Stéphanie Sarda

Infrared, Raman and NMR investigations of risedronate adsorption on nanocrystalline apatites

The aim of the current work was to study the physico-chemical interactions of a bisphosphonate molecule, risedronate, with a well-characterised synthetic nanocrystalline apatite (NCA) as a model bone mineral. We adopted a global approach, using complementary physico-chemical techniques such as FTIR, RAMAN and NMR spectroscopies in order to learn more about the interaction process of risedronate with the apatitic surface. The results obtained suggest that risedronate adsorption corresponds to an ion substitution reaction with phosphate ions occurring at the crystal surface. This mechanism explains the greater amount adsorbed (N) for NCA, compared to well crystallised stoichiometric hydroxyapatite, attributable to the well-developed hydrated layer at the surface of the nanocrystals. However, most calcium ions remain attached to the solid phase and the formation of insoluble risedronate calcium salts must also be considered as a competitive reaction to the adsorption. Thus a calcium risedronate salt was synthesised and fully characterised for comparison to the solids after adsorption. Following spectroscopic results, it can be concluded that a strong interaction was established between risedronate ions and calcium ions at the apatitic surface. However, under these experimental conditions there is no nucleation of a distinct calcium risedronate salt and the apatite crystals retain their integrity.

Interaction between a Bisphosphonate, Tiludronate, and Biomimetic Nanocrystalline Apatites

Bisphosphonates (BPs) are well established as successful antiresorptive agents for the prevention and treatment of bone diseases such as osteoporosis and Pagets disease. The aim of this work was to clarify the reaction mechanisms between a BP molecule, tiludronate, and the nanocrystalline apatite surface. The adsorption of tiludronate on well-characterized synthetic biomimetic nanocrystalline apatites with homogeneous but different compositions and surface characteristics was investigated to determine the effect of the nanocrystalline apatite substrate on the adsorption behavior. The results show that the adsorption of tiludronate on nanocrystalline biomimetic apatite surfaces varies over a large range. The most immature apatitic samples exhibited the highest affinity and the greatest amount adsorbed at saturation. Maturation of the nanocrystals induces a decrease of these values. The amount of phosphate ion released per adsorbed BP molecule varied, depending on the nanocrystalline substrate considered. The adsorption mechanism, although associated with a release of phosphate ions, cannot be considered as a simple ion exchange process involving one or two phosphate ions on the surface. A two-step process is proposed consisting of a surface binding of BP groups to calcium ions associated with a proton release inducing the protonation of surface orthophosphate ions and their eventual solubilization.

Adsorption on apatitic calcium phosphates for drug delivery: interaction with bisphosphonate molecules

Bisphosphonates (BPs) are well established as an important class of drugs for the treatment and prevention of several bone disorders including osteoporosis. This work investigated the interaction of two bisphosphonates, risedronate and tiludronate, with several apatitic supports, a well-crystallised hydroxyapatite (HA) and nanocrystalline apatites with varying maturation times, chemical composition and surface characteristics. The purpose was to fully understand the adsorption mechanism and desorption process, by the evaluation of the effect of several physicochemical parameters (temperature, pH and concentration of calcium and phosphate ions). Whatever the nature of the BP and the structure and composition of the apatite, the adsorption of such anti-resorptive agents can be well described as an ion exchange-reaction between phosphates species on the apatitic surface and BP molecules in solution. However, the parameters of adsorption can vary depending on the physicochemical conditions of the adsorption reaction. In addition, the structure and composition of the apatitic surface also influence the adsorption properties. Finally, BPs molecules are slowly released from apatitic supports, because most of the adsorbed molecules are irreversibly bound and not spontaneously released by dilution or simple washing. Moreover, similar to their adsorption, the release of bisphosphonates is strongly affected not only by the chemical properties of the molecule, but also by the chemical and structural characteristics of the apatitic substrates. The understanding of the adsorption and release processes provides fundamental tools for the development of drug delivery systems using apatite materials.

Superparamagnetic iron-doped nanocrystalline apatite as a delivery system for doxorubicin

The development of non-toxic and biodegradable magnetic nanoparticles (NPs) that can be easily functionalized with drugs or biomolecules and employed, under magnetic fields, as targeted nano-carriers or components of scaffolds with on-demand functionalities, is a big challenge in the biomaterials research. In the present work, the feasibility of previously synthesized iron-doped superparamagnetic apatite (FeHA) NPs to bind and then release the anticancer drug doxorubicin (DOX) under an applied low-frequency pulsed electromagnetic field (PEMF) was investigated. The behavior of FeHA towards DOX has been compared to that of synthetic biomimetic apatite (HA) NPs prepared ad hoc with characteristics close to those of bone mineral. The DOX adsorption kinetics and isotherms on FeHA and HA were explored and fitted according to different mathematical models (Elovich, Sips and Freundlich) revealing enhanced uptake of DOX on FeHA than HA, due to the better interaction of the drug with the surface iron cations and formation of multi-molecular DOX assemblies. In the absence of the PEMF, the quantity of DOX released from HA was higher than that released from FeHA, in agreement with the lower affinity of DOX for HA than FeHA. Interestingly, in the presence of the PEMF, the extent of DOX released from FeHA after 3 and 6 days increased significantly. The higher DOX release from FeHA under PEMF can be explained by the mechanical shacking of superparamagnetic FeHA NPs breaking the bonding with the drug and allowing detachment of DOX assemblies from the NP surface. In vitro assays demonstrated that DOX loaded on HA and FeHA displayed cytotoxicity against the human osteosarcoma cell line (SAOS-2) at the same level as free DOX, for all the concentrations and time points tested. Confocal microscopy analyses showed that drug-loaded NPs were rapidly internalized within cells and released DOX, which accumulated in the nuclei where it exerted the desired cytotoxic activity.

Interaction between a bisphosphonate, tiludronate and nanocrystalline apatite: in vitro viability and proliferation of HOP and HBMSC cells

Nanocrystalline apatites (NCA) are the inorganic components of mineralized tissues and they have been recently proposed as biomaterials for drug delivery systems. Bisphosphonates (BPs) are currently the reference drugs used to treat diseases involving bone disorders such as osteoporosis. Nevertheless, the interaction phenomena between BP molecules and apatite nanocrystals of bone are not well understood. Therefore, the adsorption characteristics have been examined and cellular activity of tiludronate molecules on NCA as models of bone mineral has been investigated. Adsorption experiments of tiludronate onto NCA were carried out and revealed a Langmuir-type adsorption isotherm. The uptake of tiludronate molecules is associated with a release of phosphate ions, indicating that the main reaction is an ion exchange process involving surface anions. The results evidence the strong affinity of BP molecules for the apatitic surface. The interactions of NCA-tiludronate associations with human osteoprogenitor cells and human bone marrow stromal cells do not reveal any cytotoxicity and evidence the activity of adsorbed tiludronate molecules. Moreover, an evolution of the physico-chemical characteristics of the apatitic substrate during biological study was observed, highlighting the existence of dynamic interactions. This work contributes to clarifying the reaction mechanisms between BPs and biomimetic apatites.

Characterization of Calcium Phosphates Using Vibrational Spectroscopies

Vibrational spectroscopies are extensively used for the characterization of calcium phosphates either as natural biological minerals (bone, teeth, ectopic calcifications) or as biomaterials (bioceramics, coatings, composites). The present review begins with a theoretical description of expected spectra for the main calcium phosphate phases (i.e., brushite, monetite, octacalcium phosphate, tricalcium phosphates, apatites, amorphous calcium phosphate) followed by the analysis of real spectra, line positions and assignments, and observed anomalies. In the second part, the spectra of complex well-crystallized ion-substituted apatites and other calcium phosphates, as well as solid solutions, are investigated, and the information gained regarding the substitution types and ion distributions are derived. Finally, we will examine and interpret the spectra of nanocrystalline apatites considering the ion substitution effects and the existence of a surface hydrated layer. Quantification processes and spectra treatments are briefly presented and discussed. Examples of the use of vibrational spectroscopies for biomaterials and biominerals characterization will be detailed for coating evaluations, including spectroscopic imaging, following up on mineral cement setting reactions, adsorption studies, near infrared investigations of surface water, residual strains determinations in bone, orientation of apatite crystals in biological tissues, and crystallinity and maturity of bone mineral.

From crystalline to amorphous calcium pyrophosphates: A solid state Nuclear Magnetic Resonance perspective

UNLABELLED Hydrated calcium pyrophosphates (CPP, Ca2P2O7·nH2O) are a fundamental family of materials among osteoarticular pathologic calcifications. In this contribution, a comprehensive multinuclear NMR (Nuclear Magnetic Resonance) study of four crystalline and two amorphous phases of this family is presented. (1)H, (31)P and (43)Ca MAS (Magic Angle Spinning) NMR spectra were recorded, leading to informative fingerprints characterizing each compound. In particular, different (1)H and (43)Ca solid state NMR signatures were observed for the amorphous phases, depending on the synthetic procedure used. The NMR parameters of the crystalline phases were determined using the GIPAW (Gauge Including Projected Augmented Wave) DFT approach, based on first-principles calculations. In some cases, relaxed structures were found to improve the agreement between experimental and calculated values, demonstrating the importance of proton positions and pyrophosphate local geometry in this particular NMR crystallography approach. Such calculations serve as a basis for the future ab initio modeling of the amorphous CPP phases. STATEMENT OF SIGNIFICANCE The general concept of NMR crystallography is applied to the detailed study of calcium pyrophosphates (CPP), whether hydrated or not, and whether crystalline or amorphous. CPP are a fundamental family of materials among osteoarticular pathologic calcifications. Their prevalence increases with age, impacting on 17.5% of the population after the age of 80. They are frequently involved or associated with acute articular arthritis such as pseudogout. Current treatments are mainly directed at relieving the symptoms of joint inflammation but not at inhibiting CPP formation nor at dissolving these crystals. The combination of advanced NMR techniques, modeling and DFT based calculation of NMR parameters allows new original insights in the detailed structural description of this important class of biomaterials.

Structure of the calcium pyrophosphate monohydrate phase (Ca2P2O7·H2O): towards understanding the dehydration process in calcium pyrophosphate hydrates.

Calcium pyrophosphate hydrate (CPP, Ca(2)P(2)O(7) · nH2O) and calcium orthophosphate compounds (including apatite, octacalcium phosphate etc.) are among the most prevalent pathological calcifications in joints. Even though only two dihydrated forms of CPP (CPPD) have been detected in vivo (monoclinic and triclinic CPPD), investigations of other hydrated forms such as tetrahydrated or amorphous CPP are relevant to a further understanding of the physicochemistry of those phases of biological interest. The synthesis of single crystals of calcium pyrophosphate monohydrate (CPPM; Ca(2)P(2)O(7) · H2O) by diffusion in silica gel at ambient temperature and the structural analysis of this phase are reported in this paper. Complementarily, data from synchrotron X-ray diffraction on a CPPM powder sample have been fitted to the crystal parameters. Finally, the relationship between the resolved structure for the CPPM phase and the structure of the tetrahydrated calcium pyrophosphate β phase (CPPT-β) is discussed.

Crystallisation of a highly metastable hydrated calcium pyrophosphate phase

A simple and fast synthesis method was set up to obtain pure hydrated calcium pyrophosphate (CPP) phases of biological interest. This work focused on a specific phase synthesised at 25 °C and pH 4.5 in a stirred tank reactor. Powder X-ray diffraction, FTIR spectroscopy, scanning electron microscopy and thermogravimetric analyses revealed that the phase is unknown but presents similarities with a monoclinic tetrahydrated CPP phase (Ca2P2O7·4H2O, m-CPPT β phase) synthesised under the same conditions of pH and temperature. Characterisation of the unreferenced phase (u-CPP) has been performed, especially to better identify its composition, structure and stability, as well as its possible relation to the m-CPPT β phase or to other hydrated CPP phases.

Crystal structure of monoclinic calcium pyrophosphate dihydrate (m-CPPD) involved in inflammatory reactions and osteoarthritis.

Pure monoclinic calcium pyrophosphate dihydrate (m-CPPD) has been synthesized and characterized by synchrotron powder X-ray diffraction and neutron diffraction. Rietveld refinement of complementary diffraction data has, for the first time, allowed the crystal structure of m-CPPD to be solved. The monoclinic system P2(1)/n was confirmed and unit-cell parameters determined: a = 12.60842 (4), b = 9.24278 (4), c = 6.74885 (2) Å and β = 104.9916 (3)°. Neutron diffraction data especially have allowed the precise determination of the position of H atoms in the structure. The relationship between the m-CPPD crystal structure and that of the triclinic calcium pyrophosphate dihydrate (t-CPPD) phase as well as other pyrophosphate phases involving other divalent cations are discussed by considering the inflammatory potential of these phases and/or their involvement in different diseases. These original structural data represent a key step in the understanding of the mechanisms of crystal formation involved in different types of arthritis and to improve early detection of calcium pyrophosphate (CPP) phases in vivo.