Direct coupled electrical stimulation towards improved osteogenic differentiation of human mesenchymal stem/stromal cells: a comparative study of different protocols.

Electrical stimulation (ES) has been described as a promising tool for bone tissue engineering, being known to promote vital cellular processes such as cell proliferation, migration, and differentiation. Despite the high variability of applied protocol parameters, direct coupled electric fields have been successfully applied to promote osteogenic and osteoinductive processes in vitro and in vivo. Our work aims to study the viability, proliferation, and osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells when subjected to five different ES protocols. The protocols were specifically selected to understand the biological effects of different parts of the generated waveform for typical direct-coupled stimuli. In vitro culture studies evidenced variations in cell responses with different electric field magnitudes (numerically predicted) and exposure protocols, mainly regarding tissue mineralization (calcium contents) and osteogenic marker gene expression while maintaining high cell viability and regular morphology. Overall, our results highlight the importance of numerical guided experiments to optimize ES parameters towards improved in vitro osteogenesis protocols.

Synergy between 3D-extruded electroconductive scaffolds and electrical stimulation to improve bone tissue engineering strategies.

In this work, we propose a simple, reliable, and versatile strategy to create 3D electroconductive scaffolds suitable for bone tissue engineering (TE) applications with electrical stimulation (ES). The proposed scaffolds are made of 3D-extruded poly(ε-caprolactone) (PCL), subjected to alkaline treatment, and of poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS), anchored to PCL with one of two different crosslinkers: (3-glycidyloxypropyl)trimethoxysilane (GOPS) and divinyl sulfone (DVS). Both cross-linkers allowed the formation of a homogenous and continuous coating of PEDOT:PSS to PCL. We show that these PEDOT:PSS coatings are electroconductive (11.3-20.1 S cm-1), stable (up to 21 days in saline solution), and allow the immobilization of gelatin (Gel) to further improve bioactivity. In vitro mineralization of the corresponding 3D conductive scaffolds was greatly enhanced (GOPS(NaOH)-Gel - 3.1 fold, DVS(NaOH)-Gel - 2.0 fold) and cell colonization and proliferation were the highest for the DVS(NaOH)-Gel scaffold. In silico modelling of ES application in DVS(NaOH)-Gel scaffolds indicates that the electrical field distribution is homogeneous, which reduces the probability of formation of faradaic products. Osteogenic differentiation of human bone marrow derived mesenchymal stem/stromal cells (hBM-MSCs) was performed under ES. Importantly, our results clearly demonstrated a synergistic effect of scaffold electroconductivity and ES on the enhancement of MSC osteogenic differentiation, particularly on cell-secreted calcium deposition and the upregulation of osteogenic gene markers such as COL I, OC and CACNA1C. These scaffolds hold promise for future clinical applications, including manufacturing of personalized bone TE grafts for transplantation with enhanced maturation/functionality or bioelectronic devices.

Syringe irrigation in confluent canals: A sequential computational fluid dynamics assessment.

This study aims to assess the influence of root canal preparation, irrigation needle design and its placement depth in the irrigation flow of confluent canals during syringe irrigation. A mandibular molar presenting two confluent canals in its mesial root was sequentially prepared and scanned by micro-computed tomography after mechanical preparation up to ProTaper Next system sizes X2 (25/.06v), X3 (30/.07v) and X4 (40/.06v). In each of the root canal preparation models, a side-vented and an open-ended needle at 5, 3 and 2 mm from the working length were included, and irrigation flow was assessed by a validated computational fluid dynamics model. The results revealed that the irrigant flowed out of the confluent canals mainly through the canal that did not have the needle. Apical penetration and renewal of the irrigant were most efficiently achieved with the use of a 30G open-ended needle and a 30/.07v preparation.

A 3D-Printed Ceramics Innovative Firing Technique: A Numerical and Experimental Study.

Additive manufacturing (AM), also known as three-dimensional (3D) printing, allows the fabrication of complex parts, which are impossible or very expensive to produce using traditional processes. That is the case for dinnerware and artworks (stoneware, porcelain and clay-based products). After the piece is formed, the greenware is fired at high temperatures so that these pieces gain its mechanical strength and aesthetics. The conventional (gas or resistive heating elements) firing usually requires long heating cycles, presently requiring around 10 h to reach temperatures as high as 1200 °C. Searching for faster processes, 3D-printed stoneware were fired using microwave (MW) radiation. The pieces were fired within 10% of the conventional processing time. The temperature were controlled using a pyrometer and monitored using Process Temperature Control Rings (PTCRs). An error of 1.25% was calculated between the PTCR (1207 ± 15 °C) and the pyrometer (1200 °C). Microwave-fast-fired pieces show similar mechanical strength to the references and to the electrically fast-fired pieces (41, 46 and 34 (N/mm2), respectively), presenting aesthetic features closer to the reference. Total porosities of ~4%, ~5% and ~9% were determined for microwave, electrically fast-fired and reference samples. Numerical studies have shown to be essential to better understand and improve the firing process using microwave radiation. In summary, microwave heating can be employed as an alternative to stoneware conventional firing methods, not compromising the quality and features of the processed pieces, and with gains in the heating time.

Impact of modeled field of view in electroconvulsive therapy current flow simulations.

Background: The field of view (FOV) considered in MRI-guided forward models of electroconvulsive therapy (ECT) are, as expected, limited to the MRI volume collected. Therefore, there is variation in model extent considered across simulation efforts. This study examines the impact of FOV on the induced electric field (E-field) due to two common electrode placements: right unilateral (RUL) and bilateral (BL). Methods: A full-body dataset was obtained and processed for modeling relevant to ECT physics. Multiple extents were derived by truncating from the head down to four levels: upper head (whole-brain), full head, neck, and torso. All relevant stimulation and focality metrics were determined. The differences in the 99th percentile peak of stimulation strength in the brain between each extent to the full-body (reference) model were considered as the relative error (RE). We also determine the FOV beyond which the difference to a full-body model would be negligible. Results: The 2D and 3D spatial plots revealed anticipated results in line with prior efforts. The RE for BL upper head was ~50% reducing to ~2% for the neck FOV. The RE for RUL upper head was ~5% reducing to subpercentage (0.28%) for the full-head FOV. As shown previously, BL was found to stimulate a larger brain volume-but restricted to the upper head and for amplitude up to ~480 mA. To some extent, RUL stimulated a larger volume. The RUL-induced volume was larger even when considering the neural activation threshold corresponding to brief pulse BL if ECT amplitude was >270 mA. This finding is explained by the BL-induced current loss through the inferior regions as more FOV is considered. Our result is a departure from prior efforts and raises questions about the focality metric as defined and/or inter-individual differences. Conclusion: Our findings highlight that BL is impacted more than RUL with respect to FOV. It is imperative to collect full-head data at a minimum for any BL simulation and possibly more. Clinical practice resorts to using BL ECT when RUL is unsuccessful. However, the notion that BL is more efficacious on the premise of stimulating more brain volume needs to be revisited.

A Novel Approach for Design and Manufacturing of Curvature-Featuring Scaffolds for Osteochondral Repair.

Osteochondral (OC) defects affect both articular cartilage and the underlying subchondral bone. Due to limitations in the cartilage tissue's self-healing capabilities, OC defects exhibit a degenerative progression to which current therapies have not yet found a suitable long-term solution. Tissue engineering (TE) strategies aim to fabricate tissue substitutes that recreate natural tissue features to offer better alternatives to the existing inefficient treatments. Scaffold design is a key element in providing appropriate structures for tissue growth and maturation. This study presents a novel method for designing scaffolds with a mathematically defined curvature, based on the geometry of a sphere, to obtain TE constructs mimicking native OC tissue shape. The lower the designed radius, the more curved the scaffold obtained. The printability of the scaffolds using fused filament fabrication (FFF) was evaluated. For the case-study scaffold size (20.1 mm × 20.1 mm projected dimensions), a limit sphere radius of 17.064 mm was determined to ensure printability feasibility, as confirmed by scanning electron microscopy (SEM) and micro-computed tomography (µ-CT) analysis. The FFF method proved suitable to reproduce the curved designs, showing good shape fidelity and replicating the expected variation in porosity. Additionally, the mechanical behavior was evaluated experimentally and by numerical modelling. Experimentally, curved scaffolds showed strength comparable to conventional orthogonal scaffolds, and finite element analysis was used to identify the scaffold regions more susceptible to higher loads.

JANUS: an open-source 3D printable perfusion bioreactor and numerical model-based design strategy for tissue engineering.

Bioreactors have been employed in tissue engineering to sustain longer and larger cell cultures, managing nutrient transfer and waste removal. Multiple designs have been developed, integrating sensor and stimulation technologies to improve cellular responses, such as proliferation and differentiation. The variability in bioreactor design, stimulation protocols, and cell culture conditions hampered comparison and replicability, possibly hiding biological evidence. This work proposes an open-source 3D printable design for a perfusion bioreactor and a numerical model-driven protocol development strategy for improved cell culture control. This bioreactor can simultaneously deliver capacitive-coupled electric field and fluid-induced shear stress stimulation, both stimulation systems were validated experimentally and in agreement with numerical predictions. A preliminary in vitro validation confirmed the suitability of the developed bioreactor to sustain viable cell cultures. The outputs from this strategy, physical and virtual, are openly available and can be used to improve comparison, replicability, and control in tissue engineering applications.

Evaluating the Injection Moulding of Plastic Parts Using In Situ Time-Resolved Small-Angle X-ray Scattering Techniques.

In this study, we describe the design and fabrication of an industrial injection moulding system that can be mounted and used on the NCD-SWEET small-angle X-ray scattering beamline at ALBA. We show how highly useful time-resolved data can be obtained using this system. We are able to evaluate the fraction of the material in the mould cavity and identify the first material to solidify and how this varies with the injection temperature. The design follows current industrial practice and provides opportunities to collect time-resolved data at several points within the mould cavity so that we can build up a 4D perspective of the morphology and its temporal development. The quantitative data obtained will prove invaluable for the optimisation of the next generation of injection moulding techniques. This preliminary work used results from the injection moulding of a general-purpose isotactic polypropylene.

Experimental validation of a computational fluid dynamics model using micro-particle image velocimetry of the irrigation flow in confluent canals.

AIM: This study aimed to experimentally validate a computational fluid dynamics (CFD) model, using micro-particle image velocimetry (micro-PIV) measurements of the irrigation flow velocity field developed in confluent canals during irrigation with a side-vented needle. METHODOLOGY: A microchip with confluent canals, manufactured in polydimethylsiloxane was used in a micro-PIV analysis of the irrigation flow using a side-vented needle placed 3 mm from the end of the confluence of the canals. Velocity fields and profiles were recorded for flow rates of 0.017 and 0.1 ml/s and compared with those predicted in CFD numerical simulations (using a finite volume commercial code - FLUENT) for both laminar and turbulent regimes. RESULTS: The overall flow pattern, isovelocity and vector maps as well as velocity profiles showed a close agreement between the micro-PIV experimental and CFD predicted data. No relevant differences were observed between the results obtained with the laminar and turbulent flow models used. CONCLUSIONS: Results showed that the laminar CFD modelling is reliable to predict the flow in similar domains.

How to correctly estimate the electric field in capacitively coupled systems for tissue engineering: a comparative study.

Capacitively Coupled (CCoupled) electric fields are used to stimulate cell cultures in Tissue Engineering. Knowing the electric field (E-Field) magnitude in the culture medium is fundamental to establish a relationship between stimulus strength and cellular effects. We analysed eight CCoupled studies and sought to corroborate the reported estimates of the E-Field in the culture medium. First, we reviewed the basic physics underlying CCoupled stimulation and delineated three approaches to estimate the E-field. Using these approaches, we found that the reported values were overestimated in five studies, four of which were based on incorrect assumptions. In all studies, insufficient information was provided to reproduce the setup exactly. Creating electrical models of the experimental setup should improve the accuracy of the E-field estimates and enhance reproducibility. For this purpose, we developed a free open-source tool, the E-field Calculator for CCoupled systems, which is available for download from an internet hosting platform.

Changing the Paradigm-Controlling Polymer Morphology during 3D Printing Defines Properties.

Direct digital manufacturing consists of a set of techniques that enable products to be fabricated directly from their digital definition, without the use of complex tooling or moulds. This manufacturing approach streamlines prototyping and small-scale production, as well as the mass customization of parts with complex designs immediately fixed before fabrication. With broad applicability, there are clearly opportunities in the field of medical devices for its use. However, many of the developments of direct digital manufacturing focus on simply specifying the shape or the form of the product, and this limited scope throws away many of the particular advantages of direct digital manufacturing. This work is focused on remedying this situation so that the digital specification of the fabricated product includes the properties as well as the form of the product. We use in situ time-resolving small-angle X-ray scattering measurements performed at the ALBA Synchrotron Light Source in Barcelona to evaluate the control that can be exerted on the morphology of a semi-crystalline polymer during extruder-based 3D printing. We use this as a methodology for printing the patterns of the morphology of the polymer to realise the patterns of properties of the polymeric material, specifically the modulus of the polymer. We give an example of products produced in this manner that contain spatial variation in their properties.

Additive Manufactured Poly(ε-caprolactone)-graphene Scaffolds: Lamellar Crystal Orientation, Mechanical Properties and Biological Performance.

Understanding the mechano-biological coupling mechanisms of biomaterials for tissue engineering is of major importance to assure proper scaffold performance in situ. Therefore, it is of paramount importance to establish correlations between biomaterials, their processing conditions, and their mechanical behaviour, as well as their biological performance. With this work, it was possible to infer a correlation between the addition of graphene nanoparticles (GPN) in a concentration of 0.25, 0.5, and 0.75% (w/w) (GPN0.25, GPN0.5, and GPN0.75, respectively) in three-dimensional poly(ε-caprolactone) (PCL)-based scaffolds, the extrusion-based processing parameters, and the lamellar crystal orientation through small-angle X-ray scattering experiments of extruded samples of PCL and PCL/GPN. Results revealed a significant impact on the scaffold's mechanical properties to a maximum of 0.5% of GPN content, with a significant improvement in the compressive modulus of 59 MPa to 93 MPa. In vitro cell culture experiments showed the scaffold's ability to support the adhesion and proliferation of L929 fibroblasts (fold increase of 28, 22, 23, and 13 at day 13 (in relation to day 1) for PCL, GPN0.25, GPN0.5, and GPN0.75, respectively) and bone marrow mesenchymal stem/stromal cells (seven-fold increase for all sample groups at day 21 in relation to day 1). Moreover, the cells maintained high viability, regular morphology, and migration capacity in all the different experimental groups, assuring the potential of PCL/GPN scaffolds for tissue engineering (TE) applications.

Effects of Scaffold Electrical Properties on Electric Field Delivery in Bioreactors.

In tissue engineering, cell culture scaffolds have been widely used in combination with electrical stimulation to promote multiple cellular outcomes, like differentiation and proliferation. Nevertheless, the influence of scaffolds on the electric field delivered inside a bioreactor is often ignored and requires a deeper study. By performing numerical analysis in a capacitively coupled setup, this work aimed to predict the effects of the scaffold presence on the electric field, considering multiple combinations of scaffold and culture medium electrical properties. We concluded that the effect of the scaffold on the electric field in the surrounding culture medium was determined by the difference in electrical conductivity of these two materials. The numerical simulations pointed to significant variations in local electric field patterns, which could lead to different cellular outcomes and confound the interpretation of the experimental results.

Personalized Reusable Face Masks with Smart Nano-Assisted Destruction of Pathogens for COVID-19: A Visionary Road.

The Coronavirus disease 2019 (COVID-19) emergency has demonstrated that the utilization of face masks plays a critical role in limiting the outbreak. Healthcare professionals utilize masks all day long without replacing them very frequently, thus representing a source of cross-infection for patients and themselves. Nanotechnology is a powerful tool with the capability to produce nanomaterials with unique physicochemical and antipathogen properties. Here, how to realize non-disposable and highly comfortable respirators with light-triggered self-disinfection ability by bridging bioactive nanofiber properties and stimuli-responsive nanomaterials is outlined. The visionary road highlighted in this Concept is based on the possibility of developing a new generation of masks based on multifunctional membranes where the presence of nanoclusters and plasmonic nanoparticles arranged in a hierarchical structure enables the realization of a chemically driven and on-demand antipathogen activities. Multilayer electrospun membranes have the ability to dissipate humidity present within the mask, enhancing the wearability and usability. The photothermal disinfected membrane is the core of these 3D printed and reusable masks with moisture pump capability. Personalized face masks with smart nano-assisted destruction of pathogens will bring enormous advantages to the entire global community, especially for front-line personnel, and will open up great opportunities for innovative medical applications.

A Multimodal Stimulation Cell Culture Bioreactor for Tissue Engineering: A Numerical Modelling Approach.

The use of digital twins in tissue engineering (TE) applications is of paramount importance to reduce the number of in vitro and in vivo tests. To pursue this aim, a novel multimodal bioreactor is developed, combining 3D design with numerical stimulation. This approach will facilitate the reproducibility between studies and the platforms optimisation (physical and digital) to enhance TE. The new bioreactor was specifically designed to be additive manufactured, which could not be reproduced with conventional techniques. Specifically, the design suggested allows the application of dual stimulation (electrical and mechanical) of a scaffold cell culture. For the selection of the most appropriate material for bioreactor manufacturing several materials were assessed for their cytotoxicity. Numerical modelling methods were then applied to the new bioreactor using one of the most appropriate material (Polyethylene Terephthalate Glycol-modified (PETG)) to find the optimal stimulation input parameters for bone TE based on two reported in vitro studies.

Neural markers of neuropathic pain associated with maladaptive plasticity in spinal cord injury.

OBJECTIVES: Given the potential use of neural markers for the development of novel treatments in spinal cord pain, we aimed to characterize the most effective neural markers of neuropathic pain following spinal cord injury (SCI). METHODS: A systematic PubMed review was conducted, compiling studies that were published prior to April, 2014 that examined neural markers associated with neuropathic pain after SCI using electrophysiological and neuroimaging techniques. RESULTS: We identified 6 studies: Four using electroencephalogram (EEG); 1 using magnetic resonance imaging (MRI) and FDG-PET (positron emission tomography); and 1 using MR spectroscopy. The EEG recordings suggested a reduction in alpha EEG peak frequency activity in the frontal regions of SCI patients with neuropathic pain. The MRI scans showed volume loss, primarily in the gray matter of the left dorsolateral prefrontal cortex, and by FDG-PET, hypometabolism in the medial prefrontal cortex was observed in SCI patients with neuropathic pain compared with healthy subjects. In the MR spectroscopy findings, the presence of pain was associated with changes in the prefrontal cortex and anterior cingulate cortex. CONCLUSIONS: When analyzed together, the results of these studies seem to point out to a common marker of pain in SCI characterized by decreased cortical activity in frontal areas and possibly increased subcortical activity. These results may contribute to planning further mechanistic studies as to better understand the mechanisms by which neuropathic pain is modulated in patients with SCI as well as clinical studies investigating best responders of treatment.