Synthesis of amorphous calcium phosphate using various types of cyclodextrins

Synthesis of amorphous calcium phosphate usingvarious types of cyclodextrinsYanbao Li 1,Tjandra Wiliana,Kam C.Tam *Singapore-MIT Alliance,School of Mechanical and Aerospace Engineering,Nanyang Technological University,50Nanyang Avenue,Singapore 639798,SingaporeReceived 17March 2006;accepted 28August 2006Available online 10October 2006AbstractAmorphous calcium phosphate (ACP)was synthesised in aqueous solution at room temperature using cyclodextrins.Fourier transform infrared (FTIR)spectroscopy,X-ray diffraction (XRD),transmission electron microscopy (TEM),energy dispersive X-ray spectroscopy (EDX)and thermal analysis (DTA/TGA)were performed on the calcium phosphate precipitates obtained from solutions.We observed that only b -CD could stabilise the amorphous phase in the mother solution because of the lower solubility of b -CD in water and the ACP remained stable in aqueous solution for more than 24h at room temperature.The ACP particle has an initial particle size of less than 40nm,Ca/P molar ratio of 1.67and b -CD absorbed on its surface.The mechanism for the stabilisation of ACP is proposed.#2006Elsevier Ltd.All rights reserved.Keywords:A.Amorphous materials;A.Nanostructures;B.Chemical synthesis1.IntroductionAmorphous calcium phosphate (denoted as ACP)is commonly found in biological system (such as soft bone tissue and bovine milk [1]),and it was first synthesised in solution by Posner and co-workers [2,3].ACP plays a vital role in the biomineralization of bone because it is a precursor to crystalline bone apatite.Furthermore,ACP is widely used as a precursor to prepare crystalline calcium phosphates with different compositions [4].This material can be used in various applications,such as coated implant [5–7],filler [8,9]and bone cements [10],because of its high bioactivity and excellent biodegradable property.ACP formation has been reported to occur via various mechanisms,and several preparation techniques have been adopted;namely (1)synthesis of calcium phosphate in aqueous solution,with or without stabilisers [3,9,11,12],(2)synthesis in organic–water mixtures [9,13],(3)organic-mediated sol–gel processes [4,14],(4)quenching of molten calcium phosphates,and (5)physical deposition techniques [12,15].However,the last two methods are not able to control the composition of ACP materials and they may also introduce contaminated elements or other crystalline phases,such as CaO to the ACP [15].The solution-mediated method has been employed because it can provide precise control of composition and phase characteristics of ACP./locate/matresbuMaterials Research Bulletin 42(2007)820–827*Corresponding author.Tel.:+6567924062;fax:+6567905590.E-mail address:mkctam@.sg (K.C.Tam).1Present address:College of Materials and Engineering,Nanjing University of Technology,5Xin Mofan Road,Nanjing 210009,China.0025-5408/$–see front matter #2006Elsevier Ltd.All rights reserved.doi:10.1016/j.materresbull.2006.08.027Y.Li et al./Materials Research Bulletin42(2007)820–827821 ACP is a transient precursor that will transform into apatite in the mother solution through the dissolution of ACP, followed by the nucleation and growth of apatite[16].The stabilising additives should be added into the ACP mother solution to obtain an amorphous phase.The additives present included not only inorganic substance such as Mg2+[16], Zn2+[12],CO32À[7],P2O74À,FÀand ZrOCl[8],but also biomacromolecules such as nucleotide and casein phosphopeptide[1].However,the addition of such inorganic additives will alter the composition,which would enhance the negative effects in the biomedical application of ACP.Previously,we reported that synthetic polymers can be employed to stabilise ACP in solution[17].Nevertheless, the synthesis conditions were very stringent;i.e.low temperature(58C)and high polymer concentration(>10wt.%). In this paper,polysaccharides,such as cyclodextrins were employed to stabilise the amorphous phase of ACP in solution,and the synthetic conditions were improved to produce nanostructures at room temperature(258C)aided bya low organic content(<1wt.%).2.ExperimentalAll the chemicals were of analytical grade,and used as received without further purification.Calcium chloride, potassium hydrogen phosphate and sodium hydroxide were purchased from Sigma–Adlrich and cyclodextrins were purchased from Cyclodextrin Technologies Development,Inc.Calcium chloride(CaCl2)and cyclodextrins(denoted as CD)were dissolved in deionised water to form0.2M calcium chloride solution in different CD concentrations ranging from0.1to2wt.%,and kept for24h to allow the reaction of calcium and cyclodextrin to reach an equilibrium.Phosphate solution was added drop-wise into CaCl2/CD solution at Ca/ P molar ratio of1.67,which was kept at a pH of10by the addition of1M NaOH.The reaction was conducted at room temperature(258C)for several hours.The precipitates were recovered from the solution using a centrifuge,and washed repeatedly to remove soluble ions such as K+,Na+,and ClÀ.Finally,the precipitates were washed twice with ethanol,and dried at room temperature for24h,and stored in the desiccator prior to characterization.The Fourier transform infrared(FTIR)spectroscopy was conducted in a Nicolet magna-IR FTIR system550 spectrophotometer with2cmÀ1resolution from4000to400cmÀ1,using KBr pellet method.Powder X-ray diffraction patterns were recorded on a Rigaku Rotaflex diffractometer using Cu K a radiation over the range108<2u<708with the step of0.058.The morphology of the precipitates was performed on a JEOL2010Transmission microscope operating at200kV with an Oxford link EDX system.The TEM specimens were prepared by placing a drop of calcium phosphate precipitates dispersed in ethanol solution on a300-mesh copper grid with the polymeric supporting film and the solvent was removed completely at room temperature.3.Results3.1.Effect of different types of CDsFigs.1and2show the XRD and FTIR spectra of calcium phosphate synthesised in the presence of different types of CDs(a-CD,b-CD,g-CD,m-CD and h-CD).Only calcium phosphate synthesised in the presence of b-CD is amorphous with a characteristic halo whose maximum peak was located at2u=308.On the contrary,the other samples had crystalline diffraction peaks,which can be assigned to apatite(JCPDS9-432).This implies that only b-CD can stabilise the ACP phase in aqueous solution at the prevailing condition.The n4PO42Àbending mode at550cmÀ1and n3PO42Àstretching mode at about1000cmÀ1appeared in the FTIR spectra for all the samples.The n4PO42Àband at550cmÀ1is a single halo peak in the b-CD containing sample,which became two peaks at604and565cmÀ1for the other four samples. The n4PO42Àband at874cmÀ1is very weak in the b-CD containing sample,and it is pronounced for the other four samples.According to Termine and Posner[18],ACP phase shows a single peak in the region of600–500cmÀ1while the anisotropic local electricalfield of crystalline phase causes an absorption in the form of an apparent doublet.Thus,the FTIR result confirmed that ACP can be obtained the solution aided by the presence of b-CD.3.2.Effect of b-CD concentrationsFigs.3and4show the XRD and FTIR spectra of calcium phosphate synthesised for3h at room temperature in the presence of different concentrations of b-CD.Diffraction peaks at2u=25.868and31.968appeared at b -CD concentration below 0.5wt.%,which can be assigned to the apatite phase (JCPDS 9-432).A characteristic halo peak occurs at 2u =308at b -CD concentration greater than 0.5wt.%,indicating that ACP is present in the solution.From Fig.4,there is a pronounced absorption band at about 1000cm À1,due to n 3PO 43Àstretching mode.The samples synthesised at high b -CD concentration (!0.5wt.%)has Y.Li et al./Materials Research Bulletin 42(2007)820–827822Fig.1.XRD spectra of calcium phosphates synthesised in the presence of 0.5wt.%CDs at room temperature for 3h and pH of10.Fig.2.FTIR spectra of calcium phosphates synthesised in the presence of 0.5wt.%CDs at room temperature for 3h and kept pH of10.Fig.3.XRD spectra of calcium phosphates synthesised at room temperature for 3h and kept pH of 10in the presence of different concentrations of b -CD.a strong halo band at 600–500cm À1,an indicator of amorphous phase [19].The obvious absorption band at 960cm À1is assigned to n 2PO 43Àstretching mode in the spectrum of the sample synthesised at lowb -CD concentration (<0.3wt.%).The band at 873cm À1becomes more pronounced when the b -CD concentration was decreased to below 0.5wt.%,and the halo band at 600–500cm À1splits into doublet peaks at 603and 562cm À1and their intensity increased with b -CD concentration due to local anisotropic electrical field of the crystalline phase.3.3.Effect of aging timesXRD and FTIR spectra of calcium phosphate synthesised in the presence of 1wt.%b -CD at room temperature for different times are shown in Figs.5and 6.A halo peak at 2u =308is present in the samples,even when aged for 24h,while crystalline diffraction peaks at 2u =25.868and 31.968appeared and became stronger after aging for more than 24h at room temperature,and these peaks belong to (002)and (211)of apatite (JCPDS 9-432).The results from the FTIR spectra agreed with those determined from XRD measurements.The absorption band at 874cm À1did not exist for the sample aged for 3h,but appeared and became stronger with increasing aging times.The halo band at 600–500cm À1became two peaks at 603and 562cm À1after being aged for 48h.A new band at 630cm À1occurred in samples that have been aged for more than 48h,and this can be assigned to OH group,which is an indicator of the hydroxyapatite phase.Thus,we conclude that the ACP phase existed in the solution before aging of 24h.Y.Li et al./Materials Research Bulletin 42(2007)820–827823Fig.4.FTIR spectra of calcium phosphates synthesised in the presence of different concentration of b -CD at room temperature for 3h and kept pH of10.Fig.5.XRD spectra of calcium phosphates synthesised in the presence of 1wt.%b -CD at room temperature and kept at pH of 10after different aging times.3.4.Characterization of ACPACP particles were investigated by TEM,EDX and TGA/DTA techniques.The typical morphology of ACP is spherical particles with size of less than 40nm and these particles aggregated during the preparation for TEM analysis (Fig.7a),and the selected area electron diffraction (SAED)confirmed the amorphous phase,which is consistent with Y.Li et al./Materials Research Bulletin 42(2007)820–827824Fig.6.FTIR spectra of calcium phosphates prepared in the presence of 1wt.%b -CD and kept pH of 10after different agingtimes.Fig.7.(a)Morphology of typical ACP observed by TEM (the inset at the top right is the select area electron diffraction pattern and bottom right is the EDX analysis),(b)XRD spectra of typical ACP after being heated at 8008C for 3h (the black solid line is the experimental data and red line is the reference according to JCPDS 9-432)and (c)TGA/DTA curves of the typical ACP precipitates.Y.Li et al./Materials Research Bulletin42(2007)820–827825 the XRD results.The chemical composition of ACP was also examined by EDX,and only Ca,O and P elements were present with a Ca/P molar ratio of1.67.Impurities such as K,Na,and Cl were not present,which implies that the ACP comprises of pure calcium phosphate.The chemical composition was also confirmed using XRD pattern of ACP after heating at8008C for3h.Only one phase,hydroxyapatite(JCPDS9-432)with the Ca/P molar ratio of1.67(Fig.7b)is present.The organics on the surface of ACP was studied by TGA(Fig.7c).There is about22wt.%weight loss, assigned to the water present in the ACP clusters,which was also reported by Posner[2].Another weight loss of 2wt.%was observed at between300and5508C,due to decomposition of b-CD.From the DTA study,an exothermal peak at6508C was observed and this can be attributed to the crystallization of ACP.4.DiscussionAmorphous calcium phosphate(ACP)occurs widely in animal bone and plays a vital role in the biomineralization process of bone formation.On the one hand,ACP may act as short-term Ca and P storage reservoirs(precursors)for the crystalline apatite in bones.It has been reported that amorphous phase improves the mechanical properties and stability of various biominerals[20].Furthermore,ACP can be used as a precursor to produce the crystalline calcium phosphates,and has been extensively studied by a number of research groups.Pure ACP phase however is a transient phase and can be easily transformed to apatite in the mother solution by the dissolution–recrystallization process,i.e.the dissolution of ACP and nucleation and growth of apatite[2].A number of parameters can affect the transformation of ACP into apatite,including additive type,amount of additive,aging time, pH value,ACP particle size,ACP slurry concentration,temperature,stirring speed,solution composition[2,16,21]. However,thefirst three parameters are the most important,and these were investigated in the present study.Cyclodextrins and calcium chloride solution was being stirred overnight to form a Ca2+-CD complex,as reported by Nicolis et al.[22,23]and Yang[24,25].When the phosphate solution was added,calcium phosphate precipitate was produced and CD molecules are absorbed on the surface of the precipitate,a step that is critical in stabilising the ACP phase.This is confirmed by the TGA thermograms(Fig.7c),where2wt.%b-CD was found on the surface of the ACP precipitates even after repeated washing.CD molecules are absorbed on the surface of calcium phosphate precipitates, thereby reducing the solubility of ACP.This effect may vary depending on the types of CDs.Only b-CD can stabilise ACP because it is more hydrophobic than the other four CD systems,as indicated by their solubilities in water listed in Table1.In addition,the large cyclic structure of CD molecule provides steric hindrance for the atomic rearrangement during the transformation of ACP to apatite.These two effects enhance the stabilising effect of b-CD on ACP in the mother solution.The process of ACP formation can summarized as shown in Eq.(1):Ca2þþCD!Ca2þ-CD complex!ACP-CDðstabilisationÞ(1) The concentration of b-CD determines the degree of stabilisation.There is a limit for the absorption of b-CD on the surface of the ACP in the mother solution.Amorphous phase could be obtained if high b-CD concentration (>0.3wt.%)was used because the amounts of b-CD absorbed on the amorphous cluster has exceeded the limit.While the amount of b-CD absorbed on the surface of the ACP precipitates did not exceed the limit at low b-CD concentration(<0.5wt.%),it cannot stabilise the ACP in the mother solution.The pH determines the process relating to the transformation of ACP into apatite[21]:(a)ACP transforms into apatite directly if pH is greater than9.25as shown by Eq.(2);Table1Solubility in water of different CDs at room temperatureCode Chemical name Solubility(wt.%,258C) a-CD a-Cyclodextrin14.5b-CD b-Cyclodextrin 1.85g-CD g-Cyclodextrin23.2m-CD Methyl-b-cyclodextrin20h-CD Hydroxypropyl-b-cyclodextrin>50(b)The intermediate phase—octacalcium phosphate (OCP)appears and the OCP is transformed into apatite at pHvalue lower than 9.25as shown in Eq.(3):ACP !apatite ðpH >9:25Þ(2)ACP !OCP !apatite ðpH <9:25Þ(3)ACP stabilised by b -CD can remain in the mother solution for more than 24h,and then transformed directly into apatite due to the high pH (about 10).Recently,several studies have reported on the preparation of ACP that remained stable for more than 24h [9,26,27].Although they used the organic–water mixture,it is very difficult to separate ACP from the mother solution and the ACP particle concentration was very low,which limits its mass production.ACP stabilised by b -CD exists as pure calcium phosphate with molar ratio of 1.67,similar to that of hydroxyapatite,without any contaminated ions such K +,Na +and Cl À.Such approach ensures that pure ACP can be produced resulting in the formation of uncontaminated apatite that can have potential applications in biomedical field.The particle size of ACP is less than 40nm and hydroxyapatite was achieved after heated at 8008C for 3h,which indicates ACP can be used as precursor for the synthesis of the crystalline calcium phosphate phases.5.ConclusionsAmorphous calcium phosphate (ACP)was synthesised in aqueous solution at room temperature using cyclodextrins as additives.We observed that 2wt.%of b -CD was absorbed on the surface of ACP particle after repeated washing,which fulfils a vital role for the stabilisation of ACP in the mother solution.ACP is present in the mother solution for more than 24h at the prevailing condition with >0.3wt.%b -CD concentration through the reduction in the solubility of ACP,which prevents the transformation of ACP to apatite.Only b -CD can stabilise ACP due to its lower solubility in water (i.e.more 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