Clermont-Ferrand II

This thesis is part of the ANR project PNano NANOBONEFILLER. The aim of this project is the development of a new injectable bone filling system based on mesoporous calcium phosphates associated with growth factors. The thesis is composed of two main parts: the synthesis of mesoporous calcium phosphates by hard templating techniques and the adsorption and release of growth factors on these porous ceramics. This work is the first association of growth factors with mesoporous calcium phosphates for bone regeneration. Calcium phosphates are extensively used for bone regeneration due to their biocompatibility and good resorption. Their performance can, however, be improved if these materials are associated with growth factors. Growth factors are soluble proteins that stimulate cell activity, such as proliferation and differentiation. In order to control the release of growth factors from ceramic materials, the aim of this work has been the synthesis of calcium phosphates with controlled mesoporosity. Hard templating approaches have been developed for the synthesis of porous hydroxyapatites: mesocellular silica foam is obtained by the condensation of tetraethyl orthosilicate in presence of surfactant micelles. These silica foams have higher pore volumes and a pore structure that is more suitable for the replication than hexagonal type SBA-15 silica which was used as a reference material. Carbon replicas of these silica foams are obtained by chemical vapor infiltration (CVD) of propylene at 750 °C or by liquid infiltration with acidic sucrose solution. Depending on the carbon precursor, the replicas have different porosities and surface chemistries. The effect of these differences on the replication with hydroxyapatite was investigated systematically. The dissolution of the silica matrix by etching with hydrofluoric acid frees the porosity of the carbon replica. Hydroxyapatite is then produced inside the porosity both of silica and carbon templates by the infiltration of precursor solutions. The template elimination method depends on the nature of the template: in the case of silica templates, the matrix is dissolved by sodium hydroxide etching, whereas the carbon template is eliminated by selective oxidation in air. The resulting ceramics are characterized by different electron microscopy techniques as well as by X-ray diffraction and nitrogen physisorption. These techniques show that the properties of the ceramics vary with the synthesis conditions such as the calcination temperature, the nature of the template and the infiltration conditions. The hydroxyapatites are composed of strongly agglomerated nanoparticles with specific surface areas from 30 to 160m/g, depending on the synthesis conditions. Their crystallinity depends on the temperature during the heat treatment. Nitrogen physisorption experiments prove that the ceramics are mesoporous. Depending on their physico-chemical properties, six ceramics have been chosen for protein adsorption experiments. Three of these samples have been obtained from a project VII te l-0 06 85 00 6, v er si on 1 3 Ap r 2 01 2 partner. At first, two less expensive model proteins, BSA (bovine serum albumin) and Cytochrome C, are used to develop an adsorption and release protocol. The adsorption and release kinetics are evaluated by a depletion method: the supernatant of the powders is analyzed by UV/Vis absorption either directly (Cytochrome C) or after staining with Bradford reagent (BSA). Adsorption and release experiments using the growth factors TGF-β1 and VEGF followed. TGF-β1 is a cytokine able to stimulate bone regeneration whereas VEGF improves vascularisation. The combination of both factors is promising for the regeneration of bone defects. As the quantification by UV/Vis absorbance is not possible for the growth factors because they are too diluted, immunoassay techniques with specific antibodies have been chosen for quantification. These experiments show which are the most efficient powders for the adsorption and controlled release of growth factors. During the thesis, we synthesized a number of calcium phosphate samples with different physico-chemical properties and evaluated their growth factor retention capacities. This study allows to connect the physico-chemical properties of calcium phosphates to their ability to store and to release proteins progressively.