Bone cell response to additively manufactured 3D micro-architectures with controlled Poisson's ratio: Auxetic vs. non-auxetic meta-biomaterials.

The Poisson's ratio and elastic modulus are two parameters determining the elastic behavior of biomaterials. While the effects of elastic modulus on the cell response is widely studied, very little is known regarding the effects of the Poisson's ratio. The micro-architecture of meta-biomaterials determines not only the Poisson's ratio but also several other parameters that also influence cell response, such as porosity, pore size, and effective elastic modulus. It is, therefore, very challenging to isolate the effects of the Poisson's ratio from those of other micro-architectural parameters. Here, we computationally design meta-biomaterials with controlled Poisson's ratios, ranging between -0.74 and +0.74, while maintaining consistent porosity, pore size, and effective elastic modulus. The 3D meta-biomaterials were additively manufactured at the micro-scale using two-photon polymerization (2PP), and were mechanically evaluated at the meso­scale. The response of murine preosteoblasts to these meta-biomaterials was then studied using in vitro cell culture models. Meta-biomaterials with positive Poisson's ratios resulted in higher metabolic activity than those with negative values. The cells could attach and infiltrate all meta-biomaterials from the bottom to the top, fully covering the scaffolds after 17 days of culture. Interestingly, the meta-biomaterials exhibited different cell-induced deformations (e.g., shrinkage or local bending) as observed via scanning electron microscopy. The outcomes of osteogenic differentiation (i.e., Runx2 immunofluorescent staining) and matrix mineralization (i.e., Alizarin red staining) assays indicated the significant potential impact of these meta-biomaterials in the field of bone tissue engineering, paving the way for the development of advanced bone meta-implants. STATEMENT OF SIGNIFICANCE: We studied the influence of Poisson's ratio on bone cell response in meta-biomaterials. While elastic modulus effects are well-studied, the impact of Poisson's ratio, especially negative values found in architected biomaterials, remains largely unexplored. The complexity arises from intertwined micro-architectural parameters, such as porosity and elastic modulus, making it challenging to isolate the Poisson's ratio. To overcome this limitation, this study employed rational computational design to create meta-biomaterials with controlled Poisson's ratios, alongside consistent effective elastic modulus, porosity, and pore size. The study reveals that two-photon polymerized 3D meta-biomaterials with positive Poisson's ratios displayed higher metabolic activity, while all the developed meta-biomaterials supported osteogenic differentiation of preosteoblasts as well as matrix mineralization. The outcomes pave the way for the development of advanced 3D bone tissue models and meta-implants.

Auxeticity as a Mechanobiological Tool to Create Meta-Biomaterials.

Mechanical and morphological design parameters, such as stiffness or porosity, play important roles in creating orthopedic implants and bone substitutes. However, we have only a limited understanding of how the microarchitecture of porous scaffolds contributes to bone regeneration. Meta-biomaterials are increasingly used to precisely engineer the internal geometry of porous scaffolds and independently tailor their mechanical properties (e.g., stiffness and Poisson's ratio). This is motivated by the rare or unprecedented properties of meta-biomaterials, such as negative Poisson's ratios (i.e., auxeticity). It is, however, not clear how these unusual properties can modulate the interactions of meta-biomaterials with living cells and whether they can facilitate bone tissue engineering under static and dynamic cell culture and mechanical loading conditions. Here, we review the recent studies investigating the effects of the Poisson's ratio on the performance of meta-biomaterials with an emphasis on the relevant mechanobiological aspects. We also highlight the state-of-the-art additive manufacturing techniques employed to create meta-biomaterials, particularly at the micrometer scale. Finally, we provide future perspectives, particularly for the design of the next generation of meta-biomaterials featuring dynamic properties (e.g., those made through 4D printing).

Sensitivity of viscoelastic characterization in multi-harmonic atomic force microscopy.

Quantifying the nanomechanical properties of soft-matter using multi-frequency atomic force microscopy (AFM) is crucial for studying the performance of polymers, ultra-thin coatings, and biological systems. Such characterization processes often make use of cantilever's spectral components to discern nanomechanical properties within a multi-parameter optimization problem. This could inadvertently lead to an over-determined parameter estimation with no clear relation between the identified parameters and their influence on the experimental data. In this work, we explore the sensitivity of viscoelastic characterization in polymeric samples to the experimental observables of multi-frequency intermodulation AFM. By performing simulations and experiments we show that surface viscoelasticity has negligible effect on the experimental data and can lead to inconsistent and often non-physical identified parameters. Our analysis reveals that this lack of influence of the surface parameters relates to a vanishing gradient and non-convexity while minimizing the objective function. By removing the surface dependency from the model, we show that the characterization of bulk properties can be achieved with ease and without any ambiguity. Our work sheds light on the sensitivity issues that can be faced when optimizing for a large number of parameters and observables in AFM operation, and calls for the development of new viscoelastic models at the nanoscale and improved computational methodologies for nanoscale mapping of viscoelasticity using AFM.

Quantifying nanoscale forces using machine learning in dynamic atomic force microscopy.

Dynamic atomic force microscopy (AFM) is a key platform that enables topological and nanomechanical characterization of novel materials. This is achieved by linking the nanoscale forces that exist between the AFM tip and the sample to specific mathematical functions through modeling. However, the main challenge in dynamic AFM is to quantify these nanoscale forces without the use of complex models that are routinely used to explain the physics of tip-sample interaction. Here, we make use of machine learning and data science to characterize tip-sample forces purely from experimental data with sub-microsecond resolution. Our machine learning approach is first trained on standard AFM models and then showcased experimentally on a polymer blend of polystyrene (PS) and low density polyethylene (LDPE) sample. Using this algorithm we probe the complex physics of tip-sample contact in polymers, estimate elasticity, and provide insight into energy dissipation during contact. Our study opens a new route in dynamic AFM characterization where machine learning can be combined with experimental methodologies to probe transient processes involved in phase transformation as well as complex chemical and biological phenomena in real-time.

Optical microlever assisted DNA stretching.

Optical microrobotics is an emerging field that has the potential to improve upon current optical tweezer studies through avenues such as limiting the exposure of biological molecules of interest to laser radiation and overcoming the current limitations of low forces and unwanted interactions between nearby optical traps. However, optical microrobotics has been historically limited to rigid, single-body end-effectors rather than even simple machines, limiting the tasks that can be performed. Additionally, while multi-body machines such as microlevers exist in the literature, they have not yet been successfully demonstrated as tools for biological studies, such as molecule stretching. In this work we have taken a step towards moving the field forward by developing two types of microlever, produced using two-photon absorption polymerisation, to perform the first lever-assisted stretches of double-stranded DNA. The aim of the work is to provide a proof of concept for using optical micromachines for single molecule studies. Both styles of microlevers were successfully used to stretch single duplexes of DNA, and the results were analysed with the worm-like chain model to show that they were in good agreement.

Engineered 3D Polymer and Hydrogel Microenvironments for Cell Culture Applications.

The realization of biomimetic microenvironments for cell biology applications such as organ-on-chip, in vitro drug screening, and tissue engineering is one of the most fascinating research areas in the field of bioengineering. The continuous evolution of additive manufacturing techniques provides the tools to engineer these architectures at different scales. Moreover, it is now possible to tailor their biomechanical and topological properties while taking inspiration from the characteristics of the extracellular matrix, the three-dimensional scaffold in which cells proliferate, migrate, and differentiate. In such context, there is therefore a continuous quest for synthetic and nature-derived composite materials that must hold biocompatible, biodegradable, bioactive features and also be compatible with the envisioned fabrication strategy. The structure of the current review is intended to provide to both micro-engineers and cell biologists a comparative overview of the characteristics, advantages, and drawbacks of the major 3D printing techniques, the most promising biomaterials candidates, and the trade-offs that must be considered in order to replicate the properties of natural microenvironments.

Submicron Patterns-on-a-Chip: Fabrication of a Microfluidic Device Incorporating 3D Printed Surface Ornaments.

Manufacturing high throughput in vitro models resembling the tissue microenvironment is highly demanded for studying bone regeneration. Tissues such as bone have complex multiscale architectures inside which cells reside. To this end, engineering a microfluidic platform incorporated with three-dimensional (3D) microscaffolds and submicron/nanoscale topographies can provide a promising model for 3D cell cultures. There are, however, certain challenges associated with this goal, such as the need to decorate large surfaces area with high-fidelity 3D submicron structures. Here, we succeeded in fabricating a microfluidic platform embedded with a large area (mm range) of reproducible submicron pillar-based topographies. Using the two-photon polymerization (2PP) as a 3D printing technique based on direct laser writing, uniform submicron patterns were created through optimization of the process parameters and writing strategy. To demonstrate the multiscale fabrication capabilities of this approach, submicron pillars of various heights were integrated onto the surfaces of a 3D microscaffold in a single-step 2PP process. The created submicron topography was also found to improve the hydrophilicity of the surface while being able to withstand flow rates of up to 8 mL/min. The material (IP-Dip resin) used for patterning did not have cytotoxic effects against human mesenchymal stromal cells after 3 days of dynamic culture in the microfluidic device. This proof-of-principle study, therefore, marks a significant step forward in manufacturing submicron structure-on-a-chip models for bone regeneration studies.

Functional dependence of resonant harmonics on nanomechanical parameters in dynamic mode atomic force microscopy.

We present a combined theoretical and experimental study of the dependence of resonant higher harmonics of rectangular cantilevers of an atomic force microscope (AFM) as a function of relevant parameters such as the cantilever force constant, tip radius and free oscillation amplitude as well as the stiffness of the sample's surface. The simulations reveal a universal functional dependence of the amplitude of the 6th harmonic (in resonance with the 2nd flexural mode) on these parameters, which can be expressed in terms of a gun-shaped function. This analytical expression can be regarded as a practical tool for extracting qualitative information from AFM measurements and it can be extended to any resonant harmonics. The experiments confirm the predicted dependence in the explored 3-45 N/m force constant range and 2-345 GPa sample's stiffness range. For force constants around 25 N/m, the amplitude of the 6th harmonic exhibits the largest sensitivity for ultrasharp tips (tip radius below 10 nm) and polymers (Young's modulus below 20 GPa).

Highly strained graphene samples of varying thickness and comparison of their behaviour.

Mechanically straining graphene opens the possibility to exploit new properties linked to the stressed lattice of this two-dimensional material. In particular, theoretical analyses have forecast that straining graphene by more than 10% is a requirement for many novel applications that have not yet been experimentally demonstrated. Recently, we reported having achieved 12.5% strain in a trilayer graphene sample (3LG) in a controlled, reversible and non-destructive way. In this paper, we explore our method by straining samples of varying thicknesses and comparing their behavior, where strains of 14% and 11% were achieved for monolayer and four-layer graphene (4LG), respectively. For the analysis, optical tracking and the correspondent Raman spectra were taken. While doing so, we observed slippage between two layers in a bilayer sample of which one layer was clamped on one side only. The obtained results when stretching different samples to extreme strains demonstrated the exceptional elasticity of graphene, which might be essential for practical applications. Hysteretic effects observed in the partially clamped layer hints at small energy losses during slippage. This may shed new light on the superlubricity property of graphene that has been reported in the literature.

Controlled, reversible, and nondestructive generation of uniaxial extreme strains (>10%) in graphene.

Theoretical calculations have predicted that extreme strains (>10%) in graphene would result in novel applications. However, up to now the highest reported strain reached ∼1.3%. Here, we demonstrate uniaxial strains >10% by pulling graphene using a tensile-MEMS. To prevent it from slipping away it was locally clamped with epoxy using a femtopipette. The results were analyzed using Raman spectroscopy and optical tracking. Furthermore, analysis proved the process to be reversible and nondestructive for the graphene.

Implementation and characterization of a quartz tuning fork based probe consisted of discrete resonators for dynamic mode atomic force microscopy.

The quartz tuning fork based probe {e.g., Akiyama et al. [Appl. Surf. Sci. 210, 18 (2003)]}, termed "A-Probe," is a self-sensing and self-actuating (exciting) probe for dynamic mode atomic force microscope (AFM) operation. It is an oscillatory force sensor consisting of the two discrete resonators. This paper presents the investigations on an improved A-Probe: its batch fabrication and assembly, mounting on an AFM head, electrical setup, characterization, and AFM imaging. The fundamental features of the A-Probe are electrically and optically characterized in "approach-withdraw" experiments. Further investigations include the frequency response of an A-Probe to small mechanical vibrations externally applied to the tip and the effective loading force yielding between the tip and the sample during the periodic contact. Imaging of an electronic chip, a compact disk stamper, carbon nanotubes, and Si beads is demonstrated with this probe at ambient conditions in the so-called frequency modulation mode. A special probe substrate, which can snap on a receptacle fixed on an AFM head, and a special holder including a preamplifier electronic are introduced. We hope that the implementation and characterization of the A-Probe described in this paper will provide hints for new scanning probe techniques.

Sub-ppm detection of vapors using piezoresistive microcantilever array sensors.

The performance of microfabricated piezoresistive cantilever array sensors has been evaluated using various vapors of volatile organic compounds including alkanes with different chain length from 5 (n-pentane) to 14 (n-tetradecane). We demonstrate that piezoresistive microcantilever array sensors have the selectivity of discriminating individual alkanes in a homologous series as well as common volatile organic compounds according to principal component analysis. We developed a new method to evaluate the sensitivity, taking advantage of the low vapor pressures of alkanes with longer chains, such as n-dodecane, n-tridecane and n-tetradecane, under saturated vapor conditions. This method reveals sub-ppm sensitivity and the cantilever response is found to follow the mass of evaporated analytes as calculated using a quantitative model based on the Langmuir evaporation model.

Early detection of aging cartilage and osteoarthritis in mice and patient samples using atomic force microscopy.

The pathological changes in osteoarthritis--a degenerative joint disease prevalent among older people--start at the molecular scale and spread to the higher levels of the architecture of articular cartilage to cause progressive and irreversible structural and functional damage. At present, there are no treatments to cure or attenuate the degradation of cartilage. Early detection and the ability to monitor the progression of osteoarthritis are therefore important for developing effective therapies. Here, we show that indentation-type atomic force microscopy can monitor age-related morphological and biomechanical changes in the hips of normal and osteoarthritic mice. Early damage in the cartilage of osteoarthritic patients undergoing hip or knee replacements could similarly be detected using this method. Changes due to aging and osteoarthritis are clearly depicted at the nanometre scale well before morphological changes can be observed using current diagnostic methods. Indentation-type atomic force microscopy may potentially be developed into a minimally invasive arthroscopic tool to diagnose the early onset of osteoarthritis in situ.

The measurement of biomechanical properties of porcine articular cartilage using atomic force microscopy.

We have recently demonstrated that indentation-type atomic force microscopy (IT-AFM) is capable of detecting early onset osteoarthritis (OA) (Stolz, 2009). This study was based on biopsies, using a desk-top commercial atomic force microscope (AFM). However, cartilage analysis in the knee joints needs to be non-destructive to avoid new seeding points for OA by the taking of biopsies. This requires bringing the probe tip in contact with the articular cartilage (AC) surface inside the joint. Here we present our recent progress towards a medical instrument for performing such IT-AFM measurements for in-vivo knee diagnostics. The scanning force arthroscope (SFA) integrates a miniaturized AFM into a standard arthroscopic sleeve, and is used for direct, quantitative, in situ inspection of AC (Imer et al., 2006). The stabilization and the positioning of the instrument relative to the surface under investigation were performed by means of eight inflatable balloons. An integrated three-dimensional, piezoelectric scanner allowed raster scanning and probing of a small area of cartilage around the point of insertion. An AFM probe with an integrated deflection sensor was mounted at the distal end of the instrument. Using this instrument, several measurements were performed on agarose gel and on porcine cartilage samples. The load-displacement curves obtained were analyzed and the dynamic elastic moduli | E(*) | were calculated. A good correlation between these values and those published in the scientific literature was found. Therefore, we concluded that the SFA can provide quantitative measurements to detect early pathological changes in OA.

Label-free detection of single protein molecules and protein-protein interactions using synthetic nanopores.

Nanofabricated pores in 20 nm-thick silicon nitride membranes were used to probe various protein analytes as well as to perform an antigen-antibody binding assay. A two-compartment electrochemical cell was separated by a single nanopore, 28 nm in diameter. Adding proteins to one compartment caused current perturbations in the ion current flowing through the pore. These perturbations correlated with both the charge and the size of the protein or of a protein-protein complex. The potential of this nanotechnology for studying protein-protein interactions is highlighted with the sensitive detection of beta-human chorionic gonadotropin, a hormone and clinical biomarker of pregnancy, by monitoring in real time and at a molecular level the formation of a complex between hormones and antibodies in solution. In this form, the assay compared advantageously to immunoassays, with the important difference that labels, immobilization, or amplification steps were no longer needed. In conclusion, we present proof-of-principle that properties of proteins and their interactions can be investigated in solution using synthetic nanopores and that these interactions can be exploited to measure protein concentrations accurately.

Conductive supports for combined AFM-SECM on biological membranes.

Four different conductive supports are analysed regarding their suitability for combined atomic force and scanning electrochemical microscopy (AFM-SECM) on biological membranes. Highly oriented pyrolytic graphite (HOPG), MoS(2), template stripped gold, and template stripped platinum are compared as supports for high resolution imaging of reconstituted membrane proteins or native membranes, and as electrodes for transferring electrons from or to a redox molecule. We demonstrate that high resolution topographs of the bacterial outer membrane protein F can be recorded by contact mode AFM on all four supports. Electrochemical feedback experiments with conductive cantilevers that feature nanometre-scale electrodes showed fast re-oxidation of the redox couple Ru(NH(3))(6)(3+/2+) with the two metal supports after prolonged immersion in electrolyte. In contrast, the re-oxidation rates decayed quickly to unpractical levels with HOPG or MoS(2) under physiological conditions. On HOPG we observed heterogeneity in the re-oxidation rate of the redox molecules with higher feedback currents at step edges. The latter results demonstrate the capability of conductive cantilevers with small electrodes to measure minor variations in an SECM signal and to relate them to nanometre-scale features in a simultaneously recorded AFM topography. Rapid decay of re-oxidation rate and surface heterogeneity make HOPG or MoS(2) less attractive for combined AFM-SECM experiments on biological membranes than template stripped gold or platinum supports.

Characterization of microfabricated probes for combined atomic force and high-resolution scanning electrochemical microscopy.

A combined atomic force and scanning electrochemical microscope probe is presented. The probe is electrically insulated except at the very apex of the tip, which has a radius of curvature in the range of 10-15 nm. Steady-state cyclic voltammetry measurements for the reduction of Ru(NH3)6Cl3 and feedback experiments showed a distinct and reproducible response of the electrode. These experimental results agreed with finite element simulations for the corresponding diffusion process. Sequentially topographical and electrochemical studies of Pt lines deposited onto Si3N4 and spaced 100 nm apart (edge to edge) showed a lateral electrochemical resolution of 10 nm.

Filling kinetics of liquids in nanochannels as narrow as 27 nm by capillary force.

We report the filling kinetics of different liquids in nanofabricated capillaries with rectangular cross-section by capillary force. Three sets of channels with different geometry were employed for the experiments. The smallest dimension of the channel cross-section was respectively 27, 50, and 73 nm. Ethanol, isopropanol, water and binary mixtures of ethanol and water spontaneously filled nanochannels with inner walls exposing silanol groups. For all the liquids the position of the moving liquid meniscus was observed to be proportional to the square root of time, which is in accordance with the classical Washburn kinetics. The velocity of the meniscus decreased both with the dimension of the channel and the ratio between the surface tension and the viscosity. In the case of water, air-bubbles were spontaneously trapped as channels were filled. For a binary mixture of 40% ethanol and water, no trapping of air was observed anymore. The filling rate was higher than expected, which also corresponds to the dynamic contact angle for the mixture being lower than that of pure ethanol. Nanochannels and porous materials share many physicochemical properties, e.g., the comparable pores size and extremely high surface to volume ratio. These similarities suggest that our nanochannels could be used as an idealized model to study mass transport mechanisms in systems where surface phenomena dominate.

Design and fabrication of nanofluidic devices by surface micromachining.

Using surface micromachining technology, we fabricated nanofluidic devices with channels down to 10 nm deep, 200 nm wide and up to 8 cm long. We demonstrated that different materials, such as silicon nitride, polysilicon and silicon dioxide, combined with variations of the fabrication procedure, could be used to make channels both on silicon and glass substrates. Critical channel design parameters were also examined. With the channels as the basis, we integrated equivalent elements which are found on micro total analysis (µTAS) chips for electrokinetic separations. On-chip platinum electrodes enabled electrokinetic liquid actuation. Micro-moulded polydimethylsiloxane (PDMS) structures bonded to the devices served as liquid reservoirs for buffers and sample. Ionic conductance measurements showed Ohmic behaviour at ion concentrations above 10 mM, and surface charge governed ion transport below 5 mM. Low device to device conductance variation (1%) indicated excellent channel uniformity on the wafer level. As proof of concept, we demonstrated electrokinetic injections using an injection cross with volume below 50 attolitres (10(-18) l).

Optical properties of microfabricated fully-metal-coated near-field probes in collection mode.

A study of the optical properties of microfabricated, fully-metal-coated quartz probes collecting longitudinal and transverse optical fields is presented. The measurements are performed by raster scanning the focal plane of an objective, focusing azimuthally and radially polarized beams by use of two metal-coated quartz probes with different metal coatings. A quantitative estimation of the collection efficiencies and spatial resolutions in imaging both longitudinal and transverse fields is made. Longitudinally polarized fields are collected with a resolution approximately 1.5 times higher as compared with transversely polarized fields, and this behavior is almost independent of the roughness of the probe's metal coating. Moreover, the coating roughness is a critical parameter in the relative collection efficiency of the two field orientations.