Michele Bianchi

Control of neuronal cell adhesion on single-walled carbon nanotube 3D patterns†

Carbon nanotubes are emerging substrates for guiding neuronal cell growth. Here we investigate the influence of multiscale 3D architecture of single-walled carbon nanotubes (SWCNT) on the adhesion of human neuronal cells (neuroblastoma SHSY5Y). 3D patterns of SWCNT were fabricated by a templating process to yield a hexagonal array of interconnected SWCNT semicapsules with controlled multiscale porosity, and integrated between Pt electrodes. Neuronal cells adhered preferentially to the SWCNT semicapsules with respect to either silicon oxide or disordered networks of SWCNT. Morphological cell features (size and shape) were evaluated upon application of an electric field across SWCNT pattern. Cell adhesion is enhanced by an electric field above 1 V cm−1, whereas it is completely depleted at 5 V cm−1. This shows the possibility to tune neuronal cell adhesion across different regimes by means of 3D pattern and voltage-biasing SWCNT.

Substrate geometry directs the in vitro mineralization of calcium phosphate ceramics

Repetitive concavities on the surface of bone implants have recently been demonstrated to foster bone formation when implanted at ectopic locations in vivo. The current study aimed to evaluate the effect of surface concavities on the surface mineralization of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) ceramics in vitro. Hemispherical concavities with different diameters were prepared at the surface of HA and β-TCP sintered disks: 1.8mm (large concavity), 0.8mm (medium concavity) and 0.4mm (small concavity). HA and β-TCP disks were sintered at 1100 or 1200°C and soaked in simulated body fluid for 28 days at 37°C; the mineralization process was followed by scanning electron microscopy, energy-dispersive spectroscopy, X-ray diffraction and calcium quantification analyses. The results showed that massive mineralization occurred exclusively at the surface of HA disks treated at 1200°C and that nucleation of large aggregates of calcium phosphate started specifically inside small concavities instead of on the planar surface of the disks. Regarding the effect of concavity diameter size on surface mineralization, it was observed that small concavities induce 124- and 10-fold increased mineralization compared to concavities of large or medium size, respectively. The results of this study demonstrated that (i) in vitro surface mineralization of calcium phosphate ceramics with surface concavities starts preferentially within the concavities and not on the planar surface, and (ii) concavity size is an effective parameter to control the spatial position and extent of mineralization in vitro.

Pulsed plasma deposition of zirconia thin films on UHMWPE: proof of concept of a novel approach for joint prosthetic implants

Wear of ultra-high molecular weight polyethylene (UHMWPE) has been recognized as the main cause for long-term revision in joint arthroplasty. A new approach to overcome this detrimental issue is here presented: zirconia (ZrO2) thin films were directly deposited onto the surface of UHMWPE by Pulsed Plasma Deposition (PPD) technique. The obtained films were structurally, morphologically and mechanically characterized by X-ray diffraction, scanning electron microscopy and nanoindentation tests, respectively. The critical fracture load was estimated by the analysis of the indenter footprints, while the adhesion degree was evaluated by a cross-cut tape test. Zirconia films exhibited a fully cubic structure, with densely packed grains, whereas mechanical tests showed that hard, tough and well-adherent films were deposited. These preliminary results suggested the feasibility of pursuing this alternative route to improve UHMPWE performances while preserving its well-established mechanical properties.

Neural cell alignment by patterning gradients of the extracellular matrix protein laminin

Anisotropic orientation and accurate positioning of neural cells is achieved by patterning stripes of the extracellular matrix protein laminin on the surface of polystyrene tissue culture dishes by micromoulding in capillaries (MIMICs). Laminin concentration decreases from the entrance of the channels in contact with the reservoir towards the end. Immunofluorescence analysis of laminin shows a decreasing gradient of concentration along the longitudinal direction of the stripes. The explanation is the superposition of diffusion and convection of the solute, the former dominating at length scales near the entrance (characteristic length around 50 μm), the latter further away (length scale in excess of 900 μm). These length scales are independent of the channel width explored from about 15 to 45 μm. Neural cells are randomly seeded and selectively adhere to the pattern, leaving the unpatterned areas depleted even upon 6 days of incubation. Cell alignment was assessed by the orientation of the long axis of the 4′,6-diamidino-2-phenylindole-stained nuclei. Samples on patterned the laminin area exhibit a large orientational order parameter. As control, cells on the unpatterned laminin film exhibit no preferential orientation. This implies that the anisotropy of laminin stripes is an effective chemical stimulus for cell recruiting and alignment.

Tribological characterization of zirconia coatings deposited on Ti6Al4V components for orthopedic applications.

One of the most important issues leading to the failure of total joint arthroplasty is related to the wear of the plastic components, which are generally made of ultra high molecular weight polyethylene (UHMWPE). Therefore, the reduction of joint wear represents one of the main challenges the research in orthopedics is called to address nowadays. Surface treatments and coatings have been recognized as innovative methods to improve tribological properties, also in the orthopedic field. This work investigated the possibility to realize hard ceramic coatings on the metal component of a prosthesis, by means of Pulsed Plasma Deposition, in order to reduce friction and wear in the standard coupling against UHMWPE. Ti6Al4V substrates were coated with a 2 μm thick yttria-stabilized zirconia (YSZ) layer. The mechanical properties of the YSZ coatings were assessed by nanoindentation tests performed on flat Ti6Al4V substrates. Tribological performance was evaluated using a ball-on-disk tribometer in dry and lubricated (i.e. with fetal bovine serum) highly-stressing conditions, up to an overall distance of 10 km. Tribology was characterized in terms of coefficient of friction (CoF) and wear rate of the UHMWPE disk. After testing, specimens were analyzed through optical microscopy and SEM images, in order to check the wear degradation mechanisms. Progressive loading scratch tests were also performed in dry and wet conditions to determine the effects of the environment on the adhesion of the coating. Our results supported the beneficial effect of YSZ coating on metal components. In particular, the proposed solution significantly reduced UHMWPE wear rate and friction. At 10 km of sliding distance, a wear rate reduction of about 18% in dry configuration and of 4% in presence of serum, was obtained by the coated group compared to the uncoated group. As far as friction in dry condition is concerned, the coating allowed to maintain low CoF values until the end of the tests, with an overall difference of about 40% compared to the uncoated balls. In wet conditions, the friction values were found to be comparable between coated and uncoated materials, mainly due to a premature delamination of the coating. Scratch tests in wet showed in fact a reduction of the critical load required to a complete delamination due to a formation of blister, although no change or damage occurred at the coating during the soaking period. Although conditions of high values of contact pressure were considered, further analyses are however required to fully understand the behavior of YSZ coatings in wet environment and additional research on the deposition process will be mandatory in order to improve the coating tribological performance at long distances addressing orthopedic applications.

Tough and adhesive nanostructured calcium phosphate thin films deposited by the pulsed plasma deposition method

Calcium phosphate (CaP) coatings are commonly employed to improve the bioactivity of bone and dental metal implants, due to the chemical–physical similarity with the mineral phase of bone, envisaged to enhance the integration with the surrounding bone tissue. However, growing concerns about the use of commercial thick CaP coatings, mainly related to their weak mechanical properties, poor interface strength and micro-structural inhomogeneity, are leading the investigation of new and alternative CaP deposition techniques. In this study the feasibility to deposit tough and well adherent CaP thin films by a novel Pulsed Plasma Deposition (PPD) method on Ti alloy was investigated. Microstructural, chemical and morphological properties of the coatings as well as the nano-mechanical properties and their adhesion to the Ti-alloy substrate were extensively characterized. In vitro biocompatibility was also preliminary assessed evaluating the adhesion and proliferation of primary mouse osteoblasts. As-deposited CaP films were amorphous and exhibited dense and uniform surface composed of sub-micrometric aggregated globular particles. Noteworthy, mechanical properties of as-deposited films were comparable to the ones of commercial plasma-sprayed coatings despite the significant difference of thickness (a few hundred nanometers vs. tens of micrometers). After the thermal annealing of the as deposited films at 600 °C for 1 h in air, a transformation from amorphous calcium phosphate to crystalline hydroxyapatite (HA) phase occurred. The mechanical properties as well as the adhesion to substrate of the annealed films strongly improved respect to those of the as deposited films, displaying interesting high hardness, elastic strain to failure and plastic deformation resistance values. Finally, biological in vitro tests indicated good biocompatibility of both as-deposited and annealed films, with this latter showing better cells adhesion and proliferation compared to the former.

NANOMECHANICAL CHARACTERIZATION OF ZIRCONIA THIN FILMS DEPOSITED ON UHMWPE BY PULSED PLASMA DEPOSITION

Plastic deformation and wear of the ultra-high molecular weight polyethylene (UHMWPE) insert have been pointed out as major issues relating to the long-term stability of an orthopaedic implant. The deposition of protective hard, tough and well-adhered zirconia (ZrO2) thin films directly on the surface of the UHMWPE component via the Pulsed Plasma Deposition (PPD) technique has been already demonstrated to be a feasible way to approach this problem. In the current study, the tribo-mechanical properties of ZrO2-coated UHMWPE with respect to pristine UHMWPE were investigated in detail. Specifically, strength to local plastic deformation, indentation work portioning and creep behavior were evaluated through nanoindentation and micro-scratch tests. Further, preliminary wear data (i.e., rate and volume) were obtained by tribology tests mating coated and pristine UHMWPE with an alumina ball under dry conditions. The results of the mechanical tests evidenced a strong reduction of plastic deformation under both normal and tangential local loads and a drop of the 80% of the creep phenomenon for coated UHMWPE compared to pristine UHMWPE. Despite tribological tests showed similar wear data for coated and pristine UHMWPE, a different wear mechanism was detected between the two groups. The reported results supported the possibility to pursue this novel approach of depositing ZrO2 thin film to protect the UHWMPE insert and enhance the long-term stability of the orthopaedic implants.

CERAMIC THIN FILMS REALIZED BY MEANS OF PULSED PLASMA DEPOSITION TECHNIQUE: APPLICATIONS FOR ORTHOPEDICS

Joint prosthesis are usually subjected to several failing mechanisms, including wear of the ultra-high molecular weight polyethylene (UHMWPE) insert. The main goal of this study was to assess the possibility to improve the tribological properties of titanium component by depositing zirconia thin films on its surface by pulsed plasma deposition (PPD) method. Zirconia-coated titanium spheres were tested against UHMWPE disks, both in dry and wet conditions. Zirconia films exhibited a homogenous sub-micrometric grain size distribution and low roughness. Interestingly, zirconia-coated titanium spheres showed lower wear rate of the UHMWPE component, compared to uncoated titanium spheres, supporting the feasibility of the proposed approach.

Cell fluidics: Producing cellular streams on micropatterned synthetic surfaces

Patterning cell-adhesive molecules on material surfaces provides a powerful tool for controlling and guiding cell locomotion and migration. Here we report fast, reliable, easy to implement methods to fabricate large patterns of proteins on synthetic substrates to control the direction and speed of cells. Two common materials exhibiting very different protein adsorption capacities, namely, polystyrene and Teflon, were functionalized with micrometric stripes of laminin. The protein was noncovalently immobilized onto the surface by following either lithographically controlled wetting (LCW) or micromolding in capillaries (MIMIC). These techniques proved to be sufficiently mild so as not to interfere with the protein adhesion capability. Cells adhered onto the functionalized stripes and remained viable for more than 20 h. During this time frame, cells migrated along the lanes and the dynamics of motion was strongly affected by the substrate surface chemistry and culturing conditions. In particular, enhanced mismatches of cell adhesive properties obtained by the juxtaposition of bare and laminin-functionalized Teflon caused cells to move slowly and their movement to be highly confined. The patterning procedure was also effective at guiding migration on conventional cell culture dishes that were functionalized with laminin patterns, even in the presence of serum proteins, although to a lesser extent compared to that for Teflon. This work demonstrates the possibility of creating well-defined, long-range cellular streams on synthetic substrates by pursuing straightforward functionalizing techniques that can be implemented for a broad class of materials under conventional, long-time cell-culturing conditions. The procedure effectively confines cells to migrate along predefined patterns and can be implemented in different biomedical and biotechnological applications.

Magnetic hydroxyapatite coatings as a new tool in medicine: A scanning probe investigation

Hydroxyapatite films enriched with magnetite have been fabricated via a Pulsed Plasma Deposition (PPD) system with the final aim of representing a new platform able to disincentivate bacterial adhesion and biofilm formation. The chemical composition and magnetic properties of films were respectively examined by X-ray photoelectron spectroscopy (XPS) and Superconducting Quantum Interference Device (SQUID) measurements. The morphology and conductive properties of the magnetic films were investigated via a combination of scanning probe technologies including atomic force microscopy (AFM), electrostatic force microscopy (EFM), and scanning tunneling microscopy (STM). Interestingly, the range of adopted techniques allowed determining the preservation of the chemical composition and magnetic properties of the deposition target material while STM analysis provided new insights on the presence of surface inhomogeneities, revealing the presence of magnetite-rich islands over length scales compatible with the applications. Finally, preliminary results of bacterial adhesion tests, indicated a higher ability of magnetic hydroxyapatite films to reduce Escherichia coli adhesion at 4h from seeding compared to control hydroxyapatite films.