Adaptable test bench for ASTM-compliant permeability measurement of porous scaffolds for tissue engineering.

Intrinsic permeability describes the ability of a porous medium to be penetrated by a fluid. Considering porous scaffolds for tissue engineering (TE) applications, this macroscopic variable can strongly influence the transport of oxygen and nutrients, the cell seeding process, and the transmission of fluid forces to the cells, playing a crucial role in determining scaffold efficacy. Thus, accurately measuring the permeability of porous scaffolds could represent an essential step in their optimization process. In literature, several methods have been proposed to characterize scaffold permeability. Most of the currently adopted approaches to assess permeability limit their applicability to specific scaffold structures, hampering protocols standardization, and ultimately leading to incomparable results among different laboratories. The content of novelty of this study is in the proposal of an adaptable test bench and in defining a specific testing protocol, compliant with the ASTM International F2952-22 guidelines, for reliable and repeatable measurements of the intrinsic permeability of TE porous scaffolds. The developed permeability test bench (PTB) exploits the pump-based method, and it is composed of a modular permeability chamber integrated within a closed-loop hydraulic circuit, which includes a peristaltic pump and pressure sensors, recirculating demineralized water. A specific testing protocol was defined for characterizing the pressure drop associated with the scaffold under test, while minimizing the effects of uncertainty sources. To assess the operational capabilities and performance of the proposed test bench, permeability measurements were conducted on PLA scaffolds with regular (PS) and random (RS) micro-architecture and on commercial bovine bone matrix-derived scaffolds (CS) for bone TE. To validate the proposed approach, the scaffolds were as well characterized using an alternative test bench (ATB) based on acoustic measurements, implementing a blind randomized testing procedure. The consistency of the permeability values measured using both the test benches demonstrated the reliability of the proposed approach. A further validation of the PTB's measurement reliability was provided by the agreement between the measured permeability values of the PS scaffolds and the theory-based predicted permeability value. Once validated the proposed PTB, the performed measurements allowed the investigation of the scaffolds' transport properties. Samples with the same structure (guaranteed by the fused-deposition modeling technique) were characterized by similar permeability values, and CS and RS scaffolds showed permeability values in agreement with the values reported in the literature for bovine trabecular bone. In conclusion, the developed PTB and the proposed testing protocol allow the characterization of the intrinsic permeability of porous scaffolds of different types and dimensions under controlled flow regimes, representing a powerful tool in view of providing a reliable and repeatable framework for characterizing and optimizing scaffolds for TE applications.

An automated 3D-printed perfusion bioreactor combinable with pulsed electromagnetic field stimulators for bone tissue investigations.

In bone tissue engineering research, bioreactors designed for replicating the main features of the complex native environment represent powerful investigation tools. Moreover, when equipped with automation, their use allows reducing user intervention and dependence, increasing reproducibility and the overall quality of the culture process. In this study, an automated uni-/bi-directional perfusion bioreactor combinable with pulsed electromagnetic field (PEMF) stimulation for culturing 3D bone tissue models is proposed. A user-friendly control unit automates the perfusion, minimizing the user dependency. Computational fluid dynamics simulations supported the culture chamber design and allowed the estimation of the shear stress values within the construct. Electromagnetic field simulations demonstrated that, in case of combination with a PEMF stimulator, the construct can be exposed to uniform magnetic fields. Preliminary biological tests on 3D bone tissue models showed that perfusion promotes the release of the early differentiation marker alkaline phosphatase. The histological analysis confirmed that perfusion favors cells to deposit more extracellular matrix (ECM) with respect to the static culture and revealed that bi-directional perfusion better promotes ECM deposition across the construct with respect to uni-directional perfusion. Lastly, the Real-time PCR results of 3D bone tissue models cultured under bi-directional perfusion without and with PEMF stimulation revealed that the only perfusion induced a ~ 40-fold up-regulation of the expression of the osteogenic gene collagen type I with respect to the static control, while a ~ 80-fold up-regulation was measured when perfusion was combined with PEMF stimulation, indicating a positive synergic pro-osteogenic effect of combined physical stimulations.

Electrical Impedance-Based Characterization of Hepatic Tissue with Early-Stage Fibrosis.

Liver fibrosis is a key pathological precondition for hepatocellular carcinoma in which the severity is confidently correlated with liver cancer. Liver fibrosis, characterized by gradual cell loss and excessive extracellular matrix deposition, can be reverted if detected at the early stage. The gold standard for staging and diagnosis of liver fibrosis is undoubtedly biopsy. However, this technique needs careful sample preparation and expert analysis. In the present work, an ex vivo, minimally destructive, label-free characterization of liver biopsies is presented. Through a custom-made experimental setup, liver biopsies of bile-duct-ligated and sham-operated mice were measured at 8, 15, and 21 days after the procedure. Changes in impedance were observed with the progression of fibrosis, and through data fitting, tissue biopsies were approximated to an equivalent RC circuit model. The model was validated by means of 3D hepatic cell culture measurement, in which the capacitive part of impedance was proportionally associated with cell number and the resistive one was proportionally associated with the extracellular matrix. While the sham-operated samples presented a decrease in resistance with time, the bile-duct-ligated ones exhibited an increase in this parameter with the evolution of fibrosis. Moreover, since the largest difference in resistance between healthy and fibrotic tissue, of around 2 kΩ, was found at 8 days, this method presents great potential for the study of fibrotic tissue at early stages. Our data point out the great potential of exploiting the proposed needle setup in clinical applications.

A novel system for measuring visual potentials evoked by passive head-mounted display stimulators.

PURPOSE: The objective of this work is to evaluate the performances of a novel integrated device, based on passive head-mounted display (HMD), for the pattern reversal visual evoked potential (PR-VEP) clinical test. METHODS: Google Cardboard® is used as passive HMD to generate the checkerboard pattern stimuli through an Android® application. Electroencephalographic signals are retrieved and processed over 20 subjects, 12 females and 8 males between 20 and 26 years. Morphological PR-VEPs and frequency response were compared with previous literature results, to test the reproducibility and the efficacy of the proposed solution. RESULTS: PR-VEPs evoked by our novel prototype showed typical triphasic waveforms in moderate agreement with those obtained with other more expensive HMDs and standard commercial devices. Statistical analysis did not highlight strong differences among the systems over the features analyzed except for the P100 amplitude and peak time (**p < 0.005). CONCLUSION: The proposed solution opens the door for a new generation of non-invasive first-level diagnostic devices of optic nerve pathologies inexpensive and easy to access.

Accuracy of a new instrument for noninvasive evaluation of pulse wave velocity: the Arterial sTiffness faitHful tOol aSsessment project.

BACKGROUND: Large artery stiffness, assessed by carotid--femoral pulse wave velocity (cfPWV), is a major risk factor for cardiovascular events, commonly used for risk stratification. Currently, the reference device for noninvasive cfPWV is SphygmoCor but its cost and technically challenging use limit its diffusion in clinical practice. AIM: To validate a new device for noninvasive assessment of cfPWV, ATHOS (Arterial sTiffness faitHful tOol aSsessment), designed in collaboration with the Politecnico di Torino, against the reference noninvasive method represented by SphygmoCor. METHODS: Ninety healthy volunteers were recruited. In each volunteer, we assessed cfPWV, using SphygmoCor (PWVSphygmoCor) and ATHOS (PWVATHOS) devices in an alternate fashion, following the ARTERY Society guidelines. The accuracy was assessed by Bland--Altman plot, and reproducibility was assessed by interoperator correlation coefficient (ICC). RESULTS: Mean PWVATHOS and mean PWVSphygmoCor were 7.88 ±â€Š1.96 and 7.72 ±â€Š1.95 m/s, respectively. Mean difference between devices was 0.15 ±â€Š0.56 m/s, with a high correlation between measurements (r = 0.959, P < 0.001). Considering only PWV values at least 8 m/s (n = 30), mean difference was 0.1 ±â€Š0.63 m/s. The ICC was 97.7% with ATHOS. CONCLUSION: ATHOS showed an excellent level of agreement with SphygmoCor, even at high PWV values, with a good reproducibility. Its simplicity of use could help increase clinical application of PWV assessment, improving patients' cardiovascular risk stratification.

A New Noninvasive System for Clinical Pulse Wave Velocity Assessment: The Athos Device.

This paper presents a low cost, noninvasive, clinical-grade Pulse Wave Velocity evaluation device. The proposed system relies on a simultaneous acquisition of femoral and carotid pulse waves to improve estimation accuracy and correctness. The sensors used are two high precision MEMS force sensors, encapsulated in two ergonomic probes, and connected to the main unit. Data are then wirelessly transmitted to a standard laptop, where a dedicated graphical user interface (GUI) runs for analysis and recording. Besides the interface, the Athos system provides a Matlab algorithm to process the signals quickly and achieve a reliable PWV assessment. To better compare the results at the end of each analysis, a detailed report is generated, including all the relevant examination information (subject data, mean PTT, and obtained PWV). A pre-clinical study was conducted to validate the system by realizing several Pulse Wave Velocity measurements on ten heterogeneous healthy subjects of different ages. The collected results were then compared with those measured by a well-established and largely more expensive clinical device (SphygmoCor).

Impedance-based drug-resistance characterization of colon cancer cells through real-time cell culture monitoring.

Interest in impedance-based cellular assays is rising due to their remarkable advantages, including label-free, low cost, non-invasive, non-destructive, quantitative and real-time monitoring. In order to test their potential in cancer treatment decision and early detection of chemoresistance, we devised a new custom-made impedance measuring system based on electric cell-substrate impedance sensing (ECIS), optimized for long term impedance measurements. This device was employed in a proof of concept cell culture impedance analysis for the characterization of chemo-resistant colon cancer cells. Doxorubicin-resistant HT-29 cells were used for this purpose and monitored for 140 h. Analysis of impedance-based curves reveal different trends from chemo-sensitive and chemo-resistant cells. An impedance-based cytoxicity assay with different concentrations of doxorubicin was also performed using ECIS. The obtained results confirm the feasibility of ECIS in the study of drug resistance and show promises for studies of time-dependent factors related to physiological and behavioral changes in cells during resistance acquisition. The methodology presented herein, allows the continuous monitoring of cells under normal culture conditions as well as upon drug exposure. The ECIS device used, sets the basis for high-throughput early detection of resistance to drugs, administered in the clinical practice to cancer patients, and for the screening of new drugs in vitro, on patient-derived cells.

Pattern-Reversal Visual Evoked Potential on Smart Glasses.

OBJECTIVE: This paper presents an integrated device, based on smart glasses, for the pattern-reversal visual evoked potential (PR-VEP) clinical test. METHODS: Smart glasses are used to generate the checkerboard changing pattern, with its related red fixation point through an Android application. Electroencephalographic signals, for monitoring the stimulus generated by PR-VEP, were amplified close to the scalp and then transmitted wirelessly to a PC. A MATLAB real-time algorithm processed the incoming signals to extract the final PR-VEP signal. METHODS: In total, 40 eyes (from 20 subjects, 12 males and 8 females between 24 and 28 years old) were tested and results were compared, with a commercial device for VEP clinical exam, to test the reproducibility and the efficacy of the proposed solution. RESULTS: PR-VEPs generated by smart glasses showed typical triphasic waveforms: We observed promising results and components in moderate agreement with those obtained using commercial PR-VEP recorder, with potential for improvements after further refinement works. SIGNIFICANCE: The proposed device leads the way for a portable and low-cost solution.

Scaling Organic Electrochemical Transistors Down to Nanosized Channels.

The perspective of downscaling organic electrochemical transistors (OECTs) in the nanorange is approached by depositing poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) on electrodes with a nanogap designed and fabricated by electromigration induced break junction (EIBJ) technique. The electrical response of the fabricated devices is obtained by acquiring transfer characteristics in order to clarify the specific main characteristics of OECTs with sub-micrometer-sized active channels (nanogap-OECTs). On the basis of their electrical response to different scan times, the nanogap-OECT shows a maximum transconductance unaffected upon changing scan times in the time window from 1 s to 100 µs, meaning that fast varying signals can be easily acquired with unchanged amplifying performance. Hence, the scaling down of the channel size to the nanometer scale leads to a geometrical paradigm that minimizes effects on device response due to the cationic diffusion into the polymeric channel. A comprehensive study of these features is carried out by an electrochemical impedance spectroscopy (EIS) study, complemented by a quantitative analysis made by equivalent circuits. The propagation of a redox front into the polymer bulk due to ionic diffusion also known as the "intercalation pseudocapacitance" is identified as a limiting factor for the transduction dynamics.

Comparison of unusual carbon-based working electrodes for electrochemiluminescence sensors.

In this work, unconventional carbon-based materials were investigated for use in electrochemiluminescence (ECL) working electrodes. Precursors such as bamboo, pistachio shells, kevlar® fibers and camphor were differently treated and used as working electrodes in ECL experiments. After a proper process they were assembled as electrodes and tested in an electrochemical cell. Comparison among them and with a commercial glassy carbon electrode (GCE) shows a very good response for all of them thus demonstrating their potential use as disposable low-cost electrodes for early detection electrochemical analysis.

Carbon Nanotubes as an Effective Opportunity for Cancer Diagnosis and Treatment.

Despite the current progresses of modern medicine, the resistance of malignant tumors to present medical treatments points to the necessity of developing new therapeutic approaches. In recent years, numerous studies have focused their attention on the promising use of nanomaterials, like iron oxide nanowires, zinc oxide or mesoporous silica nanoparticles, for cancer and metastasis treatment with the advantage of operating directly at the bio-molecular scale. Among them, carbon nanotubes emerged as valid candidates not only for drug delivery, but also as a valuable tool in cancer imaging and physical ablation. Nevertheless, deep investigations about carbon nanotubes' potential bio-compatibility and cytotoxicity limits should be also critically addressed. In the present review, after introducing carbon nanotubes and their promising advantages and drawbacks for fighting cancer, we want to focus on the numerous and different ways in which they can assist to reach this goal. Specifically, we report on how they can be used not only for drug delivery purposes, but also as a powerful ally to develop effective contrast agents for tumors' medical or photodynamic imaging, to perform direct physical ablation of metastasis, as well as gene therapy.

A Multipurpose CMOS Platform for Nanosensing.

This paper presents a customizable sensing system based on functionalized nanowires (NWs) assembled onto complementary metal oxide semiconductor (CMOS) technology. The Micro-for-Nano (M4N) chip integrates on top of the electronics an array of aluminum microelectrodes covered with gold by means of a customized electroless plating process. The NW assembly process is driven by an array of on-chip dielectrophoresis (DEP) generators, enabling a custom layout of different nanosensors on the same microelectrode array. The electrical properties of each assembled NW are singularly sensed through an in situ CMOS read-out circuit (ROC) that guarantees a low noise and reliable measurement. The M4N chip is directly connected to an external microcontroller for configuration and data processing. The processed data are then redirected to a workstation for real-time data visualization and storage during sensing experiments. As proof of concept, ZnO nanowires have been integrated onto the M4N chip to validate the approach that enables different kind of sensing experiments. The device has been then irradiated by an external UV source with adjustable power to measure the ZnO sensitivity to UV-light exposure. A maximum variation of about 80% of the ZnO-NW resistance has been detected by the M4N system when the assembled 5 µ m × 500 nm single ZnO-NW is exposed to an estimated incident radiant UV-light flux in the range of 1 nW-229 nW. The performed experiments prove the efficiency of the platform conceived for exploiting any kind of material that can change its capacitance and/or resistance due to an external stimulus.

One-Dimensional ZnO/Gold Junction for Simultaneous and Versatile Multisensing Measurements.

The sensing capabilities of zinc oxide nano/micro-structures have been widely investigated and these structures are frequently used in the fabrication of cutting-edge sensors. However, to date, little attention has been paid to the multi-sensing abilities of this material. In this work, we present an efficient multisensor based on a single zinc oxide microwire/gold junction. The device is able to detect in real time three different stimuli, UV-VIS light, temperature and pH variations. This is thanks to three properties of zinc oxide its photoconductive response, pyroelectricity and surface functionalization with amino-propyl groups, respectively. The three stimuli can be detected either simultaneously or in a sequence/random order. A specific mathematical tool was also developed, together with a design of experiments (DoE), to predict the performances of the sensor. Our micro-device allows reliable and versatile real-time measurements of UV-VIS light, temperature and pH variations. Therefore, it shows great potential for use in the field of sensing for living cell cultures.