi lter). Standard quartz cellsof the spectrophotometer (3 cm height) with anoptical path of 0.25 cm were used. A bottom of one cell was cut by a diamond saw and a plasticcap was glued instead. Another cap was  fi xed onthe top. Holes were made in the caps, whereplastic tubes were inserted (Fig. 3). In order tokeep the crystallization conditions constant, con-tinuous  fl ow of supersaturated solutions of cal-cium phosphate through the silica cell was createdby two pumps. To do so, equal amounts of A andB solutions were mixed before entering the cell(Fig. 3). In order to decrease random variationsof ambient temperature, the spectrophotometer, afan, and a small heater set at 25  ° C were placedinside a fume cupboard. In addition to this, solu-tions A and B were kept at 25.0  0.1  ° C during2 h before starting experiments.Each experiment was preceded with an exten-sive cleaning of the cell and tubes by HCl, doubledistilled water, ethyl alcohol, detergent, and dou-ble distilled water again. Then the cell was  fi lled inwith the SBS followed by turbidity measurements.Extinction value of the SBS was measured andconsidered as a background. Later, a mixture of solutions A + B (either with or without the dis-solved polypeptides) was continuously passedthrough the cell by means of two pumps (aninjection rate was 10 ml h − 1 ). Solution turbiditywas recorded each minute.Experiments with previous adsorption of thepolypeptides were performed as follows. Initially,the cell was  fi lled in with pure (without dissolvedpolypeptides) buffer followed by the turbiditymeasurements. Later the buffer was replaced bythe buffered solution of either polypeptide (con-tinuous injection for 5  –  10 h) to provide adsorp-tion onto the silica. Then, the polypeptidesolution was replaced with the pure buffer again,followed by the turbidity measurements. This Fig. 1. XRD pattern of the precipitates obtained during the SBS preparation (1) and in the modi fi ed silica cell (2). Main diffractionpeaks of OCP are marked by the asterisk.  E  . I  .  Dorozhkina ,  S  . V  .  Dorozhkin  /   Colloids and Surfaces A :   Physicochem .  Eng  .  Aspects  203 (2002) 237  –  244  240Fig. 2. FTIR spectra of the precipitates obtained during the SBS preparation (1) and in the modi fi ed silica cell (2). Note that thecharacteristic peak of hydroxyl at 3570 cm − 1 is absent while that of HPO 42 − around 875 cm − 1 is present. washing was necessary to remove the inhibitingeffect of the dissolved polypeptides [15  –  22]. Thepresence of an adsorbed layer of either polypep-tide on silica was detected as a small (approxi-mately 0.002 extinction units of thespectrophotometer) but detectable increasing of the light absorption value. Finally, the buffer wasreplaced with the SBS followed by continuousinjection of A + B solutions (all without dissolvedpolypeptides) during 10 h.The precipitates formed inside the cell werecollected, air-dried, and analyzed by XRD, FTIR,and SEM (scanning electron microscopy) tech-niques. Then, they were dissolved in HCl andconcentrations of calcium and phosphate weremeasured. Regardless on presence of the polypep-tides, the precipitates were found to consist of thin plate-like crystals approximately 2 × 2 × 0.1  m in sizes (Fig. 4) and had Ca:P = 1.50  0.05. 3. Results and discussion The results obtained are summarized in Tables1 and 2. Three independent experiments wereperformed for each experimental condition. Num-bers in the columns represent concentrations of calcium and phosphate ions in solutions A and B(the  fi rst column) and those after mixing (thesecond column). The injection time through thecell was limited to 10 h. During this limitedexperimental time no precipitation occurred insidethe silica cell when the solutions contained lessthan 11 mM of calcium and phosphate beforemixing. However, additional experiments in  fl askson mixing of the equal amounts of calcium andphosphate solutions with concentrations of 9 + 9and 7 + 7 mM resulted in precipitations at timesof 24  –  30 h and 2  –  3 weeks, respectively (bothcases without the dissolved polypeptides).Typical results on the turbidity measurementsare shown in Fig. 5. As soon as crystallization Fig. 3. A drawing of the modi fi ed silica cell.  E  . I  .  Dorozhkina ,  S  . V  .  Dorozhkin  /   Colloids and Surfaces A :   Physicochem .  Eng  .  Aspects  203 (2002) 237  –  244   241Fig. 4. A representative SEM image of the precipitates taken from the modi fi ed silica cell. Magni fi cation is  × 20000. Bar is 1   m. starts, the solution turbidity constantly increases,which makes unclear at what moment the resultsshould be compared. To overcome the problem,the light absorption value (0.032  0.003 extinc-tion units of the spectrophotometer) was mea-sured at the end of several experiments. Tenpercent of this value was chosen as a reference forthe comparison (Fig. 5). Moments of time, whenthe solution turbidity reached 0.0032 extinctionunits above the background value, were consid-ered as the time of crystallization beginning andare given in the last column of Tables 1 and 2.Regardless of presence of the polypeptides, solu-tion supersaturation increased, as expected, result-ing in crystallization time decreasing that could beeasily measured by turbidity (Tables 1 and 2).One can see that presence of a preliminary ad-sorbed layer of poly- L -glutamic acid seemed topromote calcium phosphates crystallization, whilepresence of a similar layer of poly- L -aspartic acidhad no in fl uence (Fig. 5). Presence of the dis-solved polypeptides was found to inhibit the crys-tallization in all cases (last columns in Tables 1and 2). However, the error values in the lastcolumns of both Tables appeared to be rather big.The latter prevents us from making the strictconclusion about the promotional in fl uence of adsorbed poly- L -glutamic acid; one can say abouta probability only.Turbidity technique provides no opportunity todistinguish between the surface nucleation andthat in the bulk of the solution. Both ways seemto be possible. Therefore, this topic is not dis-cussed in details. However, based on a smallpositive effect of adsorbed poly- L -glutamic acid,we can suggest that the surface nucleationoccurred.Results on XRD and FTIR measurements re-vealed that the precipitates crystallized inside thesilica cell were similar to those obtained duringthe SBS preparation (Figs. 1 and 2) and consistedof a mixture of poor crystallized non-stoichiomet-ric HA with OCP. This can be seen from thebroad diffraction peaks in Fig. 1, as well as fromthe poor resolved phosphate absorption bands inFig. 2. However, the results of SEM always showrelatively good plate-like crystals (Fig. 4). Precipi-tates formed in presence of the dissolved polypep-
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