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Unravelling Doped Biphasic Calcium Phosphate: Synthesis to Application Subhadip Basu† and Bikramjit Basu*,†,‡ †
Materials Research Centre, Indian Institute of Science, Bangalore 560012, India Center for BioSystems Science and Engineering, Indian Institute of Science, Bangalore 560012, India
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‡
ABSTRACT: Among synthetic biomaterials, calcium phosphate (CaP)-based bioceramics, specifically hydroxyapatite (HA) and tricalcium phosphate (TCP) as well as biphasic calcium phosphate (BCP), are widely investigated in the biomedical community. The arrangement of different ions in the CaP crystal structure allows one to dope with metal ions, while tailoring the properties. The present article reviews some of the multifaceted research in the field of (multi)-ion-doped BCP, particularly on the aspects of biomedical applications. After summarizing different synthesis methods, a brief overview of experimental techniques and results to probe the dopant site and local structure surrounding the dopants has been provided, with an emphasis on the XRD-based Rietveld refinement and EXAFS (extended X-ray absorption fine structure) method. The effect of ion substitution on the functional properties, such as dielectric and magnetic behavior, of BCP has also been discussed. Importantly, in vitro and in vivo biocompatibility of doped BCP has been elucidated along with in silico studies on the biomaterial−biomolecule interaction. Toward the end, the results of the published clinical trial study and limited commercialization efforts will be illustrated. KEYWORDS: biphasic calcium phosphate, hydroxyapatite, tricalcium phosphate, ionic substitution, doping, functional properties, cytocompatibility, in vivo studies, clinical trials
1. INTRODUCTION Biomaterials are synthetic materials that interact with biological systems and are employed for therapeutic or diagnostic applications with desired clinical outcomes.1 An ideal biomaterial, which replaces the natural tissue, should mimic its properties closely and must not cause any undesired toxic effects inside the living system.2 Biomaterials science is an interdisciplinary field that aims to study the properties of and develop biomaterials for different therapeutic applications. Hard-tissue engineering or the art of developing bone/dental replacement materials is a major part of biomaterials science of growing interest because of the rise of musculoskeletal and dental diseases in the global population in the recent era. According to statistics, around 2.2 million people yearly undergo bone grafting procedures.3 Moreover, data has revealed that the global annual requirement for dental implants is in the range of 10 000−30 000.4 The quest for hard-tissue grafting material is in high demand in both academia and industry. Therefore, to develop any new hard-tissue replacement material, a fundamental understanding of different physical, chemical, and biological processes related to the material and material− biological system interaction is necessary. Hard tissues, i.e., bone and teeth, are highly mineralized in nature. Bone and dentin comprise of around 45−70 wt % of mineral, and for enamel, it reaches up to 96 wt %.5,6 The hardtissue mineral is called the bioapatite and can be idealized as the hydroxyapatite (HA, Ca10(PO4)6(OH)2) phase. The apatite phase present in the bone mineral is nonstoichiometric in nature © XXXX American Chemical Society
and is a blend of different cations and anions. Different trace elements like Sr2+, Mg2+, Na+, K+, Cl−, F−, CO32−, etc. are present (some are in the ppm level) in the bioapatite.7 For this reason, the proposed chemical formulas for the bioapatite are (Ca, Na, Mg, K, Sr, Pb)10, (PO4, CO3, SO4)6, (OH, F, Cl, CO3)2,5 or Ca8.3□1.7(PO4)4.3(HPO4 and CO3)1.7(OH and 0.5CO3)0.3□1.7 (□ denotes vacancy).8 The mean crystallite size of the bioapatite is determined to be 28 nm within the 0−25 age group.9 At the nano level, these hexagonal apatite crystals are dispersed in the collagen matrix, which provides the mechanical strength of natural bone.7 The bioapatite is reported to be OH−deficient.10 The presence of a nonapatitic environment in bone mineral has also been confirmed by spectroscopic studies.11 These nonapatitic structures correspond to hydrated layers in bone mineral, and they are thought to be responsible for a wide range of beneficiary interactions between crystal−proteins, crystal−crystal, and substrate−crystal.12,13 Due to the resemblance with the natural bone mineral, HA has been explored extensively for hard-tissue engineering applications. Between hexagonal and monoclinic phases of HA, the former has drawn more attention because of the presence of the hexagonal nanoapatite crystallites in bone mineral, as previously Special Issue: Biomaterials Research in India Received: June 5, 2019 Accepted: August 1, 2019
A
DOI: 10.1021/acsabm.9b00488 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 1. Hydroxyapatite (HA) can exist in nature in two crystallographic forms: hexagonal and monoclinic. (a) Hexagonal unit cell of HA with two distinct Ca sites: Ca(1) and Ca(2). Coordination geometry of (b) Ca(1) and (c) Ca(2). (color code: Ca = blue P = pink, O = red, and H = light pink). (The image was reproduced with permission from ref 17. Copyright 2016 Royal Society of Chemistry.)
mentioned. The hexagonal HA possesses P63/m crystallographic symmetry with lattice parameters, “a” = 9.432 Å and “c” = 6.881 Å (Figure 1a).14 The OH− groups form a columnar ion channel along the “c” axis, which play a vital role in ionic conduction.15 Hydroxy groups are arranged in such a way inside the ion channel that they cannot form hydrogen bonds among themselves.16 The presence of two distinct calcium sites, Ca(1) and Ca(2), is one of the striking features of hexagonal HA (Figure 1b,c).17 Ca(1) atoms are located at the edges of the unit cell, while the Ca(2) atoms form an equilateral triangle with the ion channel at the center.18 Phosphate ions are the largest groups present in HA, and they determine the unit cell structure.19 One of the noteworthy characteristics of HA is its potential to withstand isomorphic ion substitution inside its lattice structure. HA can retain its crystal symmetry up to a Ca/P molar ratio of 1.3 against the stoichiometric Ca/P ratio of 1.67.15 Researchers exploited this fact to synthesize heavily ion-doped HA in order to obtain better bone-mimicking material. This is driven by the hypothesis that ion-doped calcium deficit synthetic HA might closely follow physical properties of multi-ion-doped bioapatite. In general, cations replace the Ca ions of the HA crystal lattice, while anions replace the hydroxy or phosphate groups. Almost half of the elements of the periodic table can be doped inside the HA crystal.20 If electroneutrality of the apatite is hampered due to ion substitution, then it is compensated either by creation of additional vacancies or through incorporation of additional cations or anions.21,22 In the case of trivalent cation-doping, formation of lower valence metal hydroxide ions takes place to maintain the charge balance.23 Any type of ion substitution in the apatite lattice causes contraction or expansion of lattice parameters and unit cell volume. It drastically affects other material properties as well. A general wrong perception is that the larger cation replaces Ca at the Ca(2) site because of a larger available volume.24 In reality, preferred doping site for any ion depends on the interaction of that ion with the surrounding atoms.25 Due to a larger ion−oxygen distance, larger ions can also substitute Ca at Ca(1) sites, but at a higher dopant concentration, the substitution at Ca(2) sites usually takes place to partially neutralize the mutual repulsion.26 Apart from HA, many other calcium phosphate-based bioceramics have been tested by the biomaterials community.
Among them, tricalcium phosphate (TCP) is arguably the second most explored bioceramics after HA. It exists in nature as three polymorphs, β, α, and ά.27 The β-TCP phase is stable at room temperature.19 It has a rhombohedral symmetry group R3c with the lattice parameters, a = 10.3961 Å and c = 37.3756 Å.28 The β-TCP crystal lattice is made of two separable atom columns and five nonequivalent Ca sites, namely, Ca(1)−Ca(5), together with a vacancy for Ca(4).29,30 The arrangement of atoms in the columns of β-TCP lattice is shown in Figure 2. Similar to HA, different cations and anions can easily get incorporated inside the TCP crystal structure. Monovalent and bivalent cations replace Ca ions at Ca(4) and Ca(5) lattice sites.30 The preferred dopant sites for trivalent metal ions are not known completely.30 Biphasic calcium phosphate (BCP), or the mixture of HA and β-TCP, is generally considered to be a better biomaterial than any of its components in terms of osteoinductive potential without any growth factors.31,32 Ionic substitution in the BCP system is a widely investigated methodology to improve its functional and biological responses. Against this background, the present review article intends to provide a brief outlook of ongoing research activities on doped BCP and its constituents. The article is broadly divided into two parts. In the first part, diverse synthesis techniques to produce ion-substituted BCPs have been summarized, followed by the elucidation of current computational and experimental results, which depict the structures of doped HA/TCP. A brief description of different experimental methods to probe the dopant sites and local chemical structures surrounding the dopants has also been provided. The main focus of the second part is to illustrate the effects of doping on functional (electrical and magnetic) and biological responses (in vitro and in vivo) of HA/TCP/BCP. Recent progress on the simulation studies of HA−biomolecule interactions has been described. Finally, the results of the clinical trial studies and commercialization efforts have been outlined. Summarizing, the current work attempts to review current developments related to doped BCP-based bioceramics.
2. INSIGHT INTO PHASE STABILITY 2.1. Doped BCP-Synthesis Protocols. A large number of synthesis strategies have been adopted by the material scientists B
DOI: 10.1021/acsabm.9b00488 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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level.43,44 Besides this, the mechanochemical method is successfully employed to synthesize Mg-substituted and carbonated HA.45,46 The wet chemical synthesis techniques are the most widely used methods to produce doped BCP. Chemical precipitation, sol−gel synthesis, ion exchange method, and emulsion techniques are some of the well-known wet chemical synthesis routes to generate doped BCP particles. In the chemical precipitation method, calcium- and phosphorus-containing reagents are mixed with each other in a dropwise manner and the resultant precipitate is washed, filtered, and dried. During the reaction, the molar ratio of different ions is kept fixed.47 The nature of the final products depends on the reagent concentration, mixing rate, etc., and these parameters must be optimized to gain the desired yield.48,49 In general, the products obtained through the precipitation method are nonstoichiometric in nature and their degree of crystallinity is also poor.50 These limitations can be overcome by supplying temporary templates of nucleation to the reaction mixture.51,52 Different copolymers, CTAB, PEG, Tween-80, different small organic molecules like citric acid, etc. are the popular candidates used in the precipitation method to enhance crystallinity of the final products.44,53−55 The precipitation technique has been successfully employed by researchers to incorporate many kinds of dopant ions inside the HA/TCP lattice structure. Carbonate, fluoride, chloride, and different metal ions (Fe, Mg, Na, Zn, Sr, Ti, etc.) are some of such examples.56−66 Another oft-used wet chemical synthesis strategy is the sol− gel technique. In this method, a gel is formed out of the mixture of the reagents in aqueous or organic medium. The obtained gel is dried and calcined to obtain the desired compound(s).67 The calcination temperature is usually kept below 1000 °C. Sol−gel synthesis allows molecular level mixing of the precursors, thus improving the chemical homogeneity of the yield product.68,69 Maintenance of constant pH (∼10) is usually required to obtain HA particles via the sol−gel synthesis route.70 However, a report on the sol−gel synthesis of HA can be found in the literature, where the pH was not controlled.71 Like the precipitation method, the sol−gel synthesis technique is also extensively used to produce (multiple) ion-doped (Fe, Sr, Cd, Ce, Y, Zn, Mg, etc.) HA/TCP/BCP.72−78 Hydrothermal and emulsion methods are two more wet chemical synthesis routes, which have been adopted by scientists to synthesize doped BCP. In the hydrothermal method, the reaction takes place at a high temperature and pressure.79 Although the degree of crystallinity of the end products of the hydrothermal method is quite high, it is very difficult to control particle morphology and size.80,81 Precise control of pH and temperature, along with the use of surfactant, helps to regulate particle morphology and size distribution.82,83 The most popular choice of surfactant is EDTA, which is proven to promote HA crystal growth along the “c” direction.84−87 Mg-, Na-, F-, Cl-, and Zn-substituted HA particles were prepared through a hydrothermal process.88,89 On the other hand, water in oil emulsion is popularly used to synthesize HA particles with a controlled shape and size.90 It should be noted here that the emulsion technique is arguably considered to be the most effective synthesis method to control particle size and morphology.91 Besides pure HA,92 carbonated HA particles with a mean size of 10−30 nm have been synthesized through the emulsion technique.93 Apart from the above-mentioned methods, many other chemical synthesis routes have been explored by the
Figure 2. Two distinct atomic columns are observed in the crystal structure of β-TCP. (a) Projected view of β-TCP lattice on the (001) plane. Atomic arrangements in (b) column A and (c) column B. (The image was reproduced with permission from ref 29. Copyright 2003 Elsevier.)
to produce HA/TCP/BCP powders. These synthesis techniques can be broadly classified into four categories, namely, dry methods, wet chemical synthesis, high-temperature processing, and synthesis from biogenic sources. The present section outlines these synthesis methods. The dry methods require mixing of reagents without any solvent. Mainly two kinds of dry methods have been explored by researchers to obtain substituted BCP. The first is the solid state reaction. In this particular route, precursors are homogeneously mixed using the ball milling method and are processed at a high temperature (∼1000 °C).33 The precursor salts act as the source of calcium and phosphate ions. Although, the end products are highly crystalline in nature because of the high-temperature treatment, they usually suffer from a large size distribution and chemical heterogeneity.34 Due to this fact, very few studies have used this particular method to synthesize doped BCP. Reports can be found on the solid state reaction synthesis of Si-doped HA and Mg-doped β-TCP.35,36 The second kind of dry method is the mechanochemical synthesis route. In this case, the molar ratio of the precursors is precisely maintained to match the stoichiometric molar ratio of different ions present in the final compound.37 The reagents are mixed in a planetary ball mill to obtain the end product.38 Milling speed, milling media and environment, milling duration, etc. must be optimized to reach the maximum yield.39,40 This particular synthesis route provides better compositional homogeneity than that of the solid state reaction method.41,42 The mechanochemical synthesis technique is especially useful to produce fluorapatite at the industry C
DOI: 10.1021/acsabm.9b00488 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Rietveld, 117 least-squares fitting is used to match the experimentally obtained data with the calculated one. The model function for fitting consists of several parameters, and it includes contributions from both the sharp Bragg peaks from the crystalline component of the sample and the underlying smooth background.118 During the course of refinement, the scale factor for adjusting the relative intensities of reflections for the constituent phases, parameters describing the background, peak profile, and instrumental aberrations arising from particle size, preferred orientation, and strain related effects were usually optimized. The extent of the match is quantitatively described by agreement indices or R values. The weighted profile R value and expected R value are defined as119
biomaterials community to synthesize ion-substituted HA/ TCP. The ion exchange method is one such special synthesis route. In this technique, the pure HA/TCP phase is soaked in the dopant metal-ion-containing solution for a certain period and the process of ion exchange of Ca2+ by the dopant ions takes place. Sometimes, a flow is applied to enhance the exchange process.94 Time of soaking, temperature, and the concentration of the solution are some of the parameters that can be varied to tune the doping ratio.95 The main advantage of the ion exchange method is the control it provides on the bulk properties of HA/ TCP.96 Different metal ions like Zn, Sr, Na, Mn, Mg, Cu, Ni, Co, and Cr have been doped inside the HA structure by means of the ion exchange method.94,95,97 Recently, successful substitution of lanthanide ions (La3+, Sm3+, Gd3+, Ho3+, Yb3+, and Lu3+) inside the apatite lattice via the ion exchange technique has been reported.96 The third family of synthesis procedures is that of hightemperature processing procedures. In this particular method, the precursors are ignited at a high temperature to obtain the end product(s). Pyrolysis and solution combustion reaction are two well-known members of this family, which have been engaged to synthesize pure or doped BCP. By tuning the temperature and precursor concentration, one can acquire wellcrystalline HA through the pyrolysis method.98 Formation of secondary agglomeration is the major disadvantage of this method, which can be overcome by addition of specific salts to the precursors.99 On the other side, end products are generated in the solution combustion method via a self-sustaining rapid exothermic reaction among the precursors, in the presence of an organic fuel.100,101 Urea, citric acid, glucose, etc. are the popular choices of fuel.102,103 Apart from undoped HA, fluorapatite-, chlorapatite-, Sr-, and Ag-doped HA has been prepared using the solution combustion reaction.104−107 BCP powder has been prepared from biogenic sources like eggshells, bovine bone, fish scale and bone, etc.108 This approach is termed as the biomimetic synthesis method. Trace elements are always present inside the lattice structures of the final products of the biomimetic approach, and they closely resemble a natural bioapatite.109 Pure and carbonated HA with a controlled particle morphology has been synthesized via this route.110,111 Many other synthesis techniques like electrospinning,112 electrospraying,113 sonochemical,114 chemical vapor,115 and flux cooling116 have been developed and tested by the researchers. Despite that, it is still difficult to obtain HA/ TCP powder with a proper stoichiometry and good crystallinity. Combination of more than one method is now being used to improve and better control the particle size distribution, chemical composition, and morphology. Scalability is another major issue with most of the above-described synthesis strategies. Research is still ongoing to develop superior synthesis methods with a better scalability to the industry level. 2.2. Experimental Methodologies for Structural Analysis. It is the primary interest of a material scientist to quantitatively characterize the structural changes of HA/TCP crystal lattice after ion substitution. Several experimental methods are currently in use to probe the dopant site and the associated structural changes. Some of those specialized techniques will be described below. 2.2.1. Rietveld Method. X-ray diffraction (XRD) is undoubtedly the most popular method to determine the crystal structure. One of the ways to extract the quantitative structural information from the XRD pattern is the Rietveld refinement method. In this oft-called technique, first described by Hugo
1/2 l w [y (obs) − y (calc)]2 | o o o o i i i R wp = m } oΣi o o o Σiwi[yi (obs)]2 o o n ~ ÉÑ1/2 ÅÄÅ Ñ R exp = ÅÅÅÅ(N − P)/Σwi[yi (obs)]2 ÑÑÑÑ ÅÇ i ÖÑ
where yi(obs) is the observed value at step i, yi(calc) is the calculated value at step i, wi is the weight, N is the number of observations, and P the number of parameters. The ratio between the two is called the goodness of fit: χ2 =
R wp R exp
At the ideal fitting, the value of χ2 should be 1, i.e., Rwp equates to Rexp. Rexp reflects the quality of the data. The quality of fitting can also be estimated using a difference profile plot. Some precautions should be taken during the data collection for Rietveld refinement. For Bragg−Brentano geometry, it is essential to maintain a constant volume condition. Sample transparency should also be checked. It is assumed that the sample is infinitely thick and only X-ray absorption takes place in the sample. If the sample contains a considerable amount of light elements, these criteria might not be fulfilled, and under this situation, the constant volume assumption becomes invalid.119 Besides this, the 2θ value of the instrument should be calibrated using a standard sample. The ideal particle size for Rietveld refinement is 1−5 μm.119 For larger particles, there would be less crystallites in the sample and all crystallite orientations may not be represented equally. This defect cannot be corrected in the refinement stage, and to avoid this problem, it is recommended to rotate the sample during data collection.119 In order to carry out the Rietveld refinement process, one must know the phases present in the sample and their corresponding crystal symmetry. This is the most prominent limitation of this method.120 Despite this fact, it is the most frequently used technique to determine the structural changes due to ion doping in the HA/TCP lattice structure and the preferred substitution site. Most of the experimental results about the structure of substituted BCP, which will be presented in the next section, have been extracted through the Rietveld refinement method. 2.2.2. XAS/XANES/EXAFS Technique. Another useful experimental technique to locate the dopant site and to probe the local chemical environment around the dopant atom is the X-ray absorption spectroscopy (XAS). It measures the transition from core electron states of the metal to the LUMO (lowest unoccupied molecular orbital) and the continuum.121 The former one is called the X-ray absorption near edge structure D
DOI: 10.1021/acsabm.9b00488 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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contraction due to replacement of Ca by Fe at the Ca(1) site.127 Gomes et al.128 used a combination of the Rietveld refinement and EXAFS technique to determine the location of Cu ions inside the HA structure and confirmed the formation of linear O−Cu−O oxocuprate inside the crystal lattice. K-edge absorption spectra of Sr in doped HA was studied by Terra et al.,129 and they concluded an average distribution of Sr ions at Ca(1) and Ca(2) sites from EXAFS spectra. Eu LIII edge spectra were also recorded for rare-earth-element-doped HA, and the result demonstrated that the central Eu atoms were coordinated with an average of ∼8.4 O at REu−O ∼ 2.43 Å.130 Moreover, Eu incorporation was found to destabilize the HA crystal structure, and the possible substitution site was proposed to be at the grain boundaries of HA.131 EXAFS results for Zn K-edge revealed the presence of additional P and Ca neighbors that support formation of an inner-sphere Zn surface complex.132 Apart from metal ions, an EXAFS study of carbonate and iodine has also been done. The results showed a lack of order around the iodine dopants and alternation of long-range structure surrounding the carbonate groups.133,134 2.2.3. Mössbauer Spectroscopy. Other than the abovementioned methods, many other experimental techniques are in use to investigate the doped HA/TCP structure. Mössbauer spectroscopy is one of them. It provides a unique way to measure electronic, magnetic, and structural properties within the material.135 This technique is based on the well-known Mössbauer effect, and a typical Mössbauer spectrum consists of the intensity of the γ-ray absorption versus energy of a resonant nucleus. The Mössbauer effect involves γ-ray emission from an excited nucleus and the absorption of this γ-ray by a second nucleus. This phenomenon is also termed as nuclear resonant γ-ray scattering.135 Energy precision of the nuclear excited state is extremely high (∼10−8 eV), and the emitted γ-ray in general lacks such precision due to the recoil of the emitting nucleus.135 Hence, the observation of nuclear resonant excitation in the free nucleus system is extremely difficult. However, Mössbauer showed that, in a crystalline material, this recoil energy loss of emitted γ-ray is very small due to restricted motion of nucleus, and nuclear resonant excitation can be observed easily.135 If the γ-ray emitting nucleus and target nucleus are in an identical chemical environment, the resonance can be observed with both materials at rest. If there is a difference in chemical environment, then there would be a shift in nuclear energy levels and this energy shift can be compensated using the Doppler effect.136 Three kinds of hyperfine interaction can be observed using Mössbauer spectroscopy, which carry important information about the material.136 The first one is the isomer shift, which arises from interaction between the nucleus and the surrounding inner electrons. The second one is electric quadrupole splitting. It results from the interaction of the nucleus with the electric field gradient (EEG) present in the Mössbauer isotope. If the Mössbauer isotope has cubic symmetry, EEG reduces to zero and no splitting is observed.137 The last kind of hyperfine splitting is the hyperfine magnetic field splitting. It arises from the interaction between the nucleus and surrounding magnetic field, if any. Hyperfine magnetic field splitting is confined to only ferro, ferri, and antiferromagnetic materials.137 Mössbauer spectroscopy has been successfully employed to detect the exact location and amount of dopants (ferromagnetic) present inside the HA structure.138,139 Using this technique, formation of the paramagnetic-like phase due to the presence of Fe ions inside HA nanoparticles was
(XANES), and the latter is known as the extended X-ray absorption fine structure (EXAFS).121 Although both contain useful information about the local structure, it is very difficult to analyze XANES data.122 XANES data are useful to determine the oxidation state of an element. On the other hand, useful local structural information can be obtained from EXAFS data in a much easier way. The EXAFS phenomena can be described as an in situ diffraction process.122 If the incident X-ray is able to eject a photoelectron from the absorbing atom, the free photoelectron interacts with the bound electrons of the nearby atoms. A backscattered electron wave is generated as a result of this interaction, and this backscattered wave interferes with the outgoing photoelectron wave. The resulting interference pattern can be observed just above the absorption edge in the X-ray absorption spectra. It can be noted here that absorption edges correspond to a sudden increase in the energy-dependent absorption coefficient at a certain energy value, which corresponds to the binding energy of the ejected core electron.123 The EXAFS phenomena can be described using the EXAFS parameter χ(k), which is associated with the absorption coefficient μ(k) in the following manner. χ(k) =
μ(k) − μ0 (k) μ0 (k)
where μ0(k) is the absorption coefficient of the bare atom; k is the wavenumber defined as k=
2m(E − E0) h̵ 2
χ(k) is related to the local structure through the EXAFS equation. χ (k) = S02 Σ j
Nj|f j (π , k)| kRaj2
2 2
e−2σajk e−2Raj/ λj(k) × sin(2kRaj + δ(k))
where S20 is the amplitude reduction term, Nj is the number of equivalent scattering atoms at distance Raj, f j(π,k) is the backscattering amplitude, σ is Debye−Waller factor, and δ and λ are the phase shift and electron mean free path, respectively. Details of this equation and its derivation can be found elsewhere.124 As far as the experimental protocol is concerned, a synchrotron X-ray source is used to obtain EXAFS spectra because of its high intensity.122 The EXAFS spectrum can be recorded in transmission or fluorescence mode, and the obtained data consist of μ(E) as a function of E. The steps included in the analysis involve the following: 1. Pre edge, post edge, and background correction of the obtained data 2. Generation of weighted χ(k) vs k plot 3. Fourier transform of the χ(k) vs k plot and fitting it using theoretical modeling By analyzing the data, one can understand the atomic radial distribution surrounding the X-ray absorbing atom. The EXAFS technique is element specific, but it cannot resolve the scattering atoms that differ by two or three in the atomic number.122,125 The EXAFS technique is used by the researchers to study the doped HA/TCP structure. Laurencin et al.126 investigated the Mg incorporation inside the HA lattice and concluded that Ca(2) is the favored dopant site. Similarly, Fe uptake inside the HA lattice was explored and the obtained XANES and EXAFS spectra supported the previously reported bond length E
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ACS Applied Bio Materials confirmed.138 Solid state NMR spectroscopy was also used by the researchers to investigate doped HA. Laurencin et al.126 studied the change in the local environment of anions inside HA by means of the 1H MAS NMR spectrum due to Mg insertion in the lattice structure. The NMR results supported the Mg incorporation at the Ca(2) site. Besides this, time-resolved laser fluorescence spectroscopy is another used technique for structural determination.131 Among the several techniques discussed above, we shall present some published results related to X-ray-based techniques only at a later section. 2.3. Crystallographic Changes Due to Doping: Experimental Perspective. It has been mentioned before that both HA and TCP can sustain heavy doping. Ion substitution has a deep impact on crystallographic, functional, and biological properties of BCP. Structural changes (lattice parameters, crystallographic distortion) induced by the ion-substitution control mechanical response and dissolution/degradation behavior of the material.140 The in vitro/in vivo concentration of dopant ions solely depends on the dissolution rate, and it is a well-known fact that different ions regulate various cellular mechanisms. Zn2+, for example, helps in osteogenesis by stimulating osteoblastic activity.141,140 Sr2+, as well, supports osteoblastic differentiation and proliferation by positively influencing CaSR and downstream signaling pathways.142 Activation of CaSR pathways promotes production of OPG (osteoprotegerin) and inhibits RANKL (receptor activator of NF-kB ligand) induced osteoclastogenesis.143,144 Mg2+ and Cu2+ ions assist angiogenesis by changing the VEGF (vascular endothelial growth factor) expression.145,146 It has been proposed that Mn has some effect on the PTH (parathyroid hormone) signaling pathway, which is a regulator of intracellular Ca activity.147 Many other ions like Li, B, Co, and Si also play important roles in angiogenesis/osteogenesis. The effect of metal ions on bone formation is so profound that the researchers are trying to use them as alternatives of biologics.140 It is noteworthy that, above a threshold concentration, metal ions usually exhibits adverse effects on cellular functionality. Hence, it is important to tailor the degradation behavior of implant material, and structural knowledge is useful for it. Moreover, proper tuning of mechanical properties is an unavoidable step to develop any new hard-tissue replacement material, and it is very difficult to do so without enough structural knowledge. On the top of that, structural changes can be linked to the alteration of grain size, surface morphology, surface charge distribution, etc., which have a direct impact on biological response. In the following section, experimental observations on the structural changes in doped BCPs will be summarized. 2.3.1. Monoionic Substitution. Two types of single ion substitution can happen inside the apatite/TCP crystal structure: anionic substitution and cationic substitution. Silicate, carbonate, and halides (fluorine and chlorine) are the main cationic groups, whose substitution in the HA/TCP lattice structure has been extensively investigated. Among them, the most common one is carbonated HA. Inside the apatite crystal, the CO32− group can substitute either for a hydroxyl group (type A) or for a phosphate group (type B).148 A mixed type of substitution (type AB) is also possible.149 Type A substitution usually dominates at low dopant concentrations (
