Fluidic Force Microscopy and Atomic Force Microscopy Unveil New Insights into the Interactions of Preosteoblasts with 3D-Printed Submicron Patterns.

Physical patterns represent potential surface cues for promoting osteogenic differentiation of stem cells and improving osseointegration of orthopedic implants. Understanding the early cell-surface interactions and their effects on late cellular functions is essential for a rational design of such topographies, yet still elusive. In this work, fluidic force microscopy (FluidFM) and atomic force microscopy (AFM) combined with optical and electron microscopy are used to quantitatively investigate the interaction of preosteoblasts with 3D-printed patterns after 4 and 24 h of culture. The patterns consist of pillars with the same diameter (200 nm) and interspace (700 nm) but distinct heights (500 and 1000 nm) and osteogenic properties. FluidFM reveals a higher cell adhesion strength after 24 h of culture on the taller pillars (32 ± 7 kPa versus 21.5 ± 12.5 kPa). This is associated with attachment of cells partly on the sidewalls of these pillars, thus requiring larger normal forces for detachment. Furthermore, the higher resistance to shear forces observed for these cells indicates an enhanced anchorage and can be related to the persistence and stability of lamellipodia. The study explains the differential cell adhesion behavior induced by different pillar heights, enabling advancements in the rational design of osteogenic patterns.

3D-Printed Submicron Patterns Reveal the Interrelation between Cell Adhesion, Cell Mechanics, and Osteogenesis.

The surface topography of implantable devices is of crucial importance for guiding the cascade of events that starts from the initial contact of the cells with the surface and continues until the complete integration of the device in its immediate environment. There is, however, limited quantitative information available regarding the relationships between the different stages of such cascade(s) and how the design of surface topography influences them. We, therefore, used direct laser writing to 3D-print submicron pillars with precisely controlled dimensions and spatial arrangements to perform a systematic study of such relationships. Using single-cell force spectroscopy, we measured the adhesion force and the work of adhesion of the preosteoblast cells residing on the different types of surfaces. Not only the adhesion parameters (after 2-60 s) but also the formation of focal adhesions was strongly dependent on the geometry and arrangement of the pillars: sufficiently tall and dense pillars enhanced both adhesion parameters and the formation of focal adhesions. Our morphological study of the cells (after 24 h) showed that those enhancements were associated with a specific way of cell settlement onto the surface (i.e., "top state"). The cells interacting with tall and dense pillars were also characterized by numerous thick actin stress fibers in the perinuclear region and possibly high internal stresses. Furthermore, living cells with highly organized cytoskeletal networks exhibited greater values of the elastic modulus. The early responses of the cells predicted their late response including matrix mineralization: tall and dense submicron pillars significantly upregulated the expression of osteopontin after 21 days of culture under both osteogenic and nonosteogenic conditions. Our findings paint a detailed picture of at least one possible cascade of events that starts from initial cell adhesion and continues to subsequent cellular functions and eventual matrix mineralization. These observations could inform the future developments of instructive surfaces for medical devices based on physical surface cues and early markers.

On the Use of Black Ti as a Bone Substituting Biomaterial: Behind the Scenes of Dual-Functionality.

Despite the potential of small-scale pillars of black titanium (bTi) for killing the bacteria and directing the fate of stem cells, not much is known about the effects of the pillars' design parameters on their biological properties. Here, three distinct bTi surfaces are designed and fabricated through dry etching of the titanium, each featuring different pillar designs. The interactions of the surfaces with MC3T3-E1 preosteoblast cells and Staphylococcus aureus bacteria are then investigated. Pillars with different heights and spatial organizations differently influence the morphological characteristics of the cells, including their spreading area, aspect ratio, nucleus area, and cytoskeletal organization. The preferential formation of focal adhesions (FAs) and their size variations also depend on the type of topography. When the pillars are neither fully separated nor extremely tall, the colocalization of actin fibers and FAs as well as an enhanced matrix mineralization are observed. However, the killing efficiency of these pillars against the bacteria is not as high as that of fully separated and tall pillars. This study provides a new perspective on the dual-functionality of bTi surfaces and elucidates how the surface design and fabrication parameters can be used to achieve a surface topography with balanced bactericidal and osteogenic properties.

Quantitative mechanics of 3D printed nanopillars interacting with bacterial cells.

One of the methods to create sub-10 nm resolution metal-composed 3D nanopillars is electron beam-induced deposition (EBID). Surface nanotopographies (e.g., nanopillars) could play an important role in the design and fabrication of implantable medical devices by preventing the infections that are caused by the bacterial colonization of the implant surface. The mechanical properties of such nanoscale structures can influence their bactericidal efficiency. In addition, these properties are key factors in determining the fate of stem cells. In this study, we quantified the relevant mechanical properties of EBID nanopillars interacting with Staphylococcus aureus (S. aureus) using atomic force microscopy (AFM). We first determined the elastic modulus (17.7 GPa) and the fracture stress (3.0 ± 0.3 GPa) of the nanopillars using the quantitative imaging (QI) mode and contact mode (CM) of AFM. The displacement of the nanopillars interacting with the bacteria cells was measured by scanning electron microscopy (50.3 ± 9.0 nm). Finite element method based simulations were then applied to obtain the force-displacement curve of the nanopillars (considering the specified dimensions and the measured value of the elastic modulus) based on which an interaction force of 88.7 ± 36.1 nN was determined. The maximum von Mises stress of the nanopillars subjected to these forces was also determined (3.2 ± 0.3 GPa). These values were close to the maximum (i.e., fracture) stress of the pillars as measured by AFM, indicating that the nanopillars were close to their breaking point while interacting with S. aureus. These findings reveal unique quantitative data regarding the mechanical properties of nanopillars interacting with bacterial cells and highlight the possibilities of enhancing the bactericidal activity of the investigated EBID nanopillars by adjusting both their geometry and mechanical properties.

Deciphering the Roles of Interspace and Controlled Disorder in the Bactericidal Properties of Nanopatterns against Staphylococcus aureus.

Recent progress in nano-/micro-fabrication techniques has paved the way for the emergence of synthetic bactericidal patterned surfaces that are capable of killing the bacteria via mechanical mechanisms. Different design parameters are known to affect the bactericidal activity of nanopatterns. Evaluating the effects of each parameter, isolated from the others, requires systematic studies. Here, we systematically assessed the effects of the interspacing and disordered arrangement of nanopillars on the bactericidal properties of nanopatterned surfaces. Electron beam induced deposition (EBID) was used to additively manufacture nanopatterns with precisely controlled dimensions (i.e., a height of 190 nm, a diameter of 80 nm, and interspaces of 100, 170, 300, and 500 nm) as well as disordered versions of them. The killing efficiency of the nanopatterns against Gram-positive Staphylococcus aureus bacteria increased by decreasing the interspace, achieving the highest efficiency of 62 ± 23% on the nanopatterns with 100 nm interspacing. By comparison, the disordered nanopatterns did not influence the killing efficiency significantly, as compared to their ordered correspondents. Direct penetration of nanopatterns into the bacterial cell wall was identified as the killing mechanism according to cross-sectional views, which is consistent with previous studies. The findings indicate that future studies aimed at optimizing the design of nanopatterns should focus on the interspacing as an important parameter affecting the bactericidal properties. In combination with controlled disorder, nanopatterns with contrary effects on bacterial and mammalian cells may be developed.

Multiscale 3D-printing of microfluidic AFM cantilevers.

Microfluidic atomic force microscopy (AFM) cantilever probes have all the functionalities of a standard AFM cantilever along with fluid pipetting. They have a channel inside the cantilever and an aperture at the tip. Such probes are useful for precise fluid manipulation at a desired location, for example near or inside cells. They are typically made by complex microfabrication process steps, resulting in expensive probes. Here, we used two different 3D additive manufacturing techniques, stereolithography and two-photon polymerization, to directly print ready-to-use microfluidic AFM cantilever probes. This approach has considerably reduced the fabrication time and increased the design freedom. One of the probes, 564 µm long, 30 µm wide, 30 µm high, with a 25 µm diameter channel and 2.5 µm wall thickness had a spring constant of 3.7 N m-1 and the polymer fabrication material had an elastic modulus of 4.2 GPa. Using these 3D printed probes, AFM imaging of a surface, puncturing of the cell membrane, and aspiration at the single cell level have been demonstrated.

Hydrophilic Silver Nanoparticles Loaded into Niosomes: Physical-Chemical Characterization in View of Biological Applications.

Silver nanoparticles (AgNPs) are widely used as antibacterial agents and anticancer drugs, but often their low stability limits their mass production and broad applications. The use of niosomes as a carrier to protect and envelop AgNPs gives a new perspective to solve these problems. In this study, AgNPs were functionalized with sodium 3-mercapto-1-propanesulfonate (3MPS) to induce hydrophilic behavior, improving loading in Tween 20 and Span 20 niosomes (NioTw20 and NioSp20, respectively). Entrapment efficiency was evaluated by UV analyses and is around 1-4%. Dimensions were investigated by means of dynamic light scattering (DLS) (<2RH> = 140 ± 4 nm and <2RH> = 251 ± 1 nm respectively for NioTw20 + AgNPs and NioSp20 + AgNPs) and were compared with those by atomic force microscopy (AFM) and small angle X ray scattering (SAXS) analyses. Stability was assessed in water up to 90 days, and both in bovine serum and human serum for up to 8 h. In order to characterize the local structure of niosomes, SAXS measurements have been performed on Tween 20 and Span 20 empty niosomes and loaded with AgNPs. The release profiles of hydrophilic probe calcein and lipophilic probe Nile Red were performed in HEPES buffer and in human serum. All these features contribute to conclude that the two systems, NioTw20 + AgNPs and NioSp20 + AgNPs, are suitable and promising in the field of biological applications.

Identification of nanoparticles and nanosystems in biological matrices with scanning probe microscopy.

Identification of nanoparticles and nanosystems into cells and biological matrices is a hot research topic in nanobiotechnologies. Because of their capability to map physical properties (mechanical, electric, magnetic, chemical, or optical), several scanning probe microscopy based techniques have been proposed for the subsurface detection of nanomaterials in biological systems. In particular, atomic force microscopy (AFM) can be used to reveal stiff nanoparticles in cells and other soft biomaterials by probing the sample mechanical properties through the acquisition of local indentation curves or through the combination of ultrasound-based methods, like contact resonance AFM (CR-AFM) or scanning near field ultrasound holography. Magnetic force microscopy can detect magnetic nanoparticles and other magnetic (bio)materials in nonmagnetic biological samples, while electric force microscopy, conductive AFM, and Kelvin probe force microscopy can reveal buried nanomaterials on the basis of the differences between their electric properties and those of the surrounding matrices. Finally, scanning near field optical microscopy and tip-enhanced Raman spectroscopy can visualize buried nanostructures on the basis of their optical and chemical properties. Despite at a still early stage, these methods are promising for detection of nanomaterials in biological systems as they could be truly noninvasive, would not require destructive and time-consuming specific sample preparation, could be performed in vitro, on alive samples and in water or physiological environment, and by continuously imaging the same sample could be used to dynamically monitor the diffusion paths and interaction mechanisms of nanomaterials into cells and biological systems. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Chitosan Glutamate-Coated Niosomes: A Proposal for Nose-to-Brain Delivery.

The aim of this in vitro study is to prepare and characterize drug free and pentamidine loaded chitosan glutamate coated niosomes for intranasal drug delivery to reach the brain through intranasal delivery. Mucoadhesive properties and stability testing in various environments were evaluated to examine the potential of these formulations to be effective drug delivery vehicles for intranasal delivery to the brain. Samples were prepared using thin film hydration method. Changes in size and ζ-potential of coated and uncoated niosomes with and without loading of pentamidine in various conditions were assessed by dynamic light scattering (DLS), while size and morphology were also studied by atomic force microscopy (AFM). Bilayer properties and mucoadhesive behavior were investigated by fluorescence studies and DLS analyses, respectively. Changes in vesicle size and ζ-potential values were shown after addition of chitosan glutamate to niosomes, and when in contact with mucin solution. In particular, interactions with mucin were observed in both drug free and pentamidine loaded niosomes regardless of the presence of the coating. The characteristics of the proposed systems, such as pentamidine entrapment and mucin interaction, show promising results to deliver pentamidine or other possible drugs to the brain via nasal administration.

Single nanoparticles magnetization curves by controlled tip magnetization magnetic force microscopy.

The development of high spatial resolution and element sensitive magnetic characterization techniques to quantitatively measure magnetic parameters of individual nanoparticles (NPs) and deeply understand and tune their magnetic properties is a hot topic in nanomagnetism. Magnetic force microscopy (MFM), thanks to its high lateral resolution, appears as a promising technique for the magnetic characterization of single nano-sized materials although it is still limited by some drawbacks, especially by the presence of electrostatic artifacts. Recently, these limitations have been overcome by the development of a particular MFM based technique called controlled magnetization - MFM (CM-MFM) allowing, in principle, a quantifiable correlation between the measured magnetic signal and the magnetization of the object under investigation. Here we propose an experimental procedure, based on the use of CM-MFM technique, to measure the magnetization curve of single magnetic NPs individuating their saturation magnetization, magnetic field, and coercivity. We measured, for the first time, the magnetization curves of individual Fe3O4 nanoparticles with diameters in the range of 18-32 nm by using a MFM instrument. Results are in very good agreement with the quantitative data obtained by SQUID analysis on a macroscopic sample, showing the high potential of the technique in the field of nanomagnetometry.

Detection of stiff nanoparticles within cellular structures by contact resonance atomic force microscopy subsurface nanomechanical imaging.

Detecting stiff nanoparticles buried in soft biological matrices by atomic force microscopy (AFM) based techniques represents a new frontier in the field of scanning probe microscopies, originally developed as surface characterization methods. Here we report the detection of stiff (magnetic) nanoparticles (NPs) internalized in cells by using contact resonance AFM (CR-AFM) employed as a potentially non-destructive subsurface characterization tool. Magnetite (Fe3O4) NPs were internalized in microglial cells from cerebral cortices of mouse embryos of 18 days by phagocytosis. Nanomechanical imaging of cells was performed by detecting the contact resonance frequencies (CRFs) of an AFM cantilever held in contact with the sample. Agglomerates of NPs internalized in cells were visualized on the basis of the local increase in the contact stiffness with respect to the surrounding biological matrix. A second AFM-based technique for nanomechanical imaging, i.e., HarmoniX™, as well as magnetic force microscopy and light microscopy were used to confirm the CR-AFM results. Thus, CR-AFM was demonstrated as a promising technique for subsurface imaging of nanomaterials in biological samples.

Removal of electrostatic artifacts in magnetic force microscopy by controlled magnetization of the tip: application to superparamagnetic nanoparticles.

Magnetic force microscopy (MFM) has been demonstrated as valuable technique for the characterization of magnetic nanomaterials. To be analyzed by MFM techniques, nanomaterials are generally deposited on flat substrates, resulting in an additional contrast in MFM images due to unavoidable heterogeneous electrostatic tip-sample interactions, which cannot be easily distinguished from the magnetic one. In order to correctly interpret MFM data, a method to remove the electrostatic contributions from MFM images is needed. In this work, we propose a new MFM technique, called controlled magnetization MFM (CM-MFM), based on the in situ control of the probe magnetization state, which allows the evaluation and the elimination of electrostatic contribution in MFM images. The effectiveness of the technique is demonstrated through a challenging case study, i.e., the analysis of superparamagnetic nanoparticles in absence of applied external magnetic field. Our CM-MFM technique allowed us to acquire magnetic images depurated of the electrostatic contributions, which revealed that the magnetic field generated by the tip is sufficient to completely orient the superparamagnetic nanoparticles and that the magnetic tip-sample interaction is describable through simple models once the electrostatic artifacts are removed.

Magnetic force microscopy: quantitative issues in biomaterials.

Magnetic force microscopy (MFM) is an atomic force microscopy (AFM) based technique in which an AFM tip with a magnetic coating is used to probe local magnetic fields with the typical AFM spatial resolution, thus allowing one to acquire images reflecting the local magnetic properties of the samples at the nanoscale. Being a well established tool for the characterization of magnetic recording media, superconductors and magnetic nanomaterials, MFM is finding constantly increasing application in the study of magnetic properties of materials and systems of biological and biomedical interest. After reviewing these latter applications, three case studies are presented in which MFM is used to characterize: (i) magnetoferritin synthesized using apoferritin as molecular reactor; (ii) magnetic nanoparticles loaded niosomes to be used as nanocarriers for drug delivery; (iii) leukemic cells labeled using folic acid-coated core-shell superparamagnetic nanoparticles in order to exploit the presence of folate receptors on the cell membrane surface. In these examples, MFM data are quantitatively analyzed evidencing the limits of the simple analytical models currently used. Provided that suitable models are used to simulate the MFM response, MFM can be used to evaluate the magnetic momentum of the core of magnetoferritin, the iron entrapment efficiency in single vesicles, or the uptake of magnetic nanoparticles into cells.