Synthesis and characterization of Ti-27.5Nb alloy made by CLAD® additive manufacturing process for biomedical applications

Biocompatible beta-titanium alloys such as Ti-27.5(at.%)Nb are good candidates for implantology and arthroplasty applications as their particular mechanical properties, including low Young’s modulus, could significantly reduce the stress-shielding phenomenon usually occurring after surgery. The CLAD® process is a powder blown additive manufacturing process that allows the manufacture of patient specific (i.e. custom) implants. Thus, the use of Ti-27.5(at.%)Nb alloy formed by CLAD® process for biomedical applications as a mean to increase cytocompatibility and mechanical biocompatibility was investigated in this study. The microstructural properties of the CLAD-deposited alloy were studied with optical microscopy and electron back-scattered diffraction (EBSD) analysis. The conservation of the mechanical properties of the Ti-27.5Nb material after the transformation steps (ingot-powder atomisation-CLAD) were verified with tensile tests and appear to remain close to those of reference material. Cytocompatibility of the material and subsequent cell viability tests showed that no cytotoxic elements are released in the medium and that viable cells proliferated well

Size-Dependent Internalization Efficiency of Macrophages from Adsorbed Nanoparticle-Based Monolayers

Functional coatings based on the assembly of submicrometric or nanoparticles are found in many applications in the biomedical field. However, these nanoparticle-based coatings are particularly fragile since they could be exposed to cells that are able to internalize nanoparticles. Here, we studied the efficiency of RAW 264.7 murine macrophages to internalize physisorbed silica nanoparticles as a function of time and particle size. This cell internalization efficiency was evaluated from the damages induced by the cells in the nanoparticle-based monolayer on the basis of scanning electron microscopy and confocal laser scanning microscopy observations. The internalization efficiency in terms of the percentage of nanoparticles cleared from the substrate is characterized by two size-dependent regimes. Additionally, we highlighted that a delay before internalization occurs, which increases with decreasing adsorbed nanoparticle size. This internalization is characterized by a minimal threshold that corresponds to 35 nm nanoparticles that are not internalized during the 12-h incubation considered in this work

Internalization efficiency of adsorbed particles

Internalization efficiency of adsorbed particle

Stabilité de revêtements nanoparticulaires

Stabilité de revêtements nanoparticulaire

Size-Dependent Internalization Efficiency of Macrophages from Adsorbed Nanoparticle-Based Monolayers

International audienceFunctional coatings based on the assembly of submicrometric or nanoparticles are found in many applications in the biomedical field. However, these nanoparticle-based coatings are particularly fragile since they could be exposed to cells that are able to internalize nanoparticles. Here, we studied the efficiency of RAW 264.7 murine macrophages to internalize physisorbed silica nanoparticles as a function of time and particle size. This cell internalization efficiency was evaluated from the damages induced by the cells in the nanoparticle-based monolayer on the basis of scanning electron microscopy and confocal laser scanning microscopy observations. The internalization efficiency in terms of the percentage of nanoparticles cleared from the substrate is characterized by two size-dependent regimes. Additionally, we highlighted that a delay before internalization occurs, which increases with decreasing adsorbed nanoparticle size. This internalization is characterized by a minimal threshold that corresponds to 35 nm nanoparticles that are not internalized during the 12-h incubation considered in this work

Elaboration de revêtements anti-aggrégatifs à l’aide de protéines chaperons

stockage de solution de protéines dans la durée. Afin de stabiliser ces solutions et d’éviter des phénomènes de dénaturation au contact des surfaces et par conséquent la perte d’activité biologique, des additifs ou stabilisants sont incorporés dans la formulation. Ces stabilisants, qui limitent les modifications de conformation, peuvent néanmoins présenter l’inconvénient d’être injectés au patient en même temps que le principe actif. Lorsque les protéines sont adsorbées sur des surfaces, elles subissent en effet de multiples modifications de conformation, qui peuvent non seulement provoquer une perte d’activité biologique mais également initier des phénomènes d’agrégation (en particulier, en exposant les feuillets β initialement enfouis dans la structure de la protéine et favorisant par la même occasion les interactions protéine-protéine plutôt que protéine-solvant). L’intensité de la dénaturation dépend de la nature de la surface, du type de protéine mais aussi du temps de séjour au contact. Dans le milieu vivant, des protéines nommées « chaperons » ont pour but d’éviter ces changements de conformation trop drastiques, de maintenir les protéines d’intérêt dans leur conformation native et ainsi de bloquer le phénomène d’agrégation. Nous proposons d’évaluer la possibilité de réaliser des revêtements avec une protéine chaperon et de maintenir son activité biologique. La protéine n’est alors plus libre dans la solution mais greffée de manière covalente sur la surface, évitant ainsi le risque de relargage. Dans une première partie, la quantité de protéine chaperon nécessaire pour limiter le phénomène d’agrégation du lysozyme sur une durée de 48h est estimée. Pour cela, la fraction soluble de lysozyme en solution, les agrégats solubles ainsi que les agrégats adsorbés sont quantifiés par spectroscopie UV. Dans un second temps, La protéine chaperon est greffée sur des substrats de titane et de verre afin de déterminer son activité vis-à-vis de l’agrégation du lysozyme. Nous avons pu montrer que la protéine greffée possède une activité comparable à la protéine en solution à 48h et que son efficacité se maintient sur une durée de 2 semaines

Laser‐Assisted Strain Engineering of Thin Elastomer Films to Form Variable Wavy Substrates for Cell Culture

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Influence of multiscale and curved structures on the migration of stem cells

International audienceUnderstanding how topographical cues can control cell behavior is a major fundamental question which is of particular interest for implant design. Recent findings show that cell-scale curvature, as well as nanoscale topography, can affect different aspects of cell migration. However, the correlation between specific curvature radii and cell behavior, as well as the combinatorial effect of nanoscale topography and cell-scale curvature, has not yet been investigated. Herein, the authors employ a new femtosecond laser ablation method to generate multiscale topographical patterns directly on titanium surfaces. The process allows us to produce microgrooves of specific curvature imprinted with oriented nanotopographical features called Laser-Induced Periodic Surface Structures (LIPSS). The authors show that curved grooves stimulate the stem cell migration speed in comparison to flat or linear grooves. The fastest velocities are observed on 75 mum curvature radius, whereas cells migrating on 125 mum curvatures exhibit a lower speed similar to the ones migrating on straight lines. Double replicas of these grooves allow us to mask the LIPSS while keeping identical the cell-scale pattern, therefore permitting to uncouple the effect of nanoscale and microscale topographies. The authors found that the presence of nanoscale topographies improves the reading of microgrooves curvature by cells. Altogether, this work shows that the combination of specific curvatures together with nanopatterning can control the velocity of migrating stem cells and promote the use of femtosecond laser ablation in the context of surface implant design

Real-Time Imaging of Bacteria/Osteoblast Dynamic Coculture on Bone Implant Material in an in Vitro Postoperative Contamination Model

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In Vitro and In Vivo Cell-Interactions with Electrospun Poly (Lactic-Co-Glycolic Acid) (PLGA): Morphological and Immune Response Analysis

Random electrospun three-dimensional fiber membranes mimic the extracellular matrix and the interfibrillar spaces promotes the flow of nutrients for cells. Electrospun PLGA membranes were analyzed in vitro and in vivo after being sterilized with gamma radiation and bioactivated with fibronectin or collagen. Madin-Darby Canine Kidney (MDCK) epithelial cells and primary fibroblast-like cells from hamster’s cheek paunch proliferated over time on these membranes, evidencing their good biocompatibility. Cell-free irradiated PLGA membranes implanted on the back of hamsters resulted in a chronic granulomatous inflammatory response, observed after 7, 15, 30 and 90 days. Morphological analysis of implanted PLGA using light microscopy revealed epithelioid cells, Langhans type of multinucleate giant cells (LCs) and multinucleated giant cells (MNGCs) with internalized biomaterial. Lymphocytes increased along time due to undegraded polymer fragments, inducing the accumulation of cells of the phagocytic lineage, and decreased after 90 days post implantation. Myeloperoxidase+ cells increased after 15 days and decreased after 90 days. LCs, MNGCs and capillaries decreased after 90 days. Analysis of implanted PLGA after 7, 15, 30 and 90 days using transmission electron microscope (TEM) showed cells exhibiting internalized PLGA fragments and filopodia surrounding PLGA fragments. Over time, TEM analysis showed less PLGA fragments surrounded by cells without fibrous tissue formation. Accordingly, MNGC constituted a granulomatous reaction around the polymer, which resolves with time, probably preventing a fibrous capsule formation. Finally, this study confirms the biocompatibility of electrospun PLGA membranes and their potential to accelerate the healing process of oral ulcerations in hamsters’ model in association with autologous cells