Hydroxyapatite (HAp) is the major mineral constituent of vertebrate bones and teeth. It has been well documented that HAp nanoparticles can significantly increase the biocompatibility and bioactivity of man-made biomaterials. Over the past decade, HAp nanoparticles have therefore increasingly been in demand, and extensive efforts have been devoted to develop many synthetic routes, involving both scientifically and economically new features. Several investigations have also been made to determine how critical properties of HAp can be effectively controlled by varying the processing parameters. With such a wide variety of methods for the preparation of HAp nanoparticles, choosing a specific procedure to synthesize a well-defined powder can be laborious; accordingly, in the present review, we have summarized all the available information on the preparation methodologies of HAp, and highlighted the inherent advantages and disadvantages involved in each method. This article is focused on nanosized HAp, although recent articles on microsized particles, especially those assembled from nanoparticles and/or nanocrystals, have also been reviewed for comparison. We have also provided several scientific figures and discussed a number of critical issues and challenges which require further research and development.

Phase purity is a well-recognized but not well-understood variable affecting the biological integration of hydroxyapatite (HA)-based biomaterials. Minor amounts of specific, relevant impurities--calcium oxide (CaO) and tricalcium phosphate (TCP)--may often be present either as deliberate additions or as a result of decomposition during sintering. We investigated the influence of these two impurities in terms of their effects on surface morphology, weight loss/gain, and microstructural-level degradation. Phase purity variations were deliberately introduced into an otherwise-standardized HA matrix--the parent HA grain size and bulk density were relatively constant--produced using identical fabrication conditions. Stability varied markedly during exposure to mildly acidic, neutral, and pH 7.4 phosphate-buffered saline. Equivalent molar variations in the Ca/P ratio (1.62 vs 1.72) on either side of the stoichiometric ratio produce relatively small volumetric amounts of CaO (1.6 vol%) versus TCP (27 vol%) in HA. However, the relatively small amounts of CaO render the bulk more susceptible to degradation and more likely to have negative effects on a biological milieu. Interestingly, the presence of CaO is also a potent nucleating agent for the precipitation of new surface phases and detectable weight gain. The TCP-containing ceramic, in contrast, paradoxically exhibited slightly greater resistance to degradation than HA.

Abstract

Nano-hydroxyapatite (nano-HAP) may be a better candidate for an apatite substitute of bone in biomedical applications than micro-sized hydroxyapatite (m-HAP). However, size control is always difficult when synthesizing well-defined nano-HAP particles. In this study, nano-HAP particles with diameters of ∼20 nm (np20) and ∼80 nm (np80) were synthesized and characterized. The size effects of these nano-HAPs and m-HAP were studied on human osteoblast-like MG-63 cells in vitro. Our results demonstrate that both cell proliferation and cell apoptosis are related to the size of the HAP particles. Np20 has the best effect on promotion of cell growth and inhibition of cell apoptosis. This work provides an interesting view of the role of nano-HAPs as ideal biomedical materials in future clinical applications.

Hydroxyapatite (HA) is a synthetic biomaterial and has been found to promote new bone formation when implanted in a bone defect site. However, its use is often limited due to its slow osteointegration rate and low antibacterial activity, particularly where HA has to be used for long term biomedical applications. This work will describe the synthesis and detailed characterization of zinc-substituted HA (ZnHA) as an alternative biomaterial to HA. ZnHA containing 1.6 wt% Zn was synthesized via a co-precipitation reaction between calcium hydroxide, orthophosphoric acid and zinc nitrate hexahydrate. Single-phase ZnHA particles with a rod-like morphology measuring ~50 nm in length and ~15 nm in width, were obtained and characterized using transmission electron microscopy and X-ray diffraction. The substitution of Zn into HA resulted in a decrease in both the a- and c-axes of the unit cell parameters, thereby causing the HA crystal structure to alter. In vitro cell culture work showed that ZnHA possessed enhanced bioactivity since an increase in the growth of human adipose-derived mesenchymal stem cells along with the bone cell differentiation markers, were observed. In addition, antibacterial work demonstrated that ZnHA exhibited antimicrobial capability since there was a significant decrease in the number of viable Staphylococcus aureus bacteria after in contact with ZnHA.

Percutaneous orthopedic and dental implants require not only good adhesion with bone but also the ability to attach and form seals with connective tissues and the skin. To solve the skin-seal problem of such implants, an electrochemical deposition method was used to modify the surfaces of metallic implants to improve their antibacterial ability and skin seals around them. A dense and uniform fluoridated calcium phosphate coating with a thickness of about 200 nm was deposited on an acid-etched pure titanium substrate by controlling the current density and reaction duration of the electrochemical process. The as-deposited amorphous fluoridated calcium phosphate transformed to fluoridated hydroxyapatite (FHA) after heat treatment at 600 degrees C in a water vapor environment for 3 h. Both single crystal diffraction patterns and high-resolution transmission electron microscope (HRTEM) images confirmed the phase of the fluoridated calcium phosphate after the heat treatment. The antibacterial activities of FHA coatings were tested against Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Porphyromonas gingivalis (P. gingivalis) with the film attachment method. The antibacterial activity of FHA coating is much higher than that of pure hydroxyapatite (HA) coating and acid-etched pure titanium surface. The promising features of FHA coating make it suitable for orthopedic and dental applications.

Fluoridated hydroxyapatite (FHA) coatings exhibit great potential for applications on implants which require good bioactivity and high antibacterial activity. This work is a comparative study of chemical stability, adhesive strength and zeta potential of a nanothick (about 200 nm) FHA coating which is densely and uniformly deposited on a titanium substrate electrochemically. The chemical stability of the nanothick coatings was evaluated using a dissolution test in a simulated physiological solution. The dissolution tests indicate that the fluorine-containing calcium phosphate (CaP) coating with an appropriate heat treatment is chemically stable in a physiological environment, even more so than its HA counterpart. The adhesive strength of FHA coatings was evaluated by the critical load obtained from nanoscratching tests. The critical load which caused coating failure in scratching was determined by a method which correlated the scratching morphology with a sudden change in friction coefficient. The adhesive strength of a nanothick FHA coating was 147% larger than that of HA coating with the same thickness level. The fluorine addition does not have a significant effect on the characteristics of the negative zeta potential of apatite coatings. The solid integrity of this nanothick FHA coating makes it an excellent candidate for biomedical applications. (C) 2011 Elsevier Ltd.

The current research is devoted to the study of the modification of the titanium implants by the micro-arc oxidation with bioactive calcium phosphate coatings containing Ag or Sr and Si elements. The coatings' microstructure, phase composition, morphology, physicochemical and biological properties were examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). Ag-containing and Sr-Si-incorporated coatings were formed in alkaline and acid electrolytes, respectively. The formation of the coatings occurred at different ranges of the applied voltages, which led to the significant difference in the coatings properties. The trace elements Ag, Sr and Si participated intensively in the plasma-chemical reactions of the micro-arc coatings formation. Ag-containing coatings demonstrated strong antibacterial effect against Staphylococcus aureus Acapital TE, Cyrilliccapital ES, Cyrilliccapital ES, Cyrillic 6538-P. MTT in vitro test with 3T3-L1 fibroblasts showed no cytotoxicity appearance on Sr-Si-incorporated coatings.

The influence of the simultaneous presence of the two inhibitors of bone degradation, strontium and zoledronate, on the direct synthesis of hydroxyapatite was explored in the range of Sr concentration up to 50 atom% at two different bisphosphonate concentrations (ZOL7 and ZOL14). The results of structural analysis indicated that HA can be obtained as a single crystalline phase up to a Sr concentration in solution of 20 and 10 atom% within the ZOL7 and ZOL14 series respectively. Both Sr substitution and ZOL incorporation affect the length of the coherently scattering crystalline domains and the dimensions of HA nanocrystals. At greater Sr content, XRD full profile fitting data indicate that zoledronate provokes the segregation of Sr in two crystalline apatitic phases, at different strontium content. Co-cultures of osteoblast-like MG63 cells and human osteoclast show that ZOL displays a greater inhibitory influence than Sr on osteoclast proliferation and activity. On the other hand, the results obtained on osteoblast surnatant and on gene expression indicate that Sr exerts a greater promotion on osteoblast proliferation and differentiation. The co-presence of Sr and ZOL has a combined effect on the differentiation markers, so that HA containing about 4 wt% ZOL and 4 Sr atom%, and even more HA containing about 4 wt% ZOL and 8 Sr atom%, result the best compromise for osteoblast promotion and osteoclast inhibition. (C) 2014 Elsevier Ltd.

Strontium-doped hydroxyapatite (Ca9Sr1(PO4)6(OH)2, Sr1-HA) nanorods with different lateral spacing (e.g., interrod spacing) values (67.3 +/- 3.8, 95.7 +/- 4.2, and 136.8 +/- 8.7 nm) and nanogranulates were grown on microarc-oxidized microporous TiO2, respectively, to form multilayer coatings. The coatings reveal two kinds of micro/nanoscaled hierarchical surfaces with a similar microscale roughness, e.g., nanogranulated 2D pattern and nanorod-shaped 3D pattern in nanotopography. When hFOB1.19 cells are employed, the proliferation and differentiation of osteoblasts on the coatings were evaluated by examining MTT assay, expressions of osteogenesis-related genes [alkaline phosphatase (ALP), runt-related transcription factor 2, osterix, osteopontin (OPN), osteocalcin (OCN), and collagen I (Col-I)], ALP activity, contents of intracellular Ca(2+), Col-I, OPN, and OCN, extracellular collagen secretion, and extracellular matrix mineralization. The results reveal that the proliferation and differentiation of osteoblasts can be directly regulated by the interrod spacing of the Sr1-HA nanorods, which are significantly enhanced on the nanorod-shaped 3D patterns with interrod spacing smaller than 96 nm and more pronounced with decreasing the interrod spacing but inhibited on the nanorods with spacing larger than 96 nm compared to the nanogranulated 2D pattern. The difference in the cellular activity is found to be related with the intracellular Ca(2+) concentrations, which are regulated by variation of the surface topology of Sr1-HA crystals. Our work provides insight to the surface structural design of a biomedical implant favoring osteointegration.

Cryogels from natural biopolymers, especially chitosan (CS) and silk fibroin (SF), have attracted significant attention for bone tissue engineering due to their good biocompatibility and tissue affinity. However, they are still not widely applied in clinics because of their poor mechanical properties, and lack of osteoinductivity and antibacterial properties. Here, by integrating a physico-chemical hybrid-crosslinking strategy and a freeze-drying method, the authors have prepared a resilient and flexible chitosan/silk cryogel with Ag and Sr co-doped hydroxyapatite (AgSrHA). The cryogel exhibits super resilience and flexibility, which guarantees the mechanical strength required for bone repair. Furthermore, the cryogel possesses long-term effective antibacterial properties and osteoinductivity due to the slow release of Ag and Sr ions. The physico-chemical hybrid-crosslinking points were introduced into a CS/SF network by treating with a mixture of alkaline and polyethylene glycol glycidyl ether (PEGDE) at 60 degrees C, which endowed the cryogel with super resilience and flexibility. Nanoscale AgSrHA was incorporated into the CS/SF network to further enhance the mechanical properties of the cryogel. In addition, Ag and Sr were doped into the HA crystal lattice, which not only prevents cytotoxicity by avoiding the ion burst release behavior, but also can achieve antibacterial and osteoinductive properties for a long period of time. In short, the AgSrHA/CS/SF cryogel with excellent mechanical properties and long-term effective dual-biofunction of antibacterial properties and osteoinductivity would be an ideal candidate for successful bone repair in clinics.

Strontium can be substituted into the calcium sublattice of hydroxyapatite without a solubility limit. However, recent ab initio simulations carried out at 0 K report endothermic nature of this process. There is also striking discrepancy between experimentally observed preference of Sr doping at Ca-II sites and the first principles calculations, which indicate that a Ca-I site is preferred energetically for the Sr substitution. In this paper we combine insights from Density Functional Theory simulations and regular configurational entropy calculations to determine the site preference of Sr doping in the range of 0-100 at% at finite temperatures. In addition, samples of Sr-HA are synthesized and refinement of the relevant structural information provides benchmark information on the experimental unit cell parameters of Sr-HA. We find that the contribution of the entropy of mixing can efficiently overcome the endothermic excess energy at a temperature typical of the calcining step in the synthesis route of hydroxyapatite (700-950 degrees C). We observe that the most preferential substitution pattern is mixed substitution of Sr regardless of the concentration. For a wet chemical method, carried out at a moderate temperature (90 degrees C), the mixed doping is still slightly favourable at higher Sr-concentrations, except the range at 20% Sr, where Site II substitution is not restricted energetically and equally possible as the mixed doping. We observe a close correspondence between our theoretical results and available experimental data. Hence it should be possible to apply this theory to other divalent dopants in HA, such as Zn(2+), Mg(2+), Pb(2+), Cu(2+), Ba(2+), Cd(2+) etc.

Magnesium (Mg) and strontium (Sr) have been widely used in the field of implanted devices because of their excellent bioactivity. However, the local high ion concentration caused by the implant affects the growth of hydroxyapatite (Ca10(PO4)6(OH)2, HA), which is the main inorganic component of bone and teeth. Many studies have investigated the effect of Mg(2+) and Sr(2+) on the growth of HA, but no systematic research has been conducted to compare these two ions in terms of the growth of HA. In this study, the substitution of a series of Sr- and Mg-substituted HA was conducted through a conventional hydrothermal method. Comprehensive characterization techniques, including X-ray diffraction, inductive coupled plasma, field emission scanning electron microscopy, transmission electron microscopy, selected-area electron diffraction, thermo gravimetric-differential scanning calorimetry, and Fourier transform infrared spectroscopy, were used to examine the effects of Sr(2+) and Mg(2+) on the phase, morphology, crystallinity, chemical composition, thermal stability, and lattice parameters of HA. The results indicated that Mg ions partially substituted for calcium (Ca) ions in the apatite structure, thus decreasing the lattice parameters, partially adsorbing on the apatite surface that formed the amorphous phase, and inhibiting the crystal growth. By contrast, Sr ions fully substituted for Ca ions and increased the lattice parameters. Both Mg and Sr ions affected the morphology of HA. Crystallinity decreased with the addition of Mg ions (transition from the crystal to amorphous phase was between 30% and 40% Mg), but it was not affected by Sr ions. Thermostability decreased with the addition of Mg (a total weight loss from 8.06 wt% for 10% Mg to 25.81 wt% for 50% Mg), but it had no significant changes in the Sr-substituted samples.

First-principles modeling combined with experimental methods were used to study hydroxyapatite in which Sr2+ is substituted for Ca2+. Detailed analyses of cation-oxygen bond distributions, cation-cation distances, and site 1-oxygen polyhedron twist angles were made in order to provide an atomic-scale interpretation of the observed structural modifications. Density functional theory periodic band-structure calculations indicate that the Ca2+ to Sr2+ substitution induces strong local distortion on the hydroxyapatite lattice: the nearest neighbor Sr-O bond structures in both cationic sites are comparable to pure SrHA, while Sr induces more distortion at site 2 than site 1. Infrared vibrational spectroscopy (FTIR) and extended X-ray absorption fine structure (EXAFS) analysis suggest increasing lattice disorder and loss of OH with increasing Sr content. Rietveld refinement of synchrotron X-ray diffraction patterns shows a preference for the Ca1 site at Sr concentrations below 1 at.%. The ideal statistical occupancy ratio Sr2/Sr1=1.5 is achieved for approximately 5 at.%; for higher Sr concentrations occupation of the Ca2 site is progressively preferred.

Abstract

The present in vitro study doped hydroxyapatite (HA) with various metal cations (Mg²⁺, Zn²⁺, La³⁺, Y³⁺, In³⁺, and Bi³⁺) in an attempt to enhance properties of HA pertinent to orthopedic and dental applications. X-ray diffraction material characterization indicated that the metal cations may have substituted for calcium in the HA crystal structure and that all of the doped HA formulations were single-phase and crystalline. Scanning electron microscopy analysis revealed a variety of grain sizes, depending on the dopant utilized. Energy-dispersive spectroscopy confirmed that the dopants added during synthesis were present and that all of the HA formulations synthesized were within the defined range of HA phase in the CaO–P₂O₅–H₂O system. Lastly, Bi-doped HA had a slower dissolution rate than either undoped HA or HA doped with other cations when exposed to simulated physiological conditions for 21 days. In terms of cell function, results provided the first evidence that osteoblasts, bone-forming cells, adhered and differentiated (as measured by alkaline phosphatase synthesis) in response to HA doped with trivalent cations (specifically, La³⁺, Y³⁺, In³⁺, Bi³⁺) at earlier time points than either HA doped with divalent cations (Mg²⁺, Zn²⁺) or undoped HA. Of the dopants examined, Bi³⁺ most enhanced osteoblast long-term calcium-containing mineral deposition. For these reasons, this study revealed for the first time the potential benefits of doping HA with Bi³⁺ according to criteria critical for bone prosthetic clinical success.

The biological HA in the body differs from that of pure HA in stoichiometry,composition, physical properties and mechanical properties, because this HA isparticularly prone to ion substitution. As a cation that can substitute forcalcium in the structure of hydroxyapatite, strontium provokes an increasinginterest because of its beneficial effect on bone formation, and prevention ofbone resorption. Here, strontium-substituted HA powders were prepared by thehydrothermal method at 180℃ for 8 h using Ca(NO₃)₂·4H₂O, (NH₄)₂HPO₄and Sr(NO₃)₂ as reagents. X-ray diffraction, Fouriertransform infrared spectroscope, transmission electron microscope, energy dispersiveX-ray, and thermogravimetric-differential thermal analysis were employed toinvestigate the crystalline phase, chemical composition, morphology, and thermalstability of the Sr-HA. And the cytotoxicity of Sr-HA was analyzed through MTT assay. The results showthat Sr is incorporated into the HA crystal structure and the crystal grainsize of the Sr-HA decreases as the Sr content (under 50wt%) is increased.Moreover, the HA thermal stability decreases. However, there’s no apparentdifference between the pure hydroxyapatite and all of the Sr-HA groups in cytotoxicity,which may have good biocompatibility.

Abstract

Recently, strontium (Sr) as ranelate compound has become increasingly popular in the treatment of osteoporosis. However, the lattice structure of bone crystal after Sr incorporation is yet to be extensively reported. In this study, we synthesized strontium-substituted hydroxyapatite (Sr-HA) with different Sr content (0.3%, 1.5% and 15% Sr-HA in mole ratio) to simulate bone crystals incorporated with Sr. The changes in chemical composition and lattice structure of apetite after synthetic incorporation of Sr were evaluated to gain insight into bone crystal changes after incorporation of Sr. X-ray diffraction (XRD) patterns revealed that 0.3% and 1.5% Sr-HA exhibited single phase spectrum, which was similar to that of HA. However, 15% Sr-HA induced the incorporation of HPO₄²⁻ and more CO₃²⁻, the crystallinity reduced dramatically. Transmission electron microscopy (TEM) images showed that the crystal length and width of 0.3% and 1.5% Sr-HA increased slightly. Meanwhile, the length and width distribution were broadened and the aspect ratio decreased from 10.68±4.00 to 7.28±2.80. The crystal size and crystallinity of 15% Sr-HA dropped rapidly, which may suggest that the fundamental crystal structure is changed. The findings from this work indicate that current clinical dosage which usually results in Sr incorporation of below 1.5% may not change chemical composition and lattice structure of bone, while it will broaden the bone crystal size distribution and strengthen the bone.

First-principles calculations are performed to investigate atomic and electronic structures of Sr(2+) ions substituting for Ca(2+) in octacalcium phosphate (OCP). The defect formation energies are evaluated from total energies of supercells and ionic chemical potentials of Sr(2+) and Ca(2+) determined under the chemical equilibrium with aqueous solution saturated with hydroxyapatite (HAp). The defect formation energy depends on the solution pH and the substitutional Ca sites in OCP, and the estimated equilibrium concentrations of Sr(2+) in OCP and HAp are in reasonable agreement with previous experimental results obtained in physiological conditions. It is also found that Sr(2+) ions can be more favorably substituted in OCP than in HAp. It is thought, therefore, that Sr(2+) plays its role to promote bone formation by being incorporated into the metastable OCP phase occurring during HAp nucleation.