Apatites nanocristallines biomimétiques comme modèles de la réactivité osseuse : étude des propriétés d'adsorption et de l'activité cellulaire d'un bisphosphonate, le tiludronate

Ce mémoire porte sur l'étude de l'interaction entre un bisphosphonate, le tiludronate et des phosphates de calcium apatitiques nanocristallins comme modèles du minéral osseux. Les bisphosphonates sont principalement utilisés dans le traitement de maladies impliquant un désordre osseux telles que l'ostéoporose et la maladie de Paget. Cependant peu d'études existent sur le mécanisme d'interaction entre les bisphosphonates et le tissu osseux et les phénomènes présents à l'interface bisphosphonate/minéral osseux restent mal connus. Notre travail a donc consisté à étudier l'interaction entre un bisphosphonate, le tiludronate, et des apatites nanocristallines comme modèle du minéral osseux afin de mieux comprendre les propriétés physico-chimiques et l'activité cellulaire de ce type d'associations. Dans un premier temps, des apatites nanocristallines à différents stades de maturation et à différentes teneurs en carbonates correspondant à des compositions d'os jeune et mature ont été synthétisées et caractérisées. L'adsorption du tiludronate sur ces apatites nanocristallines, la caractérisation des supports après adsorption et les cinétiques de libération du tiludronate ont ensuite été examinées. L'adsorption du tiludronate correspond à un isotherme de type Langmuir. Le processus d'adsorption s'explique globalement par un échange ionique entre les molécules de tiludronate en solution et les ions phosphates présents à la surface de l'apatite. Toutefois la proportion d'ions phosphates libérés par molécule de tiludronate varie suivant les supports. Une modélisation de l'interaction entre le tiludronate et la surface des nanocristaux a été proposée en nous basant sur la littérature et en considérant différents modèle de la surface des nanocristaux. Elle suggère une interaction en deux temps : l'interaction entre les groupements phosphonates de la molécule et deux calcium de la surface suivie d'une déprotonation de la molécule et de l'exclusion d'un nombre variable d'ions phosphate. Dans un deuxième temps, des supports apatitiques avec et sans tiludronate ont été élaborés par pressage uniaxial et caractérisés. L'activité biologique des cellules osseuses de la lignée ostéoblastique (HOP et HBMSC) et ostéoclastique (ostéoclastes murins) au contact des supports a été étudiée. Les résultats montrent que le tiludronate adsorbé stimule la prolifération des ostéoblastes tandis qu'il inhibe la création de lacunes de résorption. La caractérisation des modèles tout au long des tests in vitro montre une évolution physico-chimique des supports semblable à celle du minéral osseux. Ce travail permet de préciser les mécanismes mis en jeu lors des traitements utilisant des bisphosphonates et de tester la validité d'études in vitro pour le criblage de molécules actives sur le tissu osseux.Bisphosphonates (BPs) are currently administrated to treat diseases involving bone disorders such as osteoporosis or Paget's disease. However, the interactions between bisphosphonate molecules and apatite nanocrystals of bone are not well understood. The aim of this work was to study the interactions between one bisphosphonate, tiludronate and nanocrystalline apatites with different compositions as bone mineral models in order to clarify the adsorption and release of BPs on/from bone mineral analogues and to evaluate the effect of such associations on osteoblast and osteoclast cells cultures. Several nanocrystalline apatites (NCA) corresponding to young and mature bone mineral models were synthesized and characterized. The adsorption of tiludronate onto NCA, corresponded to a Langmuir type isotherm. Only a very small fraction of the adsorbed molecules was released in water. The uptake of tiludronate molecules was associated with a linear increase of phosphate ions in the adsorbing solution, indicating that the main reaction was an ion exchange process involving surface anions. The proportion of ions involved in this exchange reaction depended on the nature of the bone mineral models. Based on these results a modification of the adsorption reaction including the ion exchange with phosphate ions was proposed. A model of the adsorption process is proposed, based on the literature and several nanocrystals surface models, suggesting an interaction in two steps: a binding of phosphonates groups with calcium ions on the apatite surface associated with the loss of protons from the adsorbing molecules and the release of surface phosphate ions to maintain some surface charge equilibrium. In order to study the effects of apatite nanocrystals with different compositions and with or without adsorbed tiludronate on cells cultures, apatite pellets were obtained by uniaxial compression. Human osteoprogenitor cells (HOP), human bone marrow stromal cells (HBMSC) and murine osteoclasts were cultured on these apatitic substrates. The results indicated that osteoblasts proliferation was stimulated by adsorbed tiludronate molecules whereas creation of resorption pits by osteoclasts was inhibited. No difference in cell behaviour was noticed on the different bone analogues. Moreover, an evolution of the physico-chemical characteristics of the apatitic substrate during cell culture was observed, highlighting the existence of dynamic interactions possibly similar to bone mineral evolution. This work contributes to clarify the reaction mechanisms between bisphosphonates and biomimetic apatites and to evaluate in vitro conditions for drug delivery system

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

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

Robocasting of Single and Multi-Functional Calcium Phosphate Scaffolds and Its Hybridization with Conventional Techniques: Design, Fabrication and Characterization [Allizond V. is the co-corresponding author]

International audienceIn this work, dense, porous and, for the first time, functionally-graded bi-layer scaffolds with a cylindrical geometry were produced from a commercially available hydroxyapatite powder using the robocasting technique. The bi-layer scaffolds were made of a dense core part attached to a surrounding porous part. Subsequently, these bi-layer robocast scaffolds were joined with an outer shell of an antibacterial porous polymer layer fabricated by solvent casting/salt leaching techniques, leading to hybrid ceramic-polymer scaffolds. The antibacterial functionality was achieved through the addition of silver ions to the polymer layer. All the robocast samples, including the bi-layer ones, were first characterized through scanning electron microscopy observations, mechanical characterization in compression and preliminary bioactivity tests. Then, the hybrid bi-layer ceramic-polymer scaffolds were characterized through antimicrobial tests. After sintering at 1300 °C for 3 h, the compressive strengths of the structures were found to be equal to 29 ± 4 MPa for dense samples and 7 ± 4 MPa for lattice structures with a porosity of 34.1%. Bioactivity tests performed at 37 °C for 4 weeks showed that the precipitated layer on the robocast samples contained octacalcium phosphate. Finally, it was evidenced that the hybrid structure was effective in releasing antibacterial Ag+ ions to the surrounding medium showing its potential efficiency in limiting Staphylococcus aureus proliferation during surgery

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 nanocarriers 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 lowfrequency 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 of Folic Acid with Nanocrystalline Apatites and Extension to Methotrexate (Antifolate) in View of Anticancer Applications

Nanocrystalline apatites mimicking bone mineral represent a versatile platform for biomedical applications thanks to their similarity to bone apatite and the possibility to (multi)functionalize them so as to provide “à la carte” properties. One relevant domain is in particular oncology, where drug-loaded biomaterials and engineered nanosystems may be used for diagnosis, therapy, or both. In a previous contribution, we investigated the adsorption of doxorubicin onto two nanocrystalline apatite substrates, denoted HA and FeHA (superparamagnetic apatite doped with iron ions), and explored these drug-loaded systems against tumor cells. To widen their applicability in the oncology field, here we examine the interaction between the same two substrates and two other molecules: folic acid (FA), often used as cell targeting agent, and the anticancer drug methotrexate (MTX), an antifolate analogue. In a first stage, we investigated the adsorptive behavior of FA (or MTX) on both substrates, evidencing their specificities. At low concentration, typically under 100 mmol/L, adsorption onto HA was best described using the Sips isotherm model, while the formation of a calcium folate secondary salt was evidenced at high concentration by Raman spectroscopy. Adsorption onto FeHA was instead fitted to the Langmuir model. A larger adsorptive affinity was found for the FeHA substrate compared to HA; accordingly, a faster release was noticed from HA. In vitro tests carried out on human osteosarcoma cell line (SAOS-2) allowed us to evaluate the potential of these compounds in oncology. Finally, in vivo (subcutaneous) implantations in the mouse were run to ascertain the biocompatibility of the two substrates. These results should allow a better understanding of the interactions between FA/MTX and bioinspired nanocrystalline apatites in view of applications in the field of cancer

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 Paget's 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

Apatites nanocristallines biomimétiques comme modèles de la réactivité osseuse (étude des propriétés d'adsorption et de l'activité cellulaire d'un bisphosphonate, le tiludronate)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Des biocéramiques aux vertus réparatrices. Les substituts osseux de demain

International audienc

Advanced processing techniques for customized ceramic medical devices

International audienc