Designing Biomimetic Conductive Gelatin-Chitosan-Carbon Black Nanocomposite Hydrogels for Tissue Engineering.

Conductive nanocomposites play a significant role in tissue engineering by providing a platform to support cell growth, tissue regeneration, and electrical stimulation. In the present study, a set of electroconductive nanocomposite hydrogels based on gelatin (G), chitosan (CH), and conductive carbon black (CB) was synthesized with the aim of developing novel biomaterials for tissue regeneration application. The incorporation of conductive carbon black (10, 15 and 20 wt.%) significantly improved electrical conductivity and enhanced mechanical properties with the increased CB content. We employed an oversimplified unidirectional freezing technique to impart anisotropic morphology with interconnected porous architecture. An investigation into whether any anisotropic morphology affects the mechanical properties of hydrogel was conducted by performing compression and cyclic compression tests in each direction parallel and perpendicular to macroporous channels. Interestingly, the nanocomposite with 10% CB produced both anisotropic morphology and mechanical properties, whereas anisotropic pore morphology diminished at higher CB concentrations (15 and 20%), imparting a denser texture. Collectively, the nanocomposite hydrogels showed great structural stability as well as good mechanical stability and reversibility. Under repeated compressive cyclic at 50% deformation, the nanocomposite hydrogels showed preconditioning, characteristic hysteresis, nonlinear elasticity, and toughness. Overall, the collective mechanical behavior resembled the mechanics of soft tissues. The electrical impedance associated with the hydrogels was studied in terms of the magnitude and phase angle in dry and wet conditions. The electrical properties of the nanocomposite hydrogels conducted in wet conditions, which is more physiologically relevant, showed a decreasing magnitude with increased CB concentrations, with a resistive-like behavior in the range 1 kHz-1 MHz and a capacitive-like behavior for frequencies <1 kHz and >1 MHz. Overall, the impedance of the nanocomposite hydrogels decreased with increased CB concentrations. Together, these nanocomposite hydrogels are compositionally, morphologically, mechanically, and electrically similar to native ECMs of many tissues. These gelatin-chitosan-carbon black nanocomposite hydrogels show great promise for use as conducting substrates for the growth of electro-responsive cells in tissue engineering.

Smart Brace for Static and Dynamic Knee Laxity Measurement.

Every year in Europe more than 500 thousand injuries that involve the anterior cruciate ligament (ACL) are diagnosed. The ACL is one of the main restraints within the human knee, focused on stabilizing the joint and controlling the relative movement between the tibia and femur under mechanical stress (i.e., laxity). Ligament laxity measurement is clinically valuable for diagnosing ACL injury and comparing possible outcomes of surgical procedures. In general, knee laxity assessment is manually performed and provides information to clinicians which is mainly subjective. Only recently quantitative assessment of knee laxity through instrumental approaches has been introduced and become a fundamental asset in clinical practice. However, the current solutions provide only partial information about either static or dynamic laxity. To support a multiparametric approach using a single device, an innovative smart knee brace for knee laxity evaluation was developed. Equipped with stretchable strain sensors and inertial measurement units (IMUs), the wearable system was designed to provide quantitative information concerning the drawer, Lachman, and pivot shift tests. We specifically characterized IMUs by using a reference sensor. Applying the Bland-Altman method, the limit of agreement was found to be less than 0.06 m/s2 for the accelerometer, 0.06 rad/s for the gyroscope and 0.08 µT for the magnetometer. By using an appropriate characterizing setup, the average gauge factor of the three strain sensors was 2.169. Finally, we realized a pilot study to compare the outcomes with a marker-based optoelectronic stereophotogrammetric system to verify the validity of the designed system. The preliminary findings for the capability of the system to discriminate possible ACL lesions are encouraging; in fact, the smart brace could be an effective support for an objective and quantitative diagnosis of ACL tear by supporting the simultaneous assessment of both rotational and translational laxity. To obtain reliable information about the real effectiveness of the system, further clinical validation is necessary.

3D Electrochemical Sensor and Microstructuration Using Aerosol Jet Printing.

Electrochemical sensors are attracting great interest for their different applications. To improve their performances, basic research focuses on two main issues: improve their metrological characteristics (e.g., repeatability, reusability and sensitivity) and investigate innovative fabrication processes. In this work, we demonstrate an innovative microstructuration technique aimed at increasing electrochemical sensor sensitivity to improve electrode active area by an innovative fabrication technique. The process is empowered by aerosol jet printing (AJP), an additive-manufacturing and non-contact printing technique that allows depositing functional inks in precise patterns such as parallel lines and grids. The 3D printed microstructures increased the active surface area by up to 130% without changing the substrate occupancy. Further, electrochemical detection of ferro/ferri-cyanide was used to evaluate the sensitivity of the electrodes. This evaluation points out a sensitivity increase of 2.3-fold on average between bare and fully microstructured devices. The increase of surface area and sensitivity are well linearly correlated as expected, verifying the fitness of our production process. The proposed microstructuration is a viable solution for many applications that requires high sensitivity, and the proposed technique, since it does not require masks or complex procedures, turns out to be flexible and applicable to infinite construction geometries.

Printed Electrochemical Biosensors: Opportunities and Metrological Challenges.

Printed electrochemical biosensors have recently gained increasing relevance in fields ranging from basic research to home-based point-of-care. Thus, they represent a unique opportunity to enable low-cost, fast, non-invasive and/or continuous monitoring of cells and biomolecules, exploiting their electrical properties. Printing technologies represent powerful tools to combine simpler and more customizable fabrication of biosensors with high resolution, miniaturization and integration with more complex microfluidic and electronics systems. The metrological aspects of those biosensors, such as sensitivity, repeatability and stability, represent very challenging aspects that are required for the assessment of the sensor itself. This review provides an overview of the opportunities of printed electrochemical biosensors in terms of transducing principles, metrological characteristics and the enlargement of the application field. A critical discussion on metrological challenges is then provided, deepening our understanding of the most promising trends in order to overcome them: printed nanostructures to improve the limit of detection, sensitivity and repeatability; printing strategies to improve organic biosensor integration in biological environments; emerging printing methods for non-conventional substrates; microfluidic dispensing to improve repeatability. Finally, an up-to-date analysis of the most recent examples of printed electrochemical biosensors for the main classes of target analytes (live cells, nucleic acids, proteins, metabolites and electrolytes) is reported.

Design of multichannel potentiostat for remote and longtime monitoring of glucose concentration during yeast fermentation.

This paper presents the design of a wireless portable and multichannel potentiostat for remote monitoring in enclosed environments for long-time applications. In this paper, the proposed potentiostat is tested for monitoring the glucose concentration during the fermentation of yeast in real time for more than 24 h. The potentiostat is powered by a USB-connected battery and operated through a Bluetooth using a LabVIEW designed data monitoring and control panel. The potentiostat is capable of performing cyclic voltammetry or chronoamperometry on six biosensors simultaneously and gives the real-time response using Bluetooth connection. The potentiostat has a common counter electrode and reference electrode connection to all biosensors and independent working electrodes for all biosensors. The potentiostat was tested and validated by comparing the results obtained by a commercial potentiostat. The tests performed for monitoring the glucose concentration during the fermentation process showed a current detection limit of 180 nA and reported a standard deviation of ±2% for anodic and cathodic current peaks for cyclic voltammetry measurements when compared with the commercially available device. This study enables the novel method of monitoring the fermentation process wirelessly for days.

Impedance-Based Monitoring of Mesenchymal Stromal Cell Three-Dimensional Proliferation Using Aerosol Jet Printed Sensors: A Tissue Engineering Application.

One of the main hurdles to improving scaffolds for regenerative medicine is the development of non-invasive methods to monitor cell proliferation within three-dimensional environments. Recently, an electrical impedance-based approach has been identified as promising for three-dimensional proliferation assays. A low-cost impedance-based solution, easily integrable with multi-well plates, is here presented. Sensors were developed using biocompatible carbon-based ink on foldable polyimide substrates by means of a novel aerosol jet printing technique. The setup was tested to monitor the proliferation of human mesenchymal stromal cells into previously validated gelatin-chitosan hybrid hydrogel scaffolds. Reliability of the methodology was assessed comparing variations of the electrical impedance parameters with the outcomes of enzymatic proliferation assay. Results obtained showed a magnitude increase and a phase angle decrease at 4 kHz (maximum of 2.5 kΩ and -9 degrees) and an exponential increase of the modeled resistance and capacitance components due to the cell proliferation (maximum of 1.5 kΩ and 200 nF). A statistically significant relationship with enzymatic assay outcomes could be detected for both phase angle and electric model parameters. Overall, these findings support the potentiality of this non-invasive approach for continuous monitoring of scaffold-based cultures, being also promising in the perspective of optimizing the scaffold-culture system.

Printed Smart Devices on Cellulose-Based Materials by means of Aerosol-Jet Printing and Photonic Curing.

Printed electronics is an expanding research field that can reach the goal of reducing the environmental impact on electronics exploiting renewable and biodegradable materials, like paper. In our work, we designed and tested a new method for fabricating hybrid smart devices on cellulose substrates by aerosol jet printing (AJP) and photonic curing, also known as flash lamp annealing (FLA), capable to cure low temperature materials without any damage. Three different cellulose-based materials (chromatographic paper, photopaper, cardboard) were tested. Multilayer capability and SMDs (surface mount devices) interconnections are possible permitting high flexibility in the fabrication process. Electrical and geometrical tests were performed to analyze the behavior of printed samples. Resulted resistivities are 26.3 × 10-8 m on chromatographic paper, 22.3 × 10-8 m on photopaper and 13.1 × 10-8 m on cardboard. Profilometer and optical microscope evaluations were performed to state deposition quality and penetration of the ink in cellulose materials (thicknesses equal to 24.9, 28.5, and 51 µm respectively for chromatographic paper, photopaper, and cardboard). Furthermore, bending (only chromatographic paper did not reach the break-up) and damp environment tests (no significant variations in resistance) where performed. A final prototype of a complete functioning multilayer smart devices on cellulose 3D-substrate is shown, characterized by multilayers, capacitive sensors, SMDs interconnections.

Electrochemical detection of different p53 conformations by using nanostructured surfaces.

Protein electrochemistry represents a powerful technique for investigating the function and structure of proteins. Currently available biochemical assays provide limited information related to the conformational state of proteins and high costs. This work provides novel insights into the electrochemical investigation of the metalloprotein p53 and its redox products using label-free direct electrochemistry and label-based antibody-specific approaches. First, the redox activities of different p53 redox products were qualitatively investigated on carbon-based electrodes. Then, focusing on the open p53 isoform (denatured p53), a quantitative analysis was performed, comparing the performances of different bulk and nanostructured materials (carbon and platinum). Overall, four different p53 products could be successfully discriminated, from wild type to denatured. Label-free analysis suggested a single electron exchange with electron transfer rate constants on the order of 1 s-1. Label-based analysis showed decreasing affinity of pAb240 towards denatured, oxidized and nitrated p53. Furthermore, platinum nanostructured electrodes showed the highest enhancement of the limit of detection in the quantitative analysis (100 ng/ml). Overall, the obtained results represent a first step towards the implementation of highly requested complex integrated devices for clinical practices, with the aim to go beyond simple protein quantification.

Printed Strain Gauge on 3D and Low-Melting Point Plastic Surface by Aerosol Jet Printing and Photonic Curing.

Printing sensors and electronics directly on the objects is very attractive for producing smart devices, but it is still a challenge. Indeed, in some applications, the substrate that supports the printed electronics could be non-planar or the thermal curing of the functional inks could damage temperature-sensitive substrates such as plastics, fabric or paper. In this paper, we propose a new method for manufacturing silver-based strain sensors with arbitrary and custom geometries directly on plastic objects with curvilinear surfaces: (1) the silver lines are deposited by aerosol jet printing, which can print on non-planar or 3D surfaces; (2) photonic sintering quickly cures the deposited layer, avoiding the overheating of the substrate. To validate the manufacturing process, we printed strain gauges with conventional geometry on polyvinyl chloride (PVC) conduits. The entire manufacturing process, included sensor wiring and optional encapsulation, is performed at room temperature, compatible with the plastic surface. At the end of the process, the measured thickness of the printed sensor was 8.72 µm on average, the volume resistivity was evaluated 40 µΩ∙cm, and the thermal coefficient resistance was measured 0.150 %/°C. The average resistance was (71 ± 7) Ω and the gauge factor was found to be 2.42 on average.

3D gelatin-chitosan hybrid hydrogels combined with human platelet lysate highly support human mesenchymal stem cell proliferation and osteogenic differentiation.

Bone marrow and adipose tissue human mesenchymal stem cells were seeded in highly performing 3D gelatin-chitosan hybrid hydrogels of varying chitosan content in the presence of human platelet lysate and evaluated for their proliferation and osteogenic differentiation. Both bone marrow and adipose tissue human mesenchymal stem cells in gelatin-chitosan hybrid hydrogel 1 (chitosan content 8.1%) or gelatin-chitosan hybrid hydrogel 2 (chitosan 14.9%) showed high levels of viability (80%-90%), and their proliferation and osteogenic differentiation was significantly higher with human platelet lysate compared to fetal bovine serum, particularly in gelatin-chitosan hybrid hydrogel 1. Mineralization was detected early, after 21 days of culture, when human platelet lysate was used in the presence of osteogenic stimuli. Proteomic characterization of human platelet lysate highlighted 59 proteins mainly involved in functions related to cell adhesion, cellular repairing mechanisms, and regulation of cell differentiation. In conclusion, the combination of our gelatin-chitosan hybrid hydrogels with hPL represents a promising strategy for bone regenerative medicine using human mesenchymal stem cells.

A New Method for In Vivo Analysis of the Performances of a Heat and Moisture Exchanger (HME) in Mechanically Ventilated Patients.

AIM: To evaluate the conditioning capabilities of the DAR™ Hygrobac™ S, a Heat and Moisture Exchanger (HME), using a new device to measure the temperature (T) and the absolute humidity (AH) of the ventilated gases in vivo during mechanical ventilation in Intensive Care Unit (ICU) patients. MATERIALS AND METHODS: In 49 mechanically ventilated ICU patients, we evaluated T and AH, indicating the HME efficacy, during the inspiratory phase upstream and downstream the HME and the ratio of inspired AH to expired AH and the difference between expired T and inspired T indicated the HME efficiency. Efficacy and efficiency were assessed at three time points: at baseline (t0, HME positioning time), at 12 hours (t1), and at 24 hours (t2) using a dedicated, ad hoc built wireless device. Differences over time were evaluated using one-way ANOVA for repeated measures, whereas differences between in vivo and laboratory values (declared by the manufacturer according to UNI® EN ISO 9360 international standard) were evaluated using one-sample Student t-test. RESULTS: 49 HMEs were analysed in vivo during mechanical ventilation. T and AH means (SD) of the inspired gas (the efficacy) were 31.5°C (1.54) and 32.3 mg/l (2.60) at t0, 31.1°C (1.34) and 31.7 mg/l (2.26) at t1, and 31°C (1.29) and 31.4 mg/l (2.27) at t2. Both efficiency parameters were constant over time (inspired AH/expired AH=89%, p=0.24; and expired T-inspired T = 2.2°C, p=0.81). Compared with laboratory values, in vivo T and AH indicating efficacy were significantly lower (p<0.01), whereas the efficiency was significantly higher (p<0.01). CONCLUSIONS: HME performances can be accurately assessed for prolonged periods in vivo during routine mechanical ventilation in ICU patients. Temperature and absolute humidity of ventilated gases in vivo were maintained within the expected range and remained stable over time. HME efficacy and efficiency in vivo significantly differed from laboratory values.

Support-Material-Free Microfluidics on an Electrochemical Sensors Platform by Aerosol Jet Printing.

Printed electronics have led to new possibilities in the detection and quantification of a wide range of molecules important for medical, biotechnological, and environmental fields. The integration with microfluidics is often adopted to avoid hand-deposition of little volumes of reagents and samples on miniaturized electrodes that strongly depend on operator's skills. Here we report design, fabrication and test of an easy-to-use electrochemical sensor platform with microfluidics entirely realized with Aerosol Jet Printing (AJP). We printed a six-electrochemical-sensors platform with AJP and we explored the possibility to aerosol jet print directly on it a microfluidic structure without any support material. Thus, the sacrificial material removal and/or the assembly with sensors steps are avoided. The repeatability observed when printing both conductive and ultraviolet (UV)-curable polymer inks can be supported from the values of relative standard deviation of maximum 5% for thickness and 9% for line width. We designed the whole microfluidic platform to make the sample deposition (20 µL) independent from the operator. To validate the platform, we quantified glucose at different concentrations using a standard enzyme-mediated procedure. Both mediator and enzyme were directly aerosol jet printed on working electrodes (WEs), thus the proposed platform is entirely fabricated by AJP and ready to use. The chronoamperometric tests show limit of detection (LOD) = 2.4 mM and sensitivity = 2.2 ± 0.08 µA/mM confirming the effectiveness of mediator and enzyme directly aerosol jet printed to provide sensing in a clinically relevant range (3-10 mM). The average relative standard inter-platform deviation is about 8%. AJP technique can be used for fabricating a ready-to-use microfluidic device that does not need further processing after fabrication, but is promptly available for electrochemical sample analysis.

Aerosol Jet Printed 3D Electrochemical Sensors for Protein Detection.

The use of electrochemical sensors for the analysis of biological samples is nowadays widespread and highly demanded from diagnostic and pharmaceutical research, but the reliability and repeatability still remain debated issues. In the expanding field of printed electronics, Aerosol Jet Printing (AJP) appears promising to bring an improvement in resolution, miniaturization, and flexibility. In this paper, the use of AJP is proposed to design and fabricate customized electrochemical sensors in term of geometry, materials and 3D liquid sample confinement, reducing variability in the functionalization process. After an analysis of geometrical, electrical and surface features, the optimal layout has been selected. An electrochemical test has been then performed quantifying Interleukin-8, selected as reference protein, by means of Anodic Stripping Voltammetry. AJP sensors have been compared with standard screen-printed electrodes in terms of current density and relative standard deviation. Results from AJP sensors with Ag-based Anodic Stripping Voltammetry confirmed nanostructures capability to reduce the limit of detection (from 2.1 to 0.3 ng/mL). Furthermore, AJP appeared to bring an improvement in term of relative standard deviation from 50 to 10%, if compared to screen-printed sensors. This is promising to improve reliability and repeatability of measurement techniques integrable in several biotechnological applications.

Passive and self-powered autonomous sensors for remote measurements.

Autonomous sensors play a very important role in the environmental, structural, and medical fields. The use of this kind of systems can be expanded for several applications, for example in implantable devices inside the human body where it is impossible to use wires. Furthermore, they enable measurements in harsh or hermetic environments, such as under extreme heat, cold, humidity or corrosive conditions. The use of batteries as a power supply for these devices represents one solution, but the size, and sometimes the cost and unwanted maintenance burdens of replacement are important drawbacks. In this paper passive and self-powered autonomous sensors for harsh or hermetical environments without batteries are discussed. Their general architectures are presented. Sensing strategies, communication techniques and power management are analyzed. Then, general building blocks of an autonomous sensor are presented and the design guidelines that such a system must follow are given. Furthermore, this paper reports different proposed applications of autonomous sensors applied in harsh or hermetic environments: two examples of passive autonomous sensors that use telemetric communication are proposed, the first one for humidity measurements and the second for high temperatures. Other examples of self-powered autonomous sensors that use a power harvesting system from electromagnetic fields are proposed for temperature measurements and for airflow speeds.