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 meso‑scale. 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.

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Climate and justice are interconnected. However, simply raising ethical issues associated with the links between climate change, technology, and health is insufficient. Rather, policies and practices need to consider ethics ahead of time. If it is only added “after the fact,” policy will be less efficient and opportunities for carbon minimization will be lost. This will require the cooperation of people at many levels and can be guided by two essential ethical principles: distributive justice and environmental sustainability.@en

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).

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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.@en

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.

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Optical micromachines have the potential to improve the capabilities of optical tweezers by amplifying forces and allowing for indirect handling and probing of specimens. However, systematic design and testing of micromachine performance is still an emerging field. In this work we have designed and tested an unsupported microlever, suitable for general-purpose optical tweezer studies, that demonstrates stable trapping performance and repeatable doubling of applied forces. Stable trapping was ensured by analysing images to monitor focus shift when levers oscillated repeatedly, before the best-performing design was selected for force amplification. This study also shows that direct measurement of trap stiffness using the equipartition theorem appears to be a valid method for measuring applied forces on the spherical handles of microlevers.@en

Detected kidney stone cases are increasing globally, yet knowledge on the conditions for stone formation is lacking. Experimental approaches mimicking the micro-environmental conditions present in vivo can help sci-entists untangle intertwined physiochemical and biological phenomena leading to kidney stone formation. As crystal nucleation often initiates at liquid-solid interfaces, the interface morphology plays a significant role in the rate of nucleation. Within the nephron, the functional unit of the kidney, four segments can be distinguished that contain different surface morphologies. Particularly, the cells lining these segments contain protrusions in the shape of nanopillars that vary in length, diameter and spacing. Exploiting the opportunities provided by organ- on-a-chip technology, we designed and manufactured a proof-of-principle microfluidic device proposed to in-crease our understanding of the relation between kidney surface morphology and kidney stone crystallization. We used two-photon polymerization to fabricate biocompatible surfaces that mimic the nephron morphologies with materials properties similar to those of biological structures. The fabricated cilia were incorporated in the microfluidic device, which was designed to observe in vitro crystallization of calcium oxalate under flow@en

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.

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There is a high demand for novel flexible micro-devices for energy harvesting from low-frequency and random mechanical sources. The research of new functional designs is required to strategically enhance the performances and to increase the control on mechanical flexibility. In this work we report the fabrication and characterization of bi-stable and statically balanced thin-film piezoelectric transducers based on Aluminum Nitride (AlN). The device consists of a piezoelectric layer sandwiched between two thin Molybdenum electrodes that were deposited on a Kapton substrate by reactive sputtering and patterned by UV lithography. In order to improve the out-of-plane flexibility, the mechanical design is distinguished by a post-buckled flexure that introduces a negative stiffness to compensate the otherwise positive stiffness of the system. The buckling was introduced by a new method, called Package-Induced Preloading (PIP) where the mechanisms are laminated over a package with a geometry extending out-of-plane. The induced buckling resulted in bi-stable and statically balanced mechanisms which demonstrated an enhanced voltage output during a triggered snapping step. A preliminary study shows potential for the statically balanced designs and the PIP method for wind energy harvesting, revealing prospective applications and future improvements for the development of energy harvesters.

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Increasing the signal-to-noise ratio in dynamic atomic force microscopy plays a key role in nanomechanical mapping of materials with atomic resolution. In this work, we develop an experimental procedure for increasing the sensitivity of higher harmonics of an atomic-force-microscope cantilever without modifying the cantilever geometry but instead by utilizing dynamical mode coupling between its flexural modes of vibration. We perform experiments on different cantilevers and samples and observe that via nonlinear resonance frequency tuning we can obtain a frequency range where strong modal interactions lead to 7-fold and 16-fold increases in the sensitivity of the 6th and 17th harmonics while reducing sample indentation. We derive a numerical model that captures the observed physics and confirms that nonlinear mode coupling is the reason for the increase of the amplitude of higher harmonics during tip-sample interactions.

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Suspended microfluidic resonators enable detection of fluid density and viscosity with high sensitivity. Here, a two-legged suspended microchannel resonator that probes pico-litres of liquid is presented. The higher resonant modes (flexural and torsional) were explored for increased sensitivity and resolution. Unlike other reported microchannel resonators, this device showed an increase in the quality factor with resonant frequency value. The performance of the resonator was tested by filling the channel with three liquids, one at a time, over a density range of 779 - 1110 Kg/m^3 and a viscosity range of 0.89 - 16.2 mPa.s. The highest resolution obtained was 0.011% change in density. Measurements with torsional mode showed an improvement of about six times in sensitivity and about fifteen times in resolution compared to the first flexural mode. When the empty channel was filled with liquids of different viscosity, the quality factor of the first flexural mode remained overall constant with a variation below 3.3% between the fluids, and confirming the inherent property of suspended microchannel resonators. However, it significantly decreased for second flexural and torsional modes. No noticeable difference was observed in the quality factor between different liquid viscosities for all modes.@en

Advances in nanofabrication over the past twenty years have enabled the creation and use of ever-more interesting and useful micromachines. Optical micromachines are a particularly attractive subset of these for researchers in biological and soft-matter sciences, due to their potential to aid in optical tweezer studies of laser-sensitive samples. However, the development of multi-component micromachines is made difficult due to the dominance of surface forces at this scale, which is made all the more relevant in the high-salt concentrations used for biological studies. This study concerns the design of simple, first-class lever micromachines for use in environments with different salt concentrations, in an attempt to provide a guideline for design requirements of functional optical micromachines for use in physiological conditions.

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Fabricating large areas of geometrically complex and precisely controlled topographies is required for the studies of cell behavior on patterned surfaces. Direct laser writing (DLW) is an advanced 3D-fabrication technique, which facilitates the manufacturing of structures within various scales (from a few hundred nanometers to millimeters). However, this method requires improvements in the accuracy and reproducibility of the submicron and nanoscale features that are printed over a large area. Here, we present a scheme to both improve the uniformity of the printed submicron patterns and decrease the printing time. The effects of various processing parameters (e.g., laser power and writing field) on the dimensions and uniformity of submicron pillars as well as on their Young’s modulus and surface wettability were assessed. Decreasing the writing field to 33 × 33 μm2 significantly improved the uniformity of submicron pillars that were printed over an area of 4 mm2 in a single-step process. Preosteoblast cells (MC3T3-E1) were used to assess the cytocompatibility of the used material (IP-L780 resin) with a focus on cell morphology, cell proliferation, cytoskeletal organization, and the elastic modulus of the cells. The cells cultured for 2 days on the submicron pillars showed a polarized shape and a higher Young’s modulus of the area corresponding to the nucleus relative to those cultured on flat surfaces. Taken together, the results of the current study clearly show that the submicron patterns created using DLW are both cytocompatible and could modulate the morphology and mechanical properties of cells. This work paves the way for direct printing of submicron features with controlled Young’s moduli over large areas in a single-step process, which is necessary for systematically studying how such patterns modulate cellular functions.@en

We introduce a two-channel microfluidic atomic force microscopy (AFM) cantilever that combines the nanomechanical sensing functionality of an AFM cantilever with the ability to manipulate fluids of picolitres or smaller volumes through nanoscale apertures near the cantilever tip. Each channel is connected to a separate fluid reservoir, which can be independently controlled by pressure. Various systematic experiments with fluorescent liquids were done by either injecting the liquids from the on-chip reservoir or aspirating directly through the nanoscale apertures at the tip. A flow rate analysis of volume dosing, aspiration and concentration dosing inside the liquid medium was performed. To understand the fluid behaviour, an analytical model based on the hydrodynamic resistance, as well as numerical flow simulations of single and multi-phase conditions were performed and compared. By applying pressures between -500 mbar and 500 mbar to the reservoirs of the probe with respect to the ambient pressure, flow rates ranging from 10 fl s-1 to 83 pl s-1 were obtained inside the channels of the cantilever as predicted by the analytical model. The smallest dosing flow rate through the apertures was 720 fl s-1, which was obtained with a 10 mbar pressure on one reservoir and ambient pressure on the other. The solute concentration in the outflow could be tuned to values between 0% and 100% by pure convection and to values between 17.5% and 90% in combination with diffusion. The results prove that this new probe enables handling multiple fluids with the scope to inject different concentrations of analytes inside a single living cell and also perform regular AFM functionalities.

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Organ-on-chip (OoC) technology is increasingly used for biomedical research and to speed up the process of bringing a drug from lab to the market. The main fluidic components of an OoC device are microfluidic channels and porous membranes arranged in three dimensions. Current chips are often assembled from several parts. In the development phase a small change in design will cause a delay in the research because a new prototype has to be built and assembled again step-by-step. The research discussed in this paper addresses this point by targeting a monolithic 3D device that can be fabricated in a single lithography and development step, enabling rapid prototyping. Two-photon lithography (TPL) was used in combination with a positive photoresist AZ 4562. The exposure process was characterized, which included an experimental and theoretical study of the voxel size and shape. It was found that the voxel has an hourglass-shape for the laser power settings that were required for process stability. The smallest pores we could produce with these settings measured 250 nm in diameter. The TPL process was then used to fabricate a microfluidic device featuring two crossed channels each one on a separate height-level, connected by a membrane in the centre. Access to the channels was provided through 4 reservoirs from the top-side of the device. The device was successfully filled with water and dried to see whether it can withstand the corresponding capillary forces.

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In this work, we perform a comprehensive analysis of the robustness of attractors in tapping mode atomic force microscopy. The numerical model is based on cantilever dynamics driven in the Lennard–Jones potential. Pseudo-arc-length continuation and basins of attraction are utilized to obtain the frequency response and dynamical integrity of the attractors. The global bifurcation and response scenario maps for the system are developed by incorporating several local bifurcation loci in the excitation parameter space. Moreover, the map delineates various escape thresholds for different attractors present in the system. Our work unveils the properties of the cantilever oscillation in proximity to the sample surface, which is governed by the so-called in-contact attractor. The robustness of this attractor against operating parameters is quantified by means of integrity profiles. Our work provides a unique view into global dynamics in tapping mode atomic force microscopy and helps establishing an extended topological view of the system.

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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.

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This work aims at developing a new and unconventional Sacrificial Stencil Mask (SSM) technology by exploiting Two-Photon Polymerization (2PP) in an IP-L/SU-8 double layer resist system. The process consists of the sequential deposition of two different resists, such as SU-8 and IPL, onto the same glass substrate, followed by 2PP lithography and distinct development processes. The 2PP writing process was used to polymerize structures inside the top and bottom resist layers to form, in one single exposure process, both SSM and a permanent polymeric structure, in our case a plain pedestal. The top IPL resist was developed using Isopropyl Alcohol (IPA), which does not affect either exposed or un-exposed SU-8 regions. In this way, structures written into the bottom layer remained latent, while exposed areas of the top IPL resist, including the stencil mask, were developed. The realization of 3D stencil masks, designed to be anchored inside the un-exposed bottom layer, was combined with metal evaporation to demonstrate the deposition of a plain metal line through the stencil mask. The final development of the bottom layer led to the lift off of the sacrificial stencil mask, uncovering the underlying, permanent polymer-metal structure. The combination of sacrificial polymer structures with permanent ones opens new possibilities in 3D MEMS design, enabling the integration of distributed electronic transducers in flexible polymeric structures.

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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.@en

We report on an electrical detection method of ultrasensitive carbon nanotube mechanical resonators. The noise floor of the detection method is reduced using a RLC resonator and an amplifier based on a high electron mobility transistor cooled at 4.2 K. This allows us to resolve the resonance frequency of nanotube resonators with an unprecedented quality. We show that the noise of the resonance frequency measured at 4.2 K is limited by the resonator itself, and not by the imprecision of the measurement. The Allan deviation reaches ∼10^-5 at 125 ms integration time. When comparing the integration time dependence of the Allan deviation to a power law, the exponent approaches ∼1/4. The Allan deviation might be limited by the diffusion of particles over the surface of the nanotube. Our work holds promise for mass spectrometry and surface science experiments based on mechanical nano-resonators.

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