A digitally driven manufacturing process for high resolution patterning of cell formations.

This paper presents the engineering and validation of an enabling technology that facilitates new capabilities in in vitro cell models for high-throughput screening and tissue engineering applications. This is conducted through a computerized system that allows the design and deposition of high-fidelity microscale patterned coatings that selectively alter the chemical and topographical properties of cell culturing surfaces. Significantly, compared to alternative methods for microscale surface patterning, this is a digitally controlled and automated process thereby allowing scientists to rapidly create and explore an almost infinite range of cell culture patterns. This new capability is experimentally validated across six different cell lines demonstrating how the precise microscale deposition of these patterned coatings can influence spatiotemporal growth and movement of endothelial, fibroblast, neuronal and macrophage cells. To further demonstrate this platform, more complex patterns are then created and shown to guide the behavioral response of colorectal carcinoma cells.

Optimization and fabrication of programmable domains for soft magnetic robots: A review.

Driven by the aim of realizing functional robotic systems at the milli- and submillimetre scale for biomedical applications, the area of magnetically driven soft devices has received significant recent attention. This has resulted in a new generation of magnetically controlled soft robots with patterns of embedded, programmable domains throughout their structures. This type of programmable magnetic profiling equips magnetic soft robots with shape programmable memory and can be achieved through the distribution of discrete domains (voxels) with variable magnetic densities and magnetization directions. This approach has produced highly compliant, and often bio-inspired structures that are well suited to biomedical applications at small scales, including microfluidic transport and shape-forming surgical catheters. However, to unlock the full potential of magnetic soft robots with improved designs and control, significant challenges remain in their compositional optimization and fabrication. This review considers recent advances and challenges in the interlinked optimization and fabrication aspects of programmable domains within magnetic soft robots. Through a combination of improvements in the computational capacity of novel optimization methods with advances in the resolution, material selection and automation of existing and novel fabrication methods, significant further developments in programmable magnetic soft robots may be realized.

Micro-scale aerosol jet printing of superparamagnetic Fe3O4 nanoparticle patterns.

The opportunity to create different patterns of magnetic nanoparticles on surfaces is highly desirable across many technological and biomedical applications. In this paper, this ability is demonstrated for the first time using a computer-controlled aerosol jet printing (AJP) technology. AJP is an emerging digitally driven, non-contact and mask-less printing process which has distinguishing advantages over other patterning technologies as it offers high-resolution and versatile direct-write deposition of a wide range of materials onto a variety of substrates. This research demonstrates the ability of AJP to reliably print large-area, fine-feature patterns of superparamagnetic iron oxide nanoparticles (SPIONs) onto both rigid material (glass) and soft and flexible materials (polydimethylsiloxane (PDMS) films and poly-L-lactic acid (PLLA) nanofilms). Investigation identified and controlled influential process variables which permitted feature sizes in the region of 20 µm to be realised. This method could be employed for a wide range of applications that require a flexible and responsive process that permits high yield and rapid patterning of magnetic material over large areas. As a first proof of concept, we present patterned magnetic nanofilms with enhanced manipulability under external magnetic field gradient control and which are capable of performing complex movements such as rotation and bending, with applicability to soft robotics and biomedical engineering applications.

Digitally Driven Aerosol Jet Printing to Enable Customisable Neuronal Guidance.

Digitally driven manufacturing technologies such as aerosol jet printing (AJP) can make a significant contribution to enabling new capabilities in the field of tissue engineering disease modeling and drug screening. AJP is an emerging non-contact and mask-less printing process which has distinct advantages over other patterning technologies as it offers versatile, high-resolution, direct-write deposition of a variety of materials on planar and non-planar surfaces. This research demonstrates the ability of AJP to print digitally controlled patterns that influence neuronal guidance. These consist of patterned poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) tracks on both glass and poly(potassium 3-sulfopropyl methacrylate) (PKSPMA) coated glass surfaces, promoting selective adhesion of SH-SY5Y neuroblastoma cells. The cell attractive patterns had a maximum height ≥0.2 µm, width and half height ≥15 µm, Ra = 3.5 nm, and RMS = 4.1. The developed biocompatible PEDOT:PSS ink was shown to promote adhesion, growth and differentiation of SH-SY5Y neuronal cells. SH-SY5Y cells cultured directly onto these features exhibited increased nuclei and neuronal alignment on both substrates. In addition, the cell adhesion to the substrate was selective when cultured onto the PKSPMA surfaces resulting in a highly organized neural pattern. This demonstrated the ability to rapidly and flexibly realize intricate and accurate cell patterns by a computer controlled process.

Toward the Use of Temporary Tattoo Electrodes for Impedancemetric Respiration Monitoring and Other Electrophysiological Recordings on Skin.

The development of dry, ultra-conformable and unperceivable temporary tattoo electrodes (TTEs), based on the ink-jet printing of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) on top of commercially available temporary tattoo paper, has gained increasing attention as a new and promising technology for electrophysiological recordings on skin. In this work, we present a TTEs epidermal sensor for real time monitoring of respiration through transthoracic impedance measurements, exploiting a new design, based on the application of soft screen printed Ag ink and magnetic interlink, that guarantees a repositionable, long-term stable and robust interconnection of TTEs with external "docking" devices. The efficiency of the TTE and the proposed interconnection strategy under stretching (up to 10%) and over time (up to 96 h) has been verified on a dedicated experimental setup and on humans, fulfilling the proposed specific application of transthoracic impedance measurements. The proposed approach makes this technology suitable for large-scale production and suitable not only for the specific use case presented, but also for real time monitoring of different bio-electric signals, as demonstrated through specific proof of concept demonstrators.

On the injectability of free-standing magnetic nanofilms.

Free-standing films with sub-micrometric thickness, composed of soft polymers and functional nanostructures are promising candidates for many potential applications in the biomedical field, such as reduced port abdominal surgery. In this work, freely suspended poly(L-lactic acid) nanofilms with controlled morphology embedding superparamagnetic iron oxide nanoparticles were fabricated by spin-coating deposition. The mechanical properties of magnetic nanofilms were investigated by Strain-Induced Elastic Buckling Instability for Mechanical Measurements (SIEBIMM) test. Our results show that these freely suspended nanocomposite nanofilms are highly flexible and deformable, with Young's moduli of few GPa. Since they can be handled in liquid with syringes, a quantitative description of the nanofilms behavior during the manipulation with clinically applicable needles has been also provided. These magnetic nanofilms, remotely controllable by external electromagnetic fields, have potential applications in minimally invasive surgery as injectable nanopatches on inner organs wall. Graphical abstract ᅟ.

Toward a new generation of electrically controllable hygromorphic soft actuators.

An innovative processing strategy for fabricating soft structures that possess electric- and humidity-driven active/passive actuation capabilities along with touch- and humidity-sensing properties is reported. The intrinsically multifunctional material comprises an active thin layer of poly(3,4-ethylenedioxythiophene):poly-(styrene sulfonate) in a double-layered structure with a silicone elastomer and provides an opportunity toward developing a new class of smart structures for soft robotics.

Patterned free-standing conductive nanofilms for ultraconformable circuits and smart interfaces.

A process is presented for the fabrication of patterned ultrathin free-standing conductive nanofilms based on an all-polymer bilayer structure composed of poly(3,4-ethylenedioxythiophene)/poly(styrene sulfonate) and poly(lactic acid) (PEDOT:PSS/PLA). Based on the strategy recently introduced by our group for producing large area free-standing nanofilms of conductive polymers with ultrahigh conformability, here an inkjet subtractive patterning technique was used, with localized overoxidation of PEDOT:PSS that caused the local irreversible loss of electrical conductivity. Different pattern geometries (e.g., interdigitated electrodes with various spacing, etc.) were tested for validating the proposed process. The fabrication of individually addressable microelectrodes and simple circuits on nanofilm having thickness ∼250 nm has been demonstrated. Using this strategy, mechanically robust, conformable ultrathin polymer films could be produced that can be released in water as free-standing nanofilms and/or collected on surfaces with arbitrary shapes, topography and compliance, including human skin. The patterned bilayer nanofilms were characterized as regards their morphology, thickness, topography, conductivity, and electrochemical behavior. In addition, the electrochemical switching of surface properties has been evaluated by means of contact angle measurements. These novel conductive materials can find use as ultrathin, conformable electronic devices and in many bioelectrical applications. Moreover, by exploiting the electrochemical properties of conducting polymers, they can act as responsive smart biointerfaces and in the field of conformable bioelectronics, for example, as electrodes on tissues or smart conductive substrates for cell culturing and stimulation.

Characterization of free-standing PEDOT:PSS/iron oxide nanoparticle composite thin films and application as conformable humidity sensors.

In this study, a new simple, fast, and inexpensive technique for the preparation of free-standing nanocomposite ultrathin films based on the conductive polymer poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) and embedding iron oxide nanoparticles (NPs) is presented. These nanofilms were fabricated by a single step of spin-coated assisted deposition in conjunction with a release technique ("supporting layer technique") to detach them from the substrate. Free-standing nanofilms can be easily transferred onto several substrates due to their high conformability, preserving their functionalities. The effect of the addition of iron oxide nanoparticles on the structural and functional properties of the PEDOT:PSS nanofilms is investigated through topography, thickness, magnetic, magneto-optical activity, and conductivity characterizations. PEDOT:PSS and PEDOT:PSS/iron oxide NP nanofilms were tested as resistive humidity sensors. Their sensitivity to humidity was found to increase with increasing nanoparticle concentration. On the basis of these results, it is expected that these composites may furnish inexpensive and reliable means for relative humidity detection.

Microwrinkled conducting polymer interface for anisotropic multicellular alignment.

Surfaces with controlled micro and nanoscale topographical cues are useful as smart scaffolds and biointerfaces for cell culture. Recently, use of thin-film and surface wrinkling is emerging as a rapid unconventional method for preparing topographically patterned surfaces, especially suited for the production of smart patterns over large area surfaces. On the other hand, there is an increasing interest in employing conducting polymers as soft, biocompatible, conductive biointerfaces or as parts of bioelectronic devices. A novel convenient and versatile method is presented for producing anisotropic topographical cues at the micro- and nanoscale on conducting polymer surfaces. Micro and nanowrinkles were formed during the heat-shrinking process of a thermo-retractable polystyrene substrate. Surface wrinkling was due to the mismatch between the mechanical properties of a conducting polymer ultrathin film and the substrate. Various geometries of wrinkled structures were prepared, demonstrating the tunability of topography depending on the thickness of the conductive film. A method for patterning the conductive properties of the wrinkled substrates was also presented. Such surfaces acted as smart scaffolds for the functional alignment of cells, envisioning their electrical stimulation. Cell adhesion and proliferation were evaluated, comparing different topographies, and a preferential anisotropic alignment of C2C12 murine skeletal muscle cells along wrinkles was demonstrated. The observed trends were also confirmed concerning the formation of aligned myotubes in C2C12 differentiation stage. Furthermore, improved results in terms of aligned and mature myotube formation were obtained by co-culturing C2C12 cells with a fibroblasts feeder layer. The combination of living cells and tunable conductive nanowrinkles will represent a unique tool for the development of innovative biomedical devices.

Quantification of growth and differentiation of C2C12 skeletal muscle cells on PSS-PAH-based polyelectrolyte layer-by-layer nanofilms.

Polyelectrolyte layer-by-layer (LbL) nanofilms are interesting polymeric structures, built by alternating adsorption of positively and negatively charged polyelectrolytes. They consist of multilayer sheets with nanometric overall thickness, and they can be used as supports and surface coatings for in vitro and in vivo cell and tissue growth and regeneration. The present study focuses on nanofilms based on alternated layers of poly(sodium-4-sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) fabricated using spin-assisted LbL assembly (SA-LbL). The fabrication process used to assemble polyelectrolyte nanofilms made of up to 60 bilayers is described, and the influence of different surface charges (i.e. changing the terminal layer) and of different film composition (e.g. varying PSS molecular weight) on cell behaviour is investigated. In particular, C2C12 skeletal muscle cells' viability, proliferation and differentiation on six different typologies of polyelectrolyte nanofilms are evaluated and quantified, giving a reference for skeletal muscle regeneration capabilities on such kind of structures.

Free-standing poly(L-lactic acid) nanofilms loaded with superparamagnetic nanoparticles.

Freely suspended nanocomposite thin films based on soft polymers and functional nanostructures have been widely investigated for their potential application as active elements in microdevices. However, most studies are focused on the preparation of nanofilms composed of polyelectrolytes and charged colloidal particles. Here, a new technique for the preparation of poly(l-lactic acid) free-standing nanofilms embeddidng superparamagnetic iron oxide nanoparticles is presented. The fabrication process, based on a spin-coating deposition approach, is described, and the influence of each production parameter on the morphology and magnetic properties of the final structure is investigated. Superparamagnetic free-standing nanofilms were obtained, as evidenced by a magnetization hysteresis measurement performed with a superconducting quantum interference device (SQUID). Nanofilm surface morphology and thickness were evaluated by atomic force microscopy (AFM), and the nanoparticle dispersion inside the composites was investigated by transmission electron microscopy (TEM). These nanofilms, composed of a biodegradable polyester and remotely controllable by external magnetic fields, are promising candidates for many potential applications in the biomedical field.

Flexible polymeric ultrathin film for mesenchymal stem cell differentiation.

Ultrathin films (also called nanofilms) are two-dimensional (2-D) polymeric structures with potential application in biology, biotechnology, cosmetics and tissue engineering. Since they can be handled in liquid form with micropipettes or tweezers they have been proposed as flexible systems for cell adhesion and proliferation. In particular, with the aim of designing a novel patch for bone or tendon repair and healing, in this work the biocompatibility, adhesion and proliferation activity of Saos-2, MRC-5 and human and rat mesenchymal stem cells on poly(lactic acid) nanofilms were evaluated. The nanofilms did not impair the growth and differentiation of osteoblasts and chondrocytes. Moreover, nanofilm adhesion to rabbit joints was evident under ex vivo conditions.

Adhesion and proliferation of skeletal muscle cells on single layer poly(lactic acid) ultra-thin films.

An increasing interest in bio-hybrid systems and cell-material interactions is evident in the last years. This leads towards the development of new nano-structured devices and the assessment of their biocompatibility. In the present study, the development of free-standing single layer poly(lactic acid) (PLA) ultra-thin films is described, together with the analysis of topography and roughness properties. The biocompatibility of the PLA films has been tested in vitro, by seeding C2C12 skeletal muscle cells, and thus assessing cells shape, density and viability after 24, 48 and 72 h. The results show that free-standing flexible PLA nanofilms represent a good matrix for C2C12 cells adhesion, spreading and proliferation. Early differentiation into myotubes is also allowed. The biocompatibility of the novel ultra-thin films as substrates for cell growth promotes their application in the fields of regenerative medicine, muscle tissue engineering, drug delivery, and-in general-in the field of bio-hybrid devices.