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Amorphous calcium orthophosphate: properties, chemical and biomedical applications

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  • Save International Journal of Materials and Chemistry 2012, 2(1): 19-46 DOI: 10.5923/j.ijmc.20120201.04 Amorphous Calcium Orthophosphates: Nature, Chemistry and Biomedical Applications Sergey V. Dorozhkin Kudrinskaja sq. 1-155, Moscow 123242, Russia Abstract Amorphous calcium orthophosphates (ACPs) represent a unique class of biomedically relevant calcium or- thophosphate salts, having variable chemical but rather identical glass-like physical properties, in which there are neither translational nor orientational long-range orders of the atomic positions. Normally, ACPs are the first solid phases, precipitated after a rapid mixing of aqueous solutions containing ions of Ca2+ and PO43-; however, other production techniques are known. Interestingly, but ACPs prepared by wet-chemical techniques were found to possess a relative constancy in their chemical composition over a relatively wide range of preparation conditions, which suggested the presence of a well-defined local structural unit, presumably, with the structure of Ca9(PO4)6 – so-called a Posner’s cluster. However, the presence of similar clusters in ACPs produced by other techniques remains uncertain. All ACPs are thermodynamically unstable compounds and, unless stored in dry conditions or doped by stabilizers, spontaneously they tend to transform to crystalline calcium orthophosphates, mainly to calcium apatites. This solution instability of ACPs and their easy transformation to crystalline phases are of a great biological relevance. Namely, the initiating role ACPs play in matrix vesicle biomineralization raises the importance of ACPs from a mere laboratory curiosity to that of a reasonable key intermediate in skeletal calcification. In addition, due to great chemical and structural similarities to the calcified tissues of mammals, as well as excellent biocompatibility and bioresorbability, all types of ACPs are very promising candidates to manufacture artificial bone grafts. In this review, current knowledge on occurrence, preparation, composition, structure, major properties and biomedical applications of ACPs have been summarized. To assist the readers in looking for the specific details on ACPs, a great number of references have been collected and systematized. Keywords Amorphous, Calcium Orthophosphate, Apatite, Bone, Calcification, Posner’s Cluster, Biomaterials, Bioce- ramics, Biomineralization 1. Introduction In nature, amorphous phases exist extensively with readily moldable isotropic properties and of structure materials. For example, amorphous structures represent ~ 20% of approximately 60 different inorganic compounds and minerals formed by living organisms. These biologically formed minerals are often called biominerals, while the process of their formation is called biomineralization[1]. A recent review on the subject indicates that many biominerals are formed from amorphous precursors and, furthermore, the amorphous phases may possess fluidic properties that impart new processing capabilities to the system[2]. Among the existing biogenic amorphous minerals, those composing from calcium orthophosphates are most abundant in teeth and exoskeletal structures of marine invertebrates[2-6]. On the other hand, the existence of similar amorphous calcium * Corresponding author: (Sergey V. Dorozhkin) Published online at Copyright © 2012 Scientific & Academic Publishing. All Rights Reserved phosphate (ACP, or ACPs if plural) minerals in vertebrate organisms has not been well established experimentally except in highly specialized locations such as the inner ear structures of embryonic sharks[1], mammalian milk[7,8], dental enamel[9], as well as in mitochondria[1] and sarcoplasmic reticulum[10] of some cells. Despite the intensive efforts, accumulated evidences for ACPs as an integral mineral component in major hard tissues, such as bones and teeth, are equivocal and for many years have been the subject of considerable debates[11-24]. However, recent studies on bone and teeth formation have suggested a presence of transient amorphous mineral precursors and a universal strategy for calcium carbonate-based and calcium orthophosphatebased biomineralization in both vertebrates and invertebrates[21-29]. An interesting study on a potential role of ACPs in facilitating assembly of nano-sized particles of HA into highly ordered structures has been published recently[30]. Higher order HA architectures were detected only when the starting particles were aggregates of nanodimensional spheres with HA cores and ACP shells. Surface ACPs initially linked HA nano-sized particles in a way that allowed a parallel orienta- 20 Sergey V. Dorozhkin: Amorphous Calcium Orthophosphates: Nature, Chemistry and Biomedical Applications tion of the nano-sized particles of HA and then was incorporated into HA by phase transformation to produce more ordered architectures with the characteristic features of apatites in biologic structures. Further, it was demonstrated that enamel- and bone-like apatites could be prepared by using nanodimensional HA and ACP under the controls of different biological additives[30]. This study points to an important role of ACP might play in bone formation. However, due to a lack of undeniable proofs, the question on the occurrence of ACP phases in newly mineralized tissues of vertebrates still remains unanswered[31]. The complete list of the available calcium orthophosphates combined with their standard abbreviations, chemical formulae and major properties is given in Table 1[32,33]. Since all of them belong to calcium orthophosphates, strictly speaking, all abbreviations in Table 1 are incorrect; however, they are extensively used in literature and there is no need to modify them. As the majority of calcium orthophosphates are crystalline, this review is devoted to the detailed description of ACPs (emphasized by bold font in Table 1), which is of a great biomedical importance due to its chemical and structural similarities to the calcified tissues of mammals. Furthermore, with a few important exceptions, neither ion-substituted forms of ACP[34-47,347], nor ACP- containing biocomposites[48-75] are considered and discussed. The readers interested in either these topics and/or other types of amorphous calcium phosphates (e.g., amorphous calcium polyphosphate[76-80] and amorphous calcium metaphosphate[81]) are referred to the original publications. 2. Basic Definitions and Knowledge on the Amorphous State of Solids According to the thermodynamic laws, the perfect infinite crystals cannot exist in the real world. Various disorders in the forms of vacancies, interstitial atoms, impurities, dislocations, grain boundaries, surfaces and other interfaces disrupt the periodicity of otherwise “perfect” crystals and in many cases determine their physical properties. By contrast, highly disordered solids are those solids that are so irregular that the concept of a reference crystal lattice must be abandoned. Such highly disordered materials are called amorphous materials[82]. As Wikipedia, the free encyclopedia, has it: an amorphous (from the Greek term αμορφος, which means “shapeless” or “without form”) solid is a solid, in which there is no translational and orientational long-range order (LRO) of the atomic positions[83]. Early researchers categorized solids as amorphous or crystalline materials based on the macroscopic properties such as their external shapes, fracture mechanisms and optical properties long before X-ray diffraction techniques and other methods became available to reveal their atomic structures. Only in the past century, an understanding of the microscopic nature of amorphous materials has become possible[82]. However, there is still much debate concerning the exact nature of these materials. For example, in a recent article, Sheng, et al.,[84] have mentioned: “the atomic arrangements in amorphous alloys remain mysterious at present”. Table 1. Existing calcium orthophosphates and their major properties[32,33] Ca/P molar ratio 0.5 Compound Monocalcium phosphate monohydrate (MCPM) Formula Ca(H2PO4)2·H2O Solubility at 25℃, -log(Ks) Solubility at 25℃, g/L pH stability range in aqueous solutions at 25℃ 1.14 ~ 18 0.0 – 2.0 Monocalcium phosphate anhydrous 0.5 (MCPA or MCP) Ca(H2PO4)2 1.14 ~ 17 [c] Dicalcium phosphate dihydrate 1.0 (DCPD), mineral brushite CaHPO4·2H2O 6.59 ~ 0.088 2.0 – 6.0 Dicalcium phosphate anhydrous (DCPA 1.0 or DCP), mineral monetite CaHPO4 6.90 ~ 0.048 [c] 1.33 Octacalcium phosphate (OCP) Ca8(HPO4)2(PO4)4·5H2O 96.6 1.5 α-Tricalcium phosphate (α-TCP) α-Ca3(PO4)2 25.5 1.5 β-Tricalcium phosphate (β-TCP) β-Ca3(PO4)2 28.9 1.2 – 2.2 Amorphous calcium phosphates (ACP) CaxHy(PO4)z·nH2O, n = 3 – 4.5; 15 – 20% H2O [b] Calcium-deficient hydroxyapatite 1.5 – 1.67 (CDHA or Ca-def HA)[e] Ca10-x(HPO4)x(PO4)6-x(OH)2-x (0> α-TCP >> β-TCP > CDHA >> HA > FA [c] Stable at temperatures above 100℃ [d] Always metastable [e] Occasionally, it is called “precipitated HA (PHA)” [f] Existence of OA remains questionable International Journal of Materials and Chemistry 2012, 2(1): 19-46 21 An amorphous structure is distinctly different from a densely packed assembly of microcrystals and is closely related to the structure of a liquid phase. Ideally, an amorphous solid should be described by the model of a perfectly random structure[85]; however, this is the boundary condition. As such, the structure of amorphous solids is normally described in terms of statistical distributions. Nevertheless, prior a further description, one must specify the existing atomic length scales. The shortest length scale usually used to describe the structure of a material consists of an atom and its nearest neighbors, out to perhaps two or three atoms distant. All solids and liquids have some structure on this scale, which is called a short-range order (SRO). For crystalline solids, structural order persists over much longer distances (at least, tens or hundreds of atomic distances), such that the atoms occupy sites in a periodic three-dimensional array. Such materials are said to have a LRO and include most metals and many covalently bonded solids. Non-crystalline solids, including glasses, lack a LRO and are said to be amorphous even though they can have a SRO that is quite well defined[86]. Figure 1. A formation of skydivers illustrates disorders on an intermediate length scale. Each skydiver has a simple set of rules for bonding to the next skydiver (SRO) but there is a sufficient flexibility for different patterns of ordering to be created on the scale of a few body lengths (MRO) According to the available literature, for each particular atom of any solid there are a SRO of 2 – 5 Å, a medium-range order (MRO) of 5 – 20 Å and a LRO at distances exceeding ~ 20 Å[87,88]. In the case of covalent materials, in which a directed chemical bonding is dominant, a SRO can be characterized in terms of the well-defined coordination polyhedra, which, in many cases, appear to concur with the unit cells. The definition of a MRO is more contentious and it is helpful to subdivide MRO into 3 subcategories. At the shortest length scale (~ 5 Å), a near-MRO describes the connections among the coordination polyhedra. At the next length scale (~ 5 – 8 Å), an intermediate MRO can be associated with correlations between pairs of preferred dihedral angles for neighboring bonds. Finally, on a yet larger length scale (~ 8 – 20 Å), a far-MRO can be associated with the total dimensionality of the covalently bonded amorphous network. Thus, characterizing the nature of MRO in disordered solids is very important for understanding their structure. Fig. 1 represents an excellent visual demonstration of the differences between a SRO and a MRO[89]. Farther details on this topic are available in literature[87,88]. In covalent solids, bond angles and bond lengths, as well as a number of the nearest neighbors, are all part of the appropriate bonding scheme. Thus, due the nature of chemical bonding, even the truly amorphous materials have some structural SRO and, perhaps, some MRO. For example, MRO regions of ~ 15 Å in dimensions and comprising about 100 atoms have been directly observed in amorphous carbon[90]. Some order in two-dimensional projections of thin amorphous three-dimensional structures was found[91]. Besides, covalent amorphous solids were found to exhibit a MRO at length scales up to 20 Å or so[88]. Such MRO clusters are called paracrystals[86]. These paracrystals have a crystalline topology but the atomic positions are highly distorted from those of a perfect crystal. However, in solids there is a serious problem of very small particles. Specifically, if the crystal sizes are extremely small, it is difficult to make a distinction between the truly amorphous and crystalline solids. Namely, if a powder consists of tiny perfect crystals with dimensions of 2 nm × 2 nm × 2 nm (8 nm3) or less, both this powder and any bulk materials prepared from this powder (e.g., by compaction) will be amorphous, just due to the case their sizes are below the minimal value of LRO. Additionally, in very small crystals a large fraction of the atoms are located at/or near surface. Relaxation of the surface and various interfacial effects distort the atomic positions, decreasing the structural order. Thus, even the most advanced structural characterization techniques, such as X-ray, neutron and electron diffraction, as well as transmission electron microscopy (TEM), have difficulties in distinguishing between the amorphous and crystalline structures on these length scales[83,92]. Many studies revealed that the majority of solids could be found or prepared in an amorphous state. For example, cooling strongly reduces atomic and/or molecular mobility. Thus, in principle, given a sufficiently high cooling rate, any liquid can be transformed into an amorphous solid. As cooling is performed, the material changes from a super-cooled liquid, with properties one would expect from a liquid state material, to a solid. The temperature at which this transition occurs is called glass transition temperature. If a cooling rate is faster than the rate at which atoms and/or molecules can be organized into a more thermodynamically favorable crystalline state, then an amorphous solid will be formed. In contrast, if atoms and/or molecules have a sufficient time to be organized into structures with two- or threedimensional order, then a crystalline (at least, a semi- crystalline) solid will be formed. Furthermore, in many cases amorphous materials can be produced by additives, which interfere with the ability of the primary constituent to crystallize. For example, addition of soda to melted silicon dioxide results in amorphous window glass and addition of glycols to water results in a vitrified solid[83]. More to the point, amorphization of many solids might be achieved by 22 Sergey V. Dorozhkin: Amorphous Calcium Orthophosphates: Nature, Chemistry and Biomedical Applications applying mechanical forces, e.g., by intensive milling[93, 94], as well as by irradiation[95,96]. From a thermodynamic point of view, amorphous materials are at best metastable. Given a sufficient time, they tend to transform to crystalline phases that are thermodynamically more stable. Interestingly, but when an unstable crystalline solid is transformed to an amorphous phase, this transformation frequently exhibits features that are associated with ordinary melting. Namely, amorphization frequently begins at grain boundaries, surfaces or other defect sites, as does ordinary melting. Further, as the transformation proceeds, a sharp interface that separates the amorphous materials from the untransformed crystalline material is always observed[82]. poorly crystalline apatite (Fig. 2, middle). Dr. Tannenbaum was certain that the settings on the diffractometer were not different from those he had used previously. Instructed by Dr. Posner to repeat the experiment, he observed the same phenomenon. Immediately after being mixed, the precipitate formed was amorphous, while after several hours, it converted to poorly crystalline apatite. It seemed plausible to Dr. Posner that were such an “amorphous” material (i.e., one that did not give a crystalline diffraction pattern) present in bone, along with the apatite, it might account for the broad diffraction pattern of bone mineral”[19]. One should stress, that both L.C. Chow, et al.,[99] and E.D. Eanes[100] published corrigenda to this story by Boskey. 3. Amorphous Calcium Orthophosphates (ACPs) 3.1. History According to E.D. Eanes[20], the history of ACPs looks as this: “In 1955, Robinson and Watson[97] were the first to suggest that a substantial portion of newly formed mineral in young bone was not crystalline. Instead, they described this early mineral as being more similar in character to an amorphous-like precipitate they had prepared in a study on synthetic HA[98]. This precipitate, which appeared initially in their synthesis when sufficiently concentrated solutions of CaCl2 and Na2HPO4 were mixed at room temperature and neutral pH, had as its most distinctive features an extremely fine, non-crystalline texture when examined by TEM and no discernable electron diffraction pattern. This latter feature led them to infer that the considerably more diffuse electron diffraction pattern of newly formed bone mineral as compared to more mature bone mineral, although still apatitic in character, indicated the presence also of an amorphous component”[20]. However, A.L. Boskey[19] has reported another story: “In 1964, Dr. Paul Tannenbaum, a graduate student in periodontics at Columbia, and a research assistant in Dr. Posner’s laboratory at hospital for special surgery, was studying the effect of fluoride on apatite crystal size. He prepared a synthetic apatite by mixing high concentrations (~ 30 mM) of calcium chloride and (~ 20 mM) sodium acid phosphate in buffer, and, being anxious to confirm that the precipitate which formed was apatite, pelleted it by centrifugation, dried it with acetone and placed it on a holder for analysis by wide-angle X-ray diffraction. The pattern obtained (Fig. 2, bottom) was broad and diffuse, with a maximum at ~ 30° 2 theta, had no features, and was clearly not apatite. Dr. Posner suggested that Dr. Tannenbaum did not have the settings correct on the X-ray diffractometer, but since it was late on Friday, decided to correct the settings on Monday. On Monday, the sample, which had been left on the diffractometer over the weekend, was again subjected to X-ray diffraction analysis, but now the pattern had the appearance of a Figure 2. Comparison of X-ray diffraction patterns (Cu Kα radiation, = 0.154 nm) of synthetic ACP (bottom), poorly crystalline CDHA (middle) and well crystalline HA (top). The intensity values of the top pattern have been multiplied by a factor of 10, accounting for a high noise level In 1960’s, both X-ray diffraction and infrared spectroscopic techniques were used to obtain a quantitative estimate of the amorphous content of bone mineral and then, based on the methods used in polymer chemistry, an algorithm to estimate the ACP amount in bones was developed[101-103]. Early X-ray diffraction estimates indicated the presence of ~ 30% or more of a non-crystalline mineral in bones of several animal species. Later estimates by X-ray radial distribution analysis placed the upper limit of ~ 10% ACP in bones and brought into question whether all X-ray amorphous mineral of bones was truly non-crystalline[17,104-107]. However, further studies by higher-resolution techniques have shown that ~ 99% of the mineral in bone is a poorly crystalline ion-substituted CDHA of a biological origin[19]. Morphological evidence establishing the extent of ACP in International Journal of Materials and Chemistry 2012, 2(1): 19-46 23 skeletal tissues of mammals is equally ambiguous. Although some studies[97,108-112] report a presence of small spheroidal particles atypical of crystalline material, primarily in actively metabolizing regions, most TEM studies of bones do not even mention finding such possibly amorphous structures. Furthermore, during ageing, the amount of ACP in bones and teeth of mammals decreases while the crystalline forms of biologically formed ion-substituted CDHA increases during early stages of formation[3,11]. Since both physical and morphological evidences for ACP in skeletal tissues of mammals have been difficult to establish directly, much of our progress in clarifying the possible roles of ACP in biogenic calcification has come from both synthetic and in vitro studies[20]. 3.2. Preparation acidic pH, crystalline calcium orthophosphates normally are precipitated. However, in presence of stabilizers (magnesium and/or citrates), ACP could be precipitated at solution pH within 6.0 – 6.5[133]. No information on ACP precipitation from even more acidic aqueous solutions has been found in literature. The obtained precipitates should be collected shortly after the preparation (the sooner, the better), because in aqueous media ACP is spontaneously converted to the crystalline calcium orthophosphates, mainly to CDHA [116,134]. Furthermore, it was shown that the final calcium orthophosphate (a dry powder) would be amorphous if, beside the appropriate key factors of the synthesis (a high concentration of reagents, a basic solution pH, a rapid mixing and a low temperature), both a high addition rate and a mandatory freeze-drying of the precipitates were employed[40,116,132,135]. 3.2.1. Wet-chemistry Already in early 1970’s the researchers established that the final and stable product of a reaction between calcium and orthophosphate salts in neutral or basic aqueous solutions was crystalline stoichiometric HA. However, the stoichiometric well crystalline HA might be prepared at elevated temperatures only; thus, in the vast majority cases, in aqueous solutions CDHA is formed instead. Furthermore, during CDHA precipitation, over a broad range of the solution conditions, an ACP precursor phase is often formed [113-123], in some cases, via a short intermediate stage of OCP formation[124,125]. Data are available that CDHA crystallization from ACP simply involves a LRO increase in the structure[126]. One should stress, that already in the mid of 1970’s ACP was found to be not the mandatory precursor to CDHA. Namely, in dilute aqueous solutions CDHA was found to precipitate without going through an ACP precursor[126]. Afterwards, a model was developed to illustrate factors influencing the nature of non-stoichiometric amorphous precursor phases precipitating in highly supersaturated solutions[127]. The basic approach to synthesize ACP still consists of a spontaneous precipitation by mixing concentrated aqueous solutions of calcium and orthophosphate ions, first developed in 1953 by Watson and Robinson[98]. Another commonly used method is to prepare an acidic (pH within 0 – 4) sub-saturated aqueous solution of a calcium orthophosphate salt (e.g., DCPD, MCPM) and afterwards to induce precipitation by a rapid addition of a strong base (e.g., NaOH, KOH, NH4OH) to reach the desired solution pH, usually within 10 – 12[128]. Vigorous mixing is highly desirable. By means of both approaches, various types of ACPs have been prepared from solutions encompassing a wide range of pH (from ~ 6.5 to ~ 13), Ca/P ionic ratios (from ~ 0.1 to ~ 10), calcium and orthophosphate concentrations (from ~ 0.002 to ~ 1 mol/l), as well as at temperatures within 0℃ to 50℃. However, the Ca/P ratio of the mixing reagents (classically, Ca(NO3)2·4H2O and (NH4)2HPO4) is typically kept within 1.50 – 1.67, while a basicity of the mixing solutions is frequently created by NH4OH addition[48,116,128-132]. At Figure 3. Bright field transmission electron micrographs of ACP → CDHA transformation at reaction times of (a) 5 min, (b) 3 h, (c) 9 h, (d) 48 h. Reprinted from Ref.[136] with permission. Another set of electron micrographs of ACP → CDHA transformation might be found in Ref.[256] In all wet-precipitation techniques, the amorphous precursors, although related to the final CDHA phase, are differed from the final phase in atomic structure, particle morphology and stoichiometry. For example, the X-ray diffraction pattern of ACP (Fig. 2, bottom), if compared to those of CDHA (Fig. 2, middle) and HA (Fig. 2, top), shows a single and a very broad and diffuse diffraction peak with the maximum at ~ 25° 2 theta, typical for amorphous materials, which lack the atomic LRO characteristics of all crystalline materials, including HA[104]. The precipitated ACP phases appear to be spherical (Figs. 3 a and b) in an electron microscope (diameter ca. 30 – 100 nm), unlike the needle-like crystals of CDHA (Figs. 3 c and d). The solution pH, concentration of the mixing reagents and a preparation temperature all affect the ACP particle sizes; namely, a 24 Sergey V. Dorozhkin: Amorphous Calcium Orthophosphates: Nature, Chemistry and Biomedical Applications higher supersaturation produces smaller ACP particles[129]. Although ACP can be prepared with a Ca/P molar ratio as low as ~ 1.2 (at low pH – see Fig. 4) or as high as 1.7 (at high supersaturation), the departure from a Ca/P of ~ 1.5 has been shown to be due to surface-adsorbed soluble phases those can be washed away or to occluded Ca, respectively[104]. Besides, ACP powders might be prepared by spray drying of acidified aqueous solutions of soluble calcium orthophosphates[348]. Figure 4. Ca/P molar ratios of washed (dashed line) and unwashed (solid line) ACP precipitates as a function of their formation pH. Reprinted from Ref.[137] with permission a non-aqueous or solvent + water media[72,131,138-152]. The presence of organic compounds and/or solvents results in decreasing of a dielectric constant. Therefore, all ions in solutions appear to be less solvated than those in water. The consequence of this is a strong decrease of solubility and an increase in precipitation kinetics, which simplifies amor- phization[153]. Furthermore, in such systems, complexes of calcium with organic agents can be formed. This favors ACP formation, which is attributed to the coordinated complexing agents remaining in the structure of ACP[144]. The influ- ence of the presence of organic solvents to the amorphization degree of precipitated calcium orthophosphates is well il- lustrated in Fig. 5a. In some cases, incorporation of organic compounds into ACPs has been detected[131,150]. Inter- estingly, but a replacement of a freeze-drying stage of a wet ACP precipitate by an oven drying at 80 °C resulted in its transformation to CDHA[139]. This process was ascribed to an internal hydrolysis of a part of orthophosphate ions of ACP to those of CDHA according to the schemes[139, 154]: PO43- + H2O → HPO42- + OH- (1) Ca9(PO4)6 + H2O → Ca9(HPO4)(PO4)5OH (2) 3.2.3. Mechanical and Pressure-induced Techniques In addition to the aforementioned solution-based methods, various types of ACPs might be prepared by dry chemical techniques. For example, an ACP was prepared using a mechano-chemical method involving a dry mixture of DCPD and Ca(OH)2 reactants with a Ca/P ratio of 1.67[155]. Other authors have shown that a prolonged high-energy ball milling of either α-TCP, β-TCP powder in ethanol or a dry mixture of ACP and DCPD powders lead to ACP formation after 24 h[156-158]. Furthermore, prolonged high-energy ball milling of TTCP was found to result in a mechanical activation with the formation of undisclosed nanocrystalline and/or amorphous domains within the compound[159]. However, there is a non-negligible risk of powder contamination (ball wear) when using this processing route over extended periods to obtain an ACP[31]. In addition, a crystalline to amorphous transition has been detected for various calcium orthophosphates at very high (up to 10 GPa) pressures (Fig. 6)[160,161,347]. b Figure 5. X-ray diffraction patterns of: (a) freeze-dried precipitates prepared from aqueous solutions containing different amounts of polyethylene glycol. Please, note a shift of the center of the broadened peak from ~ 32° toward ~ 31° with increasing polyethylene glycol/Ca molar ratio, which implies some structural differences in the resulting ACPs; (b) heat-treated (800℃) ACPs prepared from aqueous solutions containing different amounts of polyethylene glycol. Reprinted from Ref.[150] with permission 3.2.2. Non-aqueous Solutions and Solvents (sol-gel) Besides, ACPs might be easily prepared in either Figure 6. Effect of pressure on X-ray diffraction patterns of HA and hydrated TCP at 1 bar and high pressures. Reprinted from Ref.[161] with permission 3.2.4. Thermal International Journal of Materials and Chemistry 2012, 2(1): 19-46 25 Furthermore, ACPs might be prepared using high energy processing at elevated temperatures. This method is based on a rapid quenching of melted calcium orthophosphates occurring, e.g., during plasma spraying of HA[162-174,349]. A plasma jet, which possesses very high temperatures (5000 – 20000℃), partly decomposes HA. Furthermore, there is an opinion, that thermal spraying produces the amorphous phase, not only due to the high cooling rate but also to the removal of hydroxyl ions which make it more difficult for the crystalline phase to form[167]. This generally leads to a mixture of calcium orthophosphate phases with variable compositions, often containing impurities, which is not convenient for preparation of pure ACPs. Interestingly, but the amorphization degree of the plasma sprayed HA coatings appeared to correlate with the presence of vacancies of hydroxyl ions in the structure of HA: the more vacancies were present in the apatite structure due to missing hydroxyl sites, the more amount of ACP was present in the resultant coatings[166]. This might be due to the fact that particles resident in plasma for a longer period of time lose more structural water. Other studies have shown more amorphous phase located adjacent to the substrate and a gradient tending towards a lower ACP content at the top of the coating[31]. To summarize, the amorphous phases in plasma sprayed HA coatings are in intimated mixtures with both crystalline calcium orthophosphates and other compounds, such as CaO [9], and up to now nobody has ever succeed to extract ACPs from the blend. However, the amorphous regions in plasma sprayed HA coatings might be mapped using a scanning cathodoluminescence microscopy technique[170]. Furthermore, due to a number of uncertainties, a reproducibility of such experiments is poor; thus, the plasma spaying technique is not considered as a valuable method to produce ACPs. A flame spray synthesis, when a liquid precursor solution is fed through a capillary into a burning methane / oxygen supporting flame, seems to be more preferable to produce ACPs at high temperatures[175,176]. Further details on and additional examples of ACP preparation might be found in literature[31]. 3.2.5. Irradiation Just a few studies are available on the amorphization of calcium orthophosphates (up to now, apatites only) by irradiation[177, 178]. However, due to the obvious risks caused by the induced radioactivity, this amorphization approach is highly unlikely to be ever used to prepare calcium orthophosphates for biomedical applications. Unfortunately, it remains unclear in what extent the structures, compositions and properties of ACPs prepared by various production approaches might be mutually compared. To conclude the preparation part, one should briefly mention on an interesting attempt to precipitate separately hydroxyapatites of Mg, Ca, Sr and Ba from basic supersaturated orthophosphate solutions containing a 10: 6 divalent cation / PO4 molar ratio[179]. In the cases of Mg, Ca and Sr, the first precipitated phase had a 3 : 2 ratio (i.e., that of a TCP), while only Ba went directly to the stable 10 : 6 HA phase. Furthermore, the precipitated magnesium orthophosphate was amorphous and remaining in the mother solution did not convert to a Mg-deficient HA. The precipitated calcium orthophosphate formed ACP, which converted to CDHA by a solution-mediated autocatalytic mechanism[130]. The precipitated strontium orthophosphate was not amorphous but poorly crystallized and readily converted in solution to a strontium HA. Thus, the smaller alkaline earth cation systems tended to form the more stable amorphous 3 : 2 compounds[179]. 3.3. Morphology of Precipitated ACPs When viewed by TEM, ACP solids precipitated from aqueous solutions usually have a curvilinear appearance rather than the faceted and angular shape of crystalline calcium orthophosphates[180, 181]. However, this curvilinear aspect has only been clearly established for dried ACP. The morphological form of highly hydrated flocculent solids that appear initially in freshly precipitated ACP suspensions is not known. What is observed when drops of these suspensions are placed on carbon-coated grids, excess solution removed and air-dried are irregularly shaped, anastomosing aggregates of low-contrast, disk-shaped particles varying widely in lateral dimensions (from ~ 0.01 µm to 5+ µm)[181, 182]. These highly flattened particles represent collapsed, de-solvated residues of the initial wet ACP flocculates. As ACP suspensions age, high contrast particles with a more spherical aspect begin to appear, initially evolving as bud-like extensions from the disks[181, 182]. With time, these spherical forms become the dominant shape for ACP. Although generally smaller (20 – 300 nm in diameter) than the disks they supplant, the spherical forms, like the disks, frequently aggregate into irregularly shaped and branching clusters. The progression from disk-shaped to ball-like particles most probably represents a spontaneous desolvation in situ or the initial gel-like flocculates into smaller, denser, less hydrated structures[183]. That the spherules are formed in suspension and are not a drying artifact is supported by the crystallization behavior of ACP preparations. Although the evolution of a spherical morphology would be favored during consolidation as this shape minimizes interfacial tension with the surrounding solution, it also requires that the contracting surface be isotropic. This is possible for uniformly curved surfaces only when the enclosed structure remains non-crystalline while desolvating[20]. 3.4. Chemical Composition Nowadays, ACPs should be recognized as a special class of biomedically relevant calcium orthophosphate salts having variable chemical but rather identical glass-like physical properties. Presumably, all calcium orthophosphates mentioned in Table 1 might somehow be fabricated in an amorphous state. Therefore, perhaps, sometime in the future people will deal with an amorphous phase corresponding to the chemical composition of MCPM (“amorphous MCPM”), 26 Sergey V. Dorozhkin: Amorphous Calcium Orthophosphates: Nature, Chemistry and Biomedical Applications an amorphous phase corresponding to the chemical composition of DCPA (“amorphous DCPA”), an amorphous phase corresponding to the chemical composition of TTCP (“amorphous TTCP”), etc. (in most cases, stabilization procedures will become necessary), as well as with various mixtures thereof. Currently the majority of such compounds remain unknown and, in the available literature, a variety of ACPs is distinguished by the Ca/P ratio only. Since the greater part of ACPs has the Ca/P ratio close to 1.5 (see below), a term “amorphous TCP” (ATCP) becomes usual in literature[31,140,154,158,175,184-191]. Other terms such as “amorphous carbonated apatite”[40,192,193], “amorphous CaHPO4”[140], which is equal to “amorphous DCPA” (Table 1), “amorphous Ca8(HPO4)2(PO4)4”[140], which is equal to “amorphous OCP” (Table 1), “amorphous OCP”[142], “amorphous Ca10(PO4)6(OH)2”[194], which is equal to “amorphous HA” (Table 1) and “amorphous dicalcium phosphate”[195] are rare but have been already mentioned. Furthermore, formation of an amorphous phase was detected during a slow dehydration at slow heating rates of brushite (DCPD) and its transformation to monetite (DCPA) and the amorphous phase was regarded as “a highly disordered monetite with some free water trapped in the structure”[350]. One should note, that ACPs with the Ca/P ratio < ~ 1.0 currently remain unknown. with a spherular morphology of the initially precipitated amorphous phase ACP1[202-204]. The same terms ACP1 and ACP2 were used in another study[145], in which the authors studied ageing of calcium orthophosphate precipitates in methanol at room temperature, finally leading to formation of a nano-sized β-TCP. Furthermore, at precipitation experiments from aqueous solutions containing polyethylene glycol, two types of ACPs were detected [150]: one with a broadened peak centered at ~ 31° and another with a broadened peak centered at ~ 32° (Fig. 5a, spectra 16:1 and 4:1, respectively). According to the authors, the first one was similar to the basic structure of β-TCP, while the second one was similar to the basic structure of HA[150]. Perhaps, one can mention on amorphous β-TCP and amorphous HA, respectively. A similar shift of the position of the amorphous maximum but obtained at different aging time of ACP precipitates (Table 2) has been detected in another study[132]. Table 2. Position of the X-ray diffraction amorphous maximum at different aging time of ACP precipitates[132] Aging time 2 min 1 h 2 h 3 h 4 h 5 h 6 h 2θ, degree 29.5 30.1 30.4 30.3 30.5 30.8 29.0 Note: The 2θ values were derived from the profile analysis of the scattering curves. Negligible changes were found for longer aged ACPs. The experimental error ± 0.5 degrees 2θ[132] 3.4.1. Precipitated ACPs Although first described in 1953[98], the quantitative chemical studies on the precipitated ACPs were not reported until 1965, when methods were devised to isolate large amounts of unstable solids for analysis. To minimize changing during sample drying, those methods utilized filtration and/or centrifugation to wash excess ions from ACP slurries, then freezing wet ACPs under high vacuum to remove any remaining entrapped solvent by sublimation[128]. Early chemical studies[134,196] on ACPs prepared at pH ~ 10.5, filtered, washed and lyophilized, showed that the Ca/PO4 molar ratio was very close to 1.5, suggesting a TCP composition (as Ca3(PO4)2·nH2O[103, 197]). No OH- ions were found in it. Furthermore, the electron spin resonance spectra of vanadyl (VO2+) ions adsorbed on ACP formed under varying conditions also revealed that ACP is either a non-crystalline form of hydrated TCP or a solid solution with the composition of Ca3(PO4)l.87(HPO4)0.2[198]. Other researchers reported a stoichiometry of the precipitated ACP more akin to OCP[138, 140, 142, 199-204], DCPA[140] and DCPD[205]; however, with a few exceptions[140, 142] the subsequent terms “an amorphous OCP” and “an amorphous DCPD” were not introduced. Namely, although the Ca/P ratio of early formed ACP phases was varied between 1.35 and 1.38, which was close to that of OCP (Table 1), the authors of Refs.[202-204] used a term ACP2 to explain initial variations in solution pH during the transformation of ACP into more crystalline phases of OCP and/or CDHA. Further, based on the results of TEM analysis, ACP2 was identified as a separate amorphous phase with a floccular morphology and no electron diffraction pattern, if compared Figure 7. Infrared spectra after baseline subtraction for ACPs prepared at: A – pH 10.0, B – pH 7.0, C – pH 6.5 and D – pH 6.0. Reprinted from Ref. [133] with permission. The complete (from 400 cm-1 to 4000 cm-1) infrared spectrum of ACP is available in Ref.[31] Figure 8. Acid phosphate content (percent of total P as HPO42-) as a function of pH for washed (dashed line) and unwashed (solid line) ACP precipitates. Reprinted from Ref.[137] with permission Additional analyzes of ACPs prepared from aqueous solutions at pH ~ 7.4 at wide variations (from 5 : 1 to 1 : 5) in International Journal of Materials and Chemistry 2012, 2(1): 19-46 27 starting Ca/PO4 molar ratios showed that the compositional Ca/P ratio decreased only slightly from ~ 1.5 due to the presence of small amounts (< 15 %) of HPO42- ions[137, 206-208]. This is an indirect confirmation of the existence of an amorphous TCP. The presence of HPO42- in ACP is easy to detect by infrared spectroscopy most notably by an absorption peak at about 890 cm-1, due to a P–O(H) stretching mode of protonated orthophosphate species (Fig. 7)[133, 140]. Furthermore, in infrared spectra of HPO42--contained ACPs there is a large band of a weak intensity near 2300 cm-1, which corresponds to H–O(P) stretching in HPO42- ions[140]. More to the point, the amount of HPO42- in ACP strongly depends on the solution pH: the lower the Ca/P ratio for any given ACP precipitate, the greater is its HPO42- content (Fig. 8). The presence of HPO42- ions in ACP has been confirmed by solid-state nuclear magnetic resonance (NMR) technique: the results revealed the presence of ~ 20 % of HPO42- in two ACP samples[209]. Furthermore, orthophosphate units close to water and a third (remained unknown) type of ortho- phosphate groups were found in the NMR spectra of the ACPs. What’s more, substantial differences were noticed between the NMR spectra of two ACP samples, donated by two different research groups[209]. In other studies, two ACP samples of different chemical composition (since they had been prepared at solution pH = 6.5 and 10.0, respectively) were found to give very similar EXAFS spectra[133, 210]. The latter means that a SRO around calcium ions of both ACPs appeared to be very similar; however, the reasons why the pH differences were not reflected in the calcium envi- ronment remained unclear. These experiments confirm the fact that ACPs are not a single chemical compound but rep- resent a special class of amorphous calcium orthophosphate salts. Thus, precipitated ACPs cannot be described by a single chemical formula; a sketch CaxHy(PO4)z·nH2O (Table 1) seems to be the most reasonable element demonstration of this class of calcium orthophosphate salts. Presumably, ACPs with the Ca/P ratio exceeding ~ 1.6 should also con- tain some amount hydroxide anions; however, no informa- tion on this point has been found in literature. However, a presence of CaO is frequently detected in ACP-containing calcium orthophosphate coatings prepared by plasma spraying. Nevertheless, in slightly alkaline aqueous solutions, ACP might have a well-defined chemical composition. For ex- ample, there is a finding that ACP slurries over the pH range 7.4 – 9.25 appeared to have a nearly constant solution ion activity product of ~ 1.6 × 10-25 when the solid-phase com- position is postulated to be Ca3(PO4)l.87(HPO4)0.2, i.e. when the local chemical unit in ACP is postulated to have a Ca/P molar ratio of 1.45, with ~ 10% HPO42-, but without OHions[207]. Thus, in spite of the absence of a LRO, this rela- tive constancy in the composition over such a wide pH range indirectly suggests that ACP should have some well-defined local chemical units. However, at solution pH exceeding 9.25, ACP does not appear to have a nearly constant solution ion activity product. This breakdown in the solution con- stancy suggests that the solubility-controlling ions of ACP are subtly dependent compositionally on the preparation conditions. Perhaps, at solution pH > 9.25 the content of HPO42- ions no more remains constant and gradually decreases with pH increasing[128]. Furthermore, at more acidic pH = 6.9, ACP precipitates with Ca/P molar ratios as low as 1.15 have been reported[211]. These latter precipitates are extremely unstable and rapidly change over into crystalline DCPD. Again, a term “amorphous DCPD” has not been introduced in that study. Even after a lyophilization, solution-matured, spheroidal ACP solids still retain ~ 15% water by weight[197,212]. A temperature programmed description analysis by Sedlak and Beebe indicated that the most part (~ 75%) of this retained water was tightly bound inside the solid, while the rest was a more loosely held surface water with different activation energies of 20.0 and 10.5 kcal/mole, respectively[213]. These results suggest that ACPs do not completely desolvate in solutions but remain partially hydrated with about 3 water molecules per formula unit. Other researchers found that water occurred in regions those were only loosely associated with calcium cations in ACP[5]. Furthermore, when prepared from carbonate-containing solutions, ACPs can readily incorporate carbonate anions[35-40,214,215]. The amount of carbonate incorporated at any given pH increases with solution carbonate concentration. At a given concentration, carbonate uptake also increases with pH. Incorporating carbonate into ACP does not affect the HPO42- content but raises the Ca/P molar ratio. At physiological pH, the carbonate content of ACP precipitated from solutions containing 30 mmol/l carbonates is ~ 3 % by weight[182]. These data suggest that ACP, if present in skeletal tissues, would contain appreciable amounts of carbonates, although less than those present in the apatitic phases of bones[215]. Two other ions that readily incorporate into the ACP structure are Mg2+[37,44,133,179,216-218] and P2O74-[215,219,220]. Ions such as P2O74-, carbonate and Mg2+ increase the solution stability of ACP and, in the case of the latter two ions, could possibly play an important role in maintaining the presence of ACP in skeletal tissue. In addition, other ionic substitutions are possible; however, such inorganic additives alter the ACP composition, which would enhance the negative effects in the biomedical application of ACP. Besides, with a few important exceptions, ion-substituted forms of ACP [34-47] are not discussed here. 3.4.2. Other Types of ACPs Little is known on the chemical composition of ACPs, prepared by other amorphization techniques. For example, various ACP samples prepared by compressing of several calcium orthophosphates at very high pressures revealed collapses of their initial crystal structures but possible changes in their chemical compositions were not investigated[160,161]. Interestingly, but the authors found that in the region below 550 cm-1 the infrared spectra of DCPD in amorphous phase resembled that of HA in the crystalline phase and conversely the spectra of DCPD in the crystalline 28 Sergey V. Dorozhkin: Amorphous Calcium Orthophosphates: Nature, Chemistry and Biomedical Applications phase resembles that of HA in the amorphous phase[161]. In the case of milling, calcium orthophosphates were found to become amorphous; however, no additional phases were detected[157,158]. Presumably, this means that during amorphization their chemical composition remained unchanged. Concerning the ACPs formed in plasma-sprayed HA coatings, the authors of one study reported that “the amorphous phase mostly consists of a dehydroxylated calcium phosphate”[174], which, presumably, meant dehydroxylated HA. If so, the chemical composition of that particular ACP should be close to amorphous OA. The authors of another study considered “that the amorphous phase substance consists of HA molecules” (Ref.[163], p. 227). However, in the next study, the same authors mentioned that “the plasmasprayed amorphous phase is an oxyapatite”[164]. No further clarification has been provided; however, all these authors have come to the conclusion on the apatitic chemical composition of the plasma-sprayed amorphous phases. Besides, these ACPs are definitively anhydrous contrary to the precipitated ACPs. To conclude the chemical part, one should mention on solubility of ACPs. Due to the chemical variations, this value cannot be measured precisely (Table 1). Several different solubility products have been proposed for various ACPs and the interested readers are referred to Table 1 of Ref.[31] for the details. done in the mid of 1960’s on a material precipitated at pH ~ 10[134, 196]. As Watson and Robinson found at neutral pH, the initial phase that spontaneously formed immediately upon mixing concentrated alkaline Ca- and PO4-containing solutions was structurally non-crystalline[98]. The X-ray diffraction pattern of this rapidly precipitated phase showed only two very broad and diffuse peaks, typical for substances that lack the periodic LRO[196]. The extreme diffuseness of the synthetic ACP pattern also provided a basis for interpreting the reduced intensity of the apatitic X-ray diffraction patterns of bones as being due to the mineral having an amorphous component[101]. The diffracted X-ray energy from this component was so uniformly dispersed that it could not be separated from the subtracted background intensity[20]. 3.5. Structure In general, determination of the atomic structure of amorphous solids is a non-trivial task. As the structure can be defined essentially only in terms of unit-cells containing an infinitely large amount of atoms (as there is no LRO periodic symmetry), a statistical description appears to be unavoidable. Thus, the structure of a particular amorphous solid can never be determined unambiguously and this uncertainty is compounded by the fact that the structure of a non- crystalline material often depends on the specific details of preparation techniques[88]. Furthermore, the chemical composition, namely the Ca/P ratio, of ACPs varies a lot (1.2 < Ca/P < 2.2 – see Table 1), which makes the task even more complicated. The latter results in the fact that different samples of ACP possess diverse properties and the data found for one particular sample appear to be inapplicable to other ACP samples. A possible solution of this problem seems to be in a wide recognition of the fact that ACP is not a single chemical compound but represents a group of calcium orthophosphates having diverse physical and chemical properties. In other words, depending on the Ca/P ratio and/or other properties, first of all, any particular sample of ACP must be ascribed to the existing crystalline phase from Table 1 and only afterwards any structural investigations should be performed. This is the only way to succeed in clarification of the ACP structures from experimental measurements in future. Current state-of-the-art on the structure of ACPs is given below. The first quantitative studies on a synthetic ACP were Figure 9. Diagrammatic representation of the SRO structure of four phosphate tetrahedra and two water molecules about calcium ions in an acidic ACP, calculated using the DCPD shell model. The positions of hydrogen atoms are not determined. Reprinted from Ref.[227] with permission Figure 10. Fragments of the infrared spectra of ACP (upper), CDHA (middle) and HA (lower). Reprinted from Ref.[129] with permission Initially, there were suggestions that a synthetic ACP was, in fact, HA of such small crystal dimensions that its X-ray diffraction pattern was widely broadened to appear amorphous in character. However, calculated X-ray diffraction patterns assuming that ACP consisted of small groups of HA

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