Response of Osteoblasts on Amine-Based Nanocoatings Correlates with the Amino Group Density.

Increased life expectancy in industrialized countries is causing an increased incidence of osteoporosis and the need for bioactive bone implants. The integration of implants can be improved physically, but mainly by chemical modifications of the material surface. It was recognized that amino-group-containing coatings improved cell attachment and intracellular signaling. The aim of this study was to determine the role of the amino group density in this positive cell behavior by developing controlled amino-rich nanolayers. This work used covalent grafting of polymer-based nanocoatings with different amino group densities. Titanium coated with the positively-charged trimethoxysilylpropyl modified poly(ethyleneimine) (Ti-TMS-PEI), which mostly improved cell area after 30 min, possessed the highest amino group density with an N/C of 32%. Interestingly, changes in adhesion-related genes on Ti-TMS-PEI could be seen after 4 h. The mRNA microarray data showed a premature transition of the MG-63 cells into the beginning differentiation phase after 24 h indicating Ti-TMS-PEI as a supportive factor for osseointegration. This amino-rich nanolayer also induced higher bovine serum albumin protein adsorption and caused the cells to migrate slower on the surface after a more extended period of cell settlement as an indication of a better surface anchorage. In conclusion, the cell spreading on amine-based nanocoatings correlated well with the amino group density (N/C).

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.

Kinetically stable sub-50 nm fluorescent block copolymer nanoparticles via photomediated RAFT dispersion polymerization for cellular imaging.

Self-assembled block copolymer nanoparticles (NPs) have emerged as major potential nanoscale vehicles for fluorescence bioimaging. The preparation of NPs with high yields possessing high kinetic stability to prevent the leakage of fluorophore molecules is crucial to their practical implementation. Here, we report a photomediated RAFT polymerization-induced self-assembly (PISA) yielding uniform and nanosized poly((oligo(ethylene glycol) acrylate)-block-poly(benzyl acrylate) particles (POEGA-b-PBzA) with a concentration of 22 wt%, over 20 times more than with micellization and nanoprecipitation. The spherical diblock copolymer nanoparticles have an average size of 10-50 nm controllable through the degree of polymerization of the stabilizing POEGA block. Subsequent dialysis against water and swelling with Nile red solution led to highly stable fluorescent NPs able to withstand the changes in concentration, ionic strength, pH or temperature. A PBzA/water interfacial tension of 48.6 mN m-1 hinders the exchange between copolymer chains, resulting in the trapping of NPs in a "kinetically frozen" state responsible for high stability. A spectroscopic study combining fluorescence and UV-vis absorption agrees with a preferential distribution of fluorophores in the outer POEGEA shell despite its hydrophobic nature. Nile red-doped POEGA-b-PBzA micelles without initiator residues and unimers but with high structural stability turn out to be noncytotoxic, and can be used for the optical imaging of cells. Real-time confocal fluorescence microscopy shows a fast cellular uptake using C2C12 cell lines in minutes, and a preferential localization in the perinuclear region, in particular in the vesicles.

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.

Actomyosin, vimentin and LINC complex pull on osteosarcoma nuclei to deform on micropillar topography.

Cell deformation occurs in many critical biological processes, including cell extravasation during immune response and cancer metastasis. These cells deform the nucleus, their largest and stiffest organelle, while passing through narrow constrictions in vivo and the underlying mechanisms still remain elusive. It is unclear which biochemical actors are responsible and whether the nucleus is pushed or pulled (or both) during deformation. Herein we use an easily-tunable poly-L-lactic acid micropillar topography, mimicking in vivo constrictions to determine the mechanisms responsible for nucleus deformation. Using biochemical tools, we determine that actomyosin contractility, vimentin and nucleo-cytoskeletal connections play essential roles in nuclear deformation, but not A-type lamins. We chemically tune the adhesiveness of the micropillars to show that pulling forces are predominantly responsible for the deformation of the nucleus. We confirm these results using an in silico cell model and propose a comprehensive mechanism for cellular and nuclear deformation during confinement. These results indicate that microstructured biomaterials are extremely versatile tools to understand how forces are exerted in biological systems and can be useful to dissect and mimic complex in vivo behaviour.

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

Endothelial and epithelial cells usually grow on a curved environment, at the surface of organs, which many techniques have tried to reproduce. Here a simple method is proposed to control curvature of the substrate. Prestrained thin elastomer films are treated by infrared laser irradiation in order to rigidify the surface of the film. Wrinkled morphologies are produced upon stress relaxation for irradiation doses above a critical value. Wrinkle wavelength and depth are controlled by the prestrain, the laser power, and the speed at which the laser scans the film surface. Stretching of elastomer substrates with a "sand clock"-width profile enables the generation of a stress gradient, which results in patterns of wrinkles with a depth gradient. Thus, different combinations of topography changes on the same substrate can be generated. The wavelength and the depth of the wrinkles, which have the characteristic values within a range of several tens of µm, can be dynamically regulated by the substrate reversible stretching. It is shown that these anisotropic features are efficient substrates to control polarization of cell shapes and orientation of their migration. With this approach a flexible tool is provided for a wide range of applications in cell biophysics studies.

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

Biomedical implants are an important part of evolving modern medicine but have a potential drawback in the form of postoperative pathogenic infection. Accordingly, the "race for surface" combat between invasive bacteria and host cells determines the fate of implants. Hence, proper in vitro systems are required to assess effective strategies to avoid infection. In this study, we developed a real time observation model, mimicking postoperative contamination, designed to follow E. coli proliferation on a titanium surface occupied by human osteoblastic progenitor cells (STRO). This model allowed us to monitor E. coli invasion of human cells on titanium surfaces coated and uncoated with fibronectin. We showed that the surface colonization of bacteria is significantly enhanced on fibronectin coated surfaces irrespective of whether areas were uncovered or covered with human cells. We further revealed that bacterial colonization of the titanium surfaces is enhanced in coculture with STRO cells. Finally, this coculture system provides a comprehensive system to describe in vitro and in situ bacterial and human cells and their localization but also to target biological mechanisms involved in adhesion as well as in interactions with surfaces, thanks to fluorescent labeling. This system is thus an efficient method for studies related to the design and function of new biomaterials.

Curvotaxis directs cell migration through cell-scale curvature landscapes.

Cells have evolved multiple mechanisms to apprehend and adapt finely to their environment. Here we report a new cellular ability, which we term "curvotaxis" that enables the cells to respond to cell-scale curvature variations, a ubiquitous trait of cellular biotopes. We develop ultra-smooth sinusoidal surfaces presenting modulations of curvature in all directions, and monitor cell behavior on these topographic landscapes. We show that adherent cells avoid convex regions during their migration and position themselves in concave valleys. Live imaging combined with functional analysis shows that curvotaxis relies on a dynamic interplay between the nucleus and the cytoskeleton-the nucleus acting as a mechanical sensor that leads the migrating cell toward concave curvatures. Further analyses show that substratum curvature affects focal adhesions organization and dynamics, nuclear shape, and gene expression. Altogether, this work identifies curvotaxis as a new cellular guiding mechanism and promotes cell-scale curvature as an essential physical cue.

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

Understanding 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 µm curvature radius, whereas cells migrating on 125 µm 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.

Cytotoxic and genotoxic responses of human lung cells to combustion smoke particles of Miscanthus straw, softwood and beech wood chips.

Inhalation of particulate matter (PM) from residential biomass combustion is epidemiologically associated with cardiovascular and pulmonary diseases. This study investigates PM0.4-1 emissions from combustion of commercial Miscanthus straw (MS), softwood chips (SWC) and beech wood chips (BWC) in a domestic-scale boiler (40 kW). The PM0.4-1 emitted during combustion of the MS, SWC and BWC were characterized by ICP-MS/OES, XRD, SEM, TEM, and DLS. Cytotoxicity and genotoxicity in human alveolar epithelial A549 and human bronchial epithelial BEAS-2B cells were assessed by the WST-1 assay and the DNA-Alkaline Unwinding Assay (DAUA). PM0.4-1 uptake/translocation in cells was investigated with a new method developed using a confocal reflection microscope. SWC and BWC had a inherently higher residual water content than MS. The PM0.4-1 emitted during combustion of SWC and BWC exhibited higher levels of Polycyclic Aromatic Hydrocarbons (PAHs), a greater variety of mineral species and a higher heavy metal content than PM0.4-1 from MS combustion. Exposure to PM0.4-1 from combustion of SWC and BWC induced cytotoxic and genotoxic effects in human alveolar and bronchial cells, whereby the strongest effect was observed for BWC and was comparable to that caused by diesel PM (SRM 2 975), In contrast, PM0.4-1 from MS combustion did not induce cellular responses in the studied lung cells. A high PAH content in PM emissions seems to be a reliable chemical marker of both combustion efficiency and particle toxicity. Residual biomass water content strongly affects particulate emissions and their toxic potential. Therefore, to minimize the harmful effects of fine PM on health, improvement of combustion efficiency (aiming to reduce the presence of incomplete combustion products bound to PM) and application of fly ash capture technology, as well as use of novel biomass fuels like Miscanthus straw is recommended.