Jean-Christophe Hornez

Prolonged local antibiotics delivery from hydroxyapatite functionalised with cyclodextrin polymers

Per-operative infection is a common complication for bone-graft surgery. Combining antiseptic agents with graft materials may offer a solution by increasing local drug concentration at target sites. Aiming to achieve a sustained local antibiotic (ATB) delivery for a widely applied bone substitute material - hydroxyapatite (HA), we attempted incorporating hydroxypropyl-beta-cyclodextrin polymer (polyHPbetaCD) into microporous HA via impregnating either in a CD monomers mixture solution or a pre-synthesized CD polymer solution, followed by thermal fixation processing. In such functionalised material (CD-HA), polyHPbetaCD could entrap ATBs and release them progressively. Infrared-spectroscopic analysis confirmed the presence of polyHPbetaCD in functionalised HA via both processing pathways; polyHPbetaCD functionalisation yields were quantitated by thermogravimetric analysis for optimising the processing regime. Ciprofloxacin (CFX) and vancomycin (VCM), commonly applied in orthopaedics, have been respectively loaded on CD-HA by dip-coating. For both ATBs, kinetic release test in phosphate buffered saline showed significantly increased initial-burst amount and prolonged release from CD-HA compared with those from non-functionalised HA. Encouragingly, ATBs loaded CD-HA also revealed a prolonged bacteriostatic activity against Staphylococcus aureus and progressively increased cytocompatibility to osteoblasts (MC3T3-E1). Overall, polyHPbetaCD functionalisation on HA could be an effective drug-delivery model for loading different drug molecules in prevention of infection.

Molecular interactions between zoledronic acid and bone: An in vitro Raman microspectroscopic study

The aim of this study was to investigate molecular interactions between a bisphosphonate (BP), zoledronic acid, and bone tissue by the use of Raman microspectroscopy. In this way, samples of hydroxyapatite (HA), as a bone model, and Wistar rat femurs were soaking in zoledronic acid solutions. Sample surfaces were studied by Environmental Scanning Electron Microscopy and Raman spectroscopy. The amount of zoledronic acid incorporated onto the samples and the inorganic phosphate released in solution were determined by (31)P NMR spectroscopy. Total carbonate content in solution was evaluated by inorganic carbon analyser. After impregnation new Raman bands with frequencies close to characteristic peaks of zoledronic acid (in particular phosphate moieties and imidazole ring of the R2 side-chain) were observed on both types of samples. Physico-chemical parameters of the bone were also significantly modified (P<0.0001). The mineral to organic ratio and the carbonate to phosphate ratio decreased and the crystallinity increased. Released inorganic phosphate and carbonate were detected in the solutions. The Raman shift of the bands corresponding to the phosphate groups and the imidazole ring of the BP highlight their implication in the binding to the mineral. The detection of released inorganic phosphate and carbonate in solution, the modifications of the mineral to phosphate ratio and the carbonate to phosphate ratio reveal that BP decrease the amount of inorganic phosphate and limit the dissolution of bone mineral. The increase of the crystallinity after BP binding shows a re-organisation of the lattice with a higher symmetry. Thus, it seems that zoledronic acid has an important contribution on the increase of crystallinity. The use of Raman spectrometry brings new and complementary information on the impact of zoledronic acid on bone composition at molecular level. Raman spectrometry could help to understand by which way BPs improve bone strength and decrease fracture risk.

The corrosion and biological behaviour of titanium alloys in the presence of human lymphoid cells and MC3T3-E1 osteoblasts

Corrosion behaviour of biomedical alloys is generally determined in mineral electrolytes: unbuffered NaCl 0.9% (pH 7.4) or artificial saliva (pH 6.8). The assays with exclusive utilization of these electrolytes are of low relevance for the biological condition, to which the alloys will be exposed once implanted in the human organism. As an approach to the biological situation regarding the interaction of proteins, electrolytes and metals, we added the RPMI cell culture medium containing foetal calf serum as a biological electrolyte (pH 7.0). The analysis of corrosion behaviour was also performed in the presence of human lymphoid cells (CEM). The rest potential (Er) and the global polarization were determined on cp-Ti, micro-arc oxidized cp-Ti (MAO-Ti), four different Ti-alloys (Ti6Al4V, Ti12Zr, Ti(AlMoZr), Ti(NbTaZr)) and 316L stainless steel. The 316L exhibited an appropriate Er and a good passive current density (Ip), but a high corrosion potential (Ec) and a very low breakdown potential (Eb) in all electrolytes. All Ti-alloys exhibited a much better electrochemical behaviour: better Er and Ec and very high Eb. No significant differences of the above parameters existed between the Ti-alloys, except for Zr-containing alloys that showed better corrosion behaviour. A remarkable difference, however, was stated with respect to the electrolytes. NaCl 0.9% induced strong variations between the Ti-alloys. More homogeneous results were obtained with artificial saliva and RPMI medium, which induced a favourable Ec and an increased Ip. The presence of cells further decreased these values. The unbuffered NaCl solution seems to be less appropriate for the analysis of corrosion of metals. Additional in vitro biological assessments with CEM cell suspensions and MC3T3-E1 osteoblasts confirmed the advantages of the Ti(AlMoZr) and Ti(NbTaZr) alloys with an improved cell proliferation and vitality rate.

Cytocompatibility and bio-corrosion of implant alloys in different cell culture systems

Any implant introduced into the human organism may undergo degradation depending on the physicio-chemical qualities of the concerned materials. Although the in vivo degradation and corrosion of implants and prostheses have largely been demonstrated in patients, very few experiments have been conducted to assess the corrosion behaviour of implant alloys under biological conditions, which tend to simulate the complex environment leading to corrosion. The present work was conducted on 5 different alloys representative for multiple biomedical use: Ti6Al4V, NiTi (49/51), NiCr (59/26), 316L, PdAg (62/28), and 2 pure metals: hp-Ni and cp-Ti. The electrolytes for the electrochemical assays were artificial saliva pH 6.8 and RPMI cell culture medium as biological milieu with and without cells. The rest potential and the global polarisation were established. The CEM line, human lymphoid cells, (ATCC-TIB95) was used for the electrochemical assays. The cell reaction was assessed by in vitro proliferation test on L132 human epithelial cells (ATCC-CCL5). Actin labelling was performed to assess their adhesion behaviour. Ion analyses by ICP-AES were carried out in the culture medium after the electrochemical assays and proliferation tests. The electrochemical assays reveal a different corrosion behaviour of all alloys in the three electrolytes: RPMI with/without cells generally increase corrosion and diminish the break-down potential. These differences are significant for 316L, NiCr and hp-Ni, less notable for PdAg and nearly absent for the Ti-base alloys. A similar classification can be obtained by the cell proliferation tests. Actin labelling revealed important and well aligned intracellular filaments on the control and the Ti6Al4V, and a little less on cp-Ti, PdAg, and 316L. These fibres were more reduced on NiTi and NiCr and absent on hp-Ni. Chemical analyses by ICP demonstrated an increased ion release from ORIGINAL ARBEITEN J.-C. Hornez: Cytocompatibility and bio-corrosion of implant alloys in different cell culture systems

Die Knorpelgrundsubstanzen Kollagene und Hyaluronsäure und ihre Bedeutung im Tissue Engineering

Cartilage is a solid and elastic, compact and flexible specialized connective tissue. It is a necessary support o f soft tissue and bone movement and the precursor for bone development. The multiple functions and the variable composition o f its fundamental substance distinguish three types: hyaline cartilage, elastic cartilage, and fibrocartilage. The three main components o f cartilage are chondrocytes, a network of collagen fibers and a proteoglycan gel with water. The cartilage collagens are mainly synthesized and secreted by chondroblasts. The principal collagen type is Type-II in all cartilage forms. Other types have been identified in lower amounts: in particular Types IX, VI and XI, types V and X are probably also existing. The ground substance is composed o f hyaluronic acid (HuA), a high molecular weight polysaccharide from the glucoseaminoglycan (GAGs) family, glucoseaminosulfate, chondroitin sulfate, and keratane sulfate, forming with HuA as central molecule and with some central and linking proteins a highly viscoelastic proteoglycan retaining water and cations. HuA is a fundamental substance o f any connective tissue; it is synthesized and secreted by multiple cell types, in particular by chondrocytes, fibroblasts and synoviocytes. The main role o f HuA is to assume the mechanical function as lubricant of cartilage and joints . HuA and collagens have two principle roles for the elaboration of composite matrices, these are the support for tissue engineered implants and the physiological action, i.e. stimulation of chondrocytes and other cell growth. HuA is the most important and suitable compound, which should be present in all scaffolds and matrices because o f its multiple functional potentials and qualities for the material physico-chemistry and the cell biology as well. HuA increases the swelling ratio and conditions the porous structure o f scaffolds and sponges; it regulates the interaction between different cell types such as chondrocytes, fibroblasts, synoviocytes and leukocytes; it stimulates chondrocyte and fibroblast proliferation by increasing tubulin synthesis and controlling the diploid DNA state; it enhances chondroitin sulfate synthesis in chondrocytes, it stimulates collagen synthesis and secretion. Hyaluronic acid has multiple other therapeutic and diagnostic means, which sensibly increase the importance o f that omnipresent macromolecule.