Christophe Drouet

Bone mineral: update on chemical composition and structure

The structure of the Ca–P solid phase in bone was first identified by deJong in 1926 as a crystalline calcium phosphate similar to geological apatite by chemical analyses and, most importantly, by X-ray diffraction [1]. The X-ray diffraction data was confirmed a few years later [2]. These findings initiated a flurry of research on a more detailed chemical composition and crystal structure of both geological and synthetic apatites and of bone mineral, initially carried out principally by geologists, crystallographers, and chemists, but later by biochemists and physiologists because of the clear potential of this new information to shed light on the biological and physiological functions of bonemineral and as indicators of disorders of the skeletal system. It soon became clear that there were significant structural and chemical compositional differences between the many different geological hydroxyapatites, synthetic hydroxyapatites, and the apatite crystals found in bone and related skeletal tissues in addition to the very large size of the geological and many of the synthetic apatite crystals, compared with the extremely small particle size of bone mineral. Further studies were directed in roughly three avenues: continued more careful and complete analytical compositional data of bone mineral, from which it was clearly established that the chemical composition of bone crystals in many ways did not correspond to the chemical compositions of stoichiometric hydroxyapatite. Indeed, the bone crystals were found to contain significant and varying amounts of carbonate and HPO4 ions. Much later, it was discovered by a variety of techniques, including solid-state NMR [3], Raman spectroscopy [4], and inelastic neutron scattering [5], that the biological bone apatites contain only a very small percentage of the total number of hydroxyl groups present in highly purified synthetic calcium hydroxyapatites. Other studies clearly pointed out that a substantial fraction of the phosphate ions are situated on the surfaces of the bone mineral crystals which are mainly protonated and in a disordered environment [6], in contrast to the phosphate ions in lattice positions, which are unprotonated. Also, other uniquely protonated phosphate ions were identified in bone mineral by phosphorus 31 NMR spectroscopy, which are not present in synthetic calcium phosphate apatites [7]. Structural studies were first carried out to determine crystal size by measuring the extent of X-ray diffraction peak broadening [8], which yielded crystal sizes varying from 31 to 290Å. More detailed structural data were obtained by the then recently introduced field of electron microscopy and electron diffraction [9–11], which revealed that the bone crystals were thin plates, approximately 500Å long, 250Å wide, and 100Å thick. However, calculations from low-angle X-ray diffraction scattering studies [12–14] were more consistent with the conclusions that the bone crystals were very much smaller than those observed by Osteoporos Int (2009) 20:1013–1021 DOI 10.1007/s00198-009-0860-y

Adsorption and release of BMP-2 on nanocrystalline apatite-coated and uncoated hydroxyapatite/β-tricalcium phosphate porous ceramics

The association of bone morphogenetic proteins (BMPs) with calcium phosphate bioceramics is known to confer them osteoinductive properties. The aim of this study was to evaluate the surface properties, especially regarding recombinant human BMP-2 (rhBMP-2) adsorption and release, of commercial sintered biphasic calcium phosphate ceramics after coating with biomimetic nanocrystalline apatite. The raw and coated ceramics exhibited similar macroporous structures but different nanometer-sized pores contents. Both types of ceramics showed Langmuir-type adsorption isotherms of rhBMP-2. The coating noticeably increased the rate of adsorption and the total amount of growth factor taken up, but the maximum coverage per surface area unit as well as the affinity constant appeared lower for coated ceramics compared with raw ceramic surfaces. The limited advantage gained by coating the ceramics can be assigned to a lower accessibility of the surface adsorption sites compared with the raw ceramics. The quantity of rhBMP-2 spontaneously released in cell culture medium during the first weeks was lower for coated samples than for uncoated ceramics and represented a minor fraction of the total adsorbed amount. In conclusion, the nanocrystalline apatite coating was found to favor the adsorption of rhBMP-2 while providing a mean to fine tune the release of the growth factor.

Surface Characteristics of Nanocrystalline Apatites: Effect of Mg Surface Enrichment on Morphology, Surface Hydration Species, and Cationic Environments

The incorporation of foreign ions, such as Mg2+, exhibiting a biological activity for bone regeneration is presently considered as a promising route for increasing the bioactivity of bone-engineering scaffolds. In this work, the morphology, structure, and surface hydration of biomimetic nanocrystalline apatites were investigated before and after surface exchange with such Mg2+ ions, by combining chemical alterations (ion exchange, H2O-D2O exchanges) and physical examinations (Fourier transform infrared spectroscopy (FTIR) and high-resolution transmission electron microscopy (HRTEM)). HRTEM data suggested that the Mg2+/Ca2+ exchange process did not affect the morphology and surface topology of the apatite nanocrystals significantly, while a new phase, likely a hydrated calcium and/or magnesium phosphate, was formed in small amount for high Mg concentrations. Near-infrared (NIR) and medium-infrared (MIR) spectroscopies indicated that the samples enriched with Mg2+ were found to retain more water at their surface than the Mg-free sample, both at the level of H2O coordinated to cations and adsorbed in the form of multilayers. Additionally, the H-bonding network in defective subsurface layers was also noticeably modified, indicating that the Mg2+/Ca2+ exchange involved was not limited to the surface. This work is intended to widen the present knowledge on Mg-enriched calcium phosphate-based bioactive materials intended for bone repair applications.

Apatite Formation: Why It May Not Work as Planned, and How to Conclusively Identify Apatite Compounds

Calcium phosphate apatites are inorganic compounds encountered in many different mineralized tissues. Bone mineral, for example, is constituted of nanocrystalline nonstoichiometric apatite, and the production of “analogs” through a variety of methods is frequently reported. In another context, the ability of solid surfaces to favor the nucleation and growth of “bone-like” apatite upon immersion in supersaturated fluids such as SFB is commonly used as one evaluation index of the “bioactivity” of such surfaces. Yet, the compounds or deposits obtained are not always thoroughly characterized, and their apatitic nature is sometimes not firmly assessed by appropriate physicochemical analyses. Of particular importance are the “actual” conditions in which the precipitation takes place. The precipitation of a white solid does not automatically indicate the formation of a “bone-like carbonate apatite layer” as is sometimes too hastily concluded: “all that glitters is not gold.” The identification of an apatite phase should be carefully demonstrated by appropriate characterization, preferably using complementary techniques. This review considers the fundamentals of calcium phosphate apatite characterization discussing several techniques: electron microscopy/EDX, XRD, FTIR/Raman spectroscopies, chemical analyses, and solid state NMR. It also underlines frequent problems that should be kept in mind when making “bone-like apatites.”

Formation and evolution of hydrated surface layers of apatites

Nanocrystalline apatites exhibit a very fragile structured hydrated surface layer which is only observed in aqueous media. This surface layer contains mobile ionic species which can be easily exchanged with ions from the surrounding fluids. Although the precise structure of this surface layer is still unknown, it presents very specific spectroscopic characteristics. The structure of the hydrated surface layer depends on the constitutive mineral ions: ion exchanges of HPO4 2- ions by CO3 2- ions or of Ca2+ by Mg2+ ions result in a de-structuration of the hydrated layer and modifies its spectroscopic characteristics. However, the original structure can be retrieved by reverse exchange reaction. These alterations do not seem to affect the apatitic lattice. Stoichiometric apatite also shows HPO4 2- on their surface due to a surface hydrolysis after contact with aqueous solutions. Ion exchange is also observed and the environments of the surface carbonate ions seem analogous to that observed in nanocrystalline apatites. The formation of a hydrated layer in aqueous media appears to be a property common to apatites which has to be taken into account in their reactivity and biological behavior.

Biomimetic nanocrystalline apatites: Emerging perspectives in cancer diagnosis and treatment

Nanocrystalline calcium phosphate apatites constitute the mineral part of hard tissues, and the synthesis of biomimetic analogs is now well-mastered at the lab-scale. Recent advances in the fine physico-chemical characterization of these phases enable one to envision original applications in the medical field along with a better understanding of the underlying chemistry and related pharmacological features. In this contribution, we specifically focused on applications of biomimetic apatites in the field of cancer diagnosis or treatment. We first report on the production and first biological evaluations (cytotoxicity, pro-inflammatory potential, internalization by ZR-75-1 breast cancer cells) of individualized luminescent nanoparticles based on Eu-doped apatites, eventually associated with folic acid, for medical imaging purposes. We then detail, in a first approach, the preparation of tridimensional constructs associating nanocrystalline apatite aqueous gels and drug-loaded pectin microspheres. Sustained releases of a fluorescein analog (erythrosin) used as model molecule were obtained over 7 days, in comparison with the ceramic or microsphere reference compounds. Such systems could constitute original bone-filling materials for in situ delivery of anticancer drugs.

Synthesis and characterization of non-stoichiometric nickel–copper manganites

Non-stoichiometric nickel–copper manganites Ni Cu Mn h O were synthesized by thermal decomposition of x y 32x2y 3d / 4 41d mixed Ni Cu Mn C O , nH O oxalates in air at low temperature (623–673 K). X-ray diffraction showed that, x / 3 y / 3 (32x2y) / 3 2 4 2 for a nickel content x

1.111 – Bioactive Ceramics: Physical Chemistry

0.1, the oxalates precipitated presented a mixed crystal structure up to a limit value of copper Ni extent, whereas the oxalates obtained with x ,0.1 were not mixed. This could be explained by the intermediate structure Ni of nickel oxalate (b orthorhombic form) between those of copper and manganese (a monoclinic form) oxalates. The structure (a or b) of the mixed oxalates obtained was also investigated and their lattice parameters are given. The Ni Cu Mn h O oxides crystallize in the spinel structure in a wide range of composition and a stabilizing effect x y 32x2y 3d / 4 41d 2 21 of copper was evidenced. They are highly divided (Sw.100 m g ) however Sw tends to decrease with increasing y . Cu The non-stoichiometry d of such nickel–copper manganites was for the first time determined by selective titration (gas chromatography) of the oxygen released during TPR experiments in argon. The technique is presented and the results, along with those obtained with manganese oxide Mn O and nickel manganites synthesized in the same conditions, showed that d 5 8 depended both on the decomposition temperature of the oxalate and on the chemical composition of the oxide. Such results should provide interesting data concerning the cationic distributions of these non-stoichiometric nickel–copper manganites.

Chemical Diversity of Apatites

The chapter mainly discusses the physical–chemical properties of calcium phosphates (Ca-P), among the most important and most used bioactive ceramics. The main calcium phosphate compounds are presented with a brief description of their synthesis methods. Their characterization, using different techniques, including chemical analyses, X-ray diffraction, Fourier transform infrared (FTIR), Raman and solid-state nuclear magnetic resonance (NMR) spectrometries, and scanning and transmission electron microscopies (SEM and TEM), is reviewed and the different information obtained are discussed. The thermal stability and the relationships between different Ca-P phases are then described. The biological properties of Ca-P are related to their behavior in solution; their solubility, transformations and hydrolysis, nucleation ability, and surface properties and reactivity (ion exchange, adsorption) are presented especially in the case of apatites. The biological response regarding bioactivity, biodegradation, and simulated body fluid (SBF) testing is discussed from the point of view of the Ca-P physical chemistry. Several examples of applications are then proposed as ceramics, coatings, cements, and composite materials. A brief presentation of other bioactive mineral compounds follows (oxides and hydroxides, calcium carbonate, calcium sulfate).

Tetracycline-Loaded Biomimetic Apatite: An Adsorption Study

Apatites can accommodate a large number of vacancies and afford multiple ionic substitutions determining their reactivity and biological properties. Unlike other biominerals they offer a unique adaptability to various biological functions. The diversity of apatites is essentially related to their structure and to their mode of formation. Special charge compensation mechanisms allow molecular insertions and ion substitutions and determine to some extent their solubility behaviour. Apatite formation at physiological pH involves a structured surface hydrated layer nourishing the development of apatite domains. This surface layer contains relatively mobile and exchangeable ions, and is mainly responsible for the surface properties of apatite crystals from a chemical (dissolution properties, ion exchange ability, ion insertions, molecule adsorption and insertions) and a physical (surface charge, interfacial energy) point of view. These characteristics are used by living organisms and can also be exploited in material science.