Dynamic Vortex Generation, Pulsed Injection, and Rapid Mixing of Blood Samples in Microfluidics Using the Tube Oscillation Mechanism.

Here, we describe the generation of dynamic vortices in micro-scale cavities at low flow rates. The system utilizes a computer-controlled audio speaker to axially oscillate the inlet tube of the microfluidic system at desired frequencies and amplitudes. The oscillation of the tube induces transiently high flow rates in the system, which facilitates the generation of dynamic vortices inside the cavity. The size of the vortices can be modulated by varying the tube oscillation frequency or amplitude. The vortices can be generated in single or serial cavities and in a wide range of cavity sizes. We demonstrate the suitability of the tube oscillation mechanism for the pulsed injection of water-based solutions or whole blood into the cavity. The injection rate can be controlled by the oscillation characteristics of the tube, enabling the injection of liquids at ultralow flow rates. The dynamic vortices facilitate the rapid mixing of the injected liquid with the main flow. The controllability and versatility of this technology allow for the development of programmable inertial microfluidic systems for performing multistep biological assays.

Deep Reinforcement Learning for Articulatory Synthesis in a Vowel-to-Vowel Imitation Task.

Articulatory synthesis is one of the approaches used for modeling human speech production. In this study, we propose a model-based algorithm for learning the policy to control the vocal tract of the articulatory synthesizer in a vowel-to-vowel imitation task. Our method does not require external training data, since the policy is learned through interactions with the vocal tract model. To improve the sample efficiency of the learning, we trained the model of speech production dynamics simultaneously with the policy. The policy was trained in a supervised way using predictions of the model of speech production dynamics. To stabilize the training, early stopping was incorporated into the algorithm. Additionally, we extracted acoustic features using an acoustic word embedding (AWE) model. This model was trained to discriminate between different words and to enable compact encoding of acoustics while preserving contextual information of the input. Our preliminary experiments showed that introducing this AWE model was crucial to guide the policy toward a near-optimal solution. The acoustic embeddings, obtained using the proposed approach, were revealed to be useful when applied as inputs to the policy and the model of speech production dynamics.

The influence of psychological and cognitive states on error-related negativity evoked during post-stroke rehabilitation movements.

BACKGROUND: Recently, error-related negativity (ERN) signals are proposed to develop an assist-as-needed robotic stroke rehabilitation program. Stroke patients' state-of-mind, such as motivation to participate and active involvement in the rehabilitation program, affects their rate of recovery from motor disability. If the characteristics of the robotic stroke rehabilitation program can be altered based on the state-of-mind of the patients, such that the patients remain engaged in the program, the rate of recovery from their motor disability can be improved. However, before that, it is imperative to understand how the states-of-mind of a participant affect their ERN signal. METHODS: This study aimed to determine the association between the ERN signal and the psychological and cognitive states of the participants. Experiments were conducted on stroke patients, which involved performing a physical rehabilitation exercise and a questionnaire to measure participants' subjective experience on four factors: motivation in participating in the experiment, perceived effort, perceived pressure, awareness of uncompleted exercise trials while performing the rehabilitation exercise. Statistical correlation analysis, EEG time-series and topographical analysis were used to assess the association between the ERN signals and the psychological and cognitive states of the participants. RESULTS: A strong correlation between the amplitude of the ERN signal and the psychological and cognitive states of the participants was observed, which indicate the possibility of estimating the said states using the amplitudes of the novel ERN signal. CONCLUSIONS: The findings pave the way for the development of an ERN based dynamically adaptive assist-as-needed robotic stroke rehabilitation program of which characteristics can be altered to keep the participants' motivation, effort, engagement in the rehabilitation program high. In future, the single-trial prediction ability of the novel ERN signals to predict the state-of-mind of stroke patients will be evaluated.

3D Printed Multiphasic Scaffolds for Osteochondral Repair: Challenges and Opportunities.

Osteochondral (OC) defects are debilitating joint injuries characterized by the loss of full thickness articular cartilage along with the underlying calcified cartilage through to the subchondral bone. While current surgical treatments can provide some relief from pain, none can fully repair all the components of the OC unit and restore its native function. Engineering OC tissue is challenging due to the presence of the three distinct tissue regions. Recent advances in additive manufacturing provide unprecedented control over the internal microstructure of bioscaffolds, the patterning of growth factors and the encapsulation of potentially regenerative cells. These developments are ushering in a new paradigm of 'multiphasic' scaffold designs in which the optimal micro-environment for each tissue region is individually crafted. Although the adoption of these techniques provides new opportunities in OC research, it also introduces challenges, such as creating tissue interfaces, integrating multiple fabrication techniques and co-culturing different cells within the same construct. This review captures the considerations and capabilities in developing 3D printed OC scaffolds, including materials, fabrication techniques, mechanical function, biological components and design.

Microfluidic Skin-on-a-Chip Models: Toward Biomimetic Artificial Skin.

The role of skin in the human body is indispensable, serving as a barrier, moderating homeostatic balance, and representing a pronounced endpoint for cosmetics and pharmaceuticals. Despite the extensive achievements of in vitro skin models, they do not recapitulate the complexity of human skin; thus, there remains a dependence on animal models during preclinical drug trials, resulting in expensive drug development with high failure rates. By imparting a fine control over the microenvironment and inducing relevant mechanical cues, skin-on-a-chip (SoC) models have circumvented the limitations of conventional cell studies. Enhanced barrier properties, vascularization, and improved phenotypic differentiation have been achieved by SoC models; however, the successful inclusion of appendages such as hair follicles and sweat glands and pigmentation relevance have yet to be realized. The present Review collates the progress of SoC platforms with a focus on their fabrication and the incorporation of mechanical cues, sensors, and blood vessels.

Tunable Harmonic Flow Patterns in Microfluidic Systems through Simple Tube Oscillation.

Generation of tunable harmonic flows at low cost in microfluidic systems is a persistent and significant obstacle to this field, substantially limiting its potential to address major scientific questions and applications. This work introduces a simple and elegant way to overcome this obstacle. Harmonic flow patterns can be generated in microfluidic structures by simply oscillating the inlet tubes. Complex rib and vortex patterns can be dynamically modulated by changing the frequency and magnitude of tube oscillation and the viscosity of liquid. Highly complex rib patterns and synchronous vortices can be generated in serially connected microfluidic chambers. Similar dynamic patterns can be generated using whole or diluted blood samples without damaging the sample. This method offers unique opportunities for studying complex fluids and soft materials, chemical synthesis of various compounds, and mimicking harmonic flows in biological systems using compact, tunable, and low-cost devices.

Oscillation and self-propulsion of Leidenfrost droplets enclosed in cylindrical cavities.

Leidenfrost droplets can be considered as soft engines capable of directly transforming heat into mechanical energy. Despite remarkable advancements in understanding the propulsion of Leidenfrost droplets on asymmetric structures, the complex dynamics of droplets in enclosed structures is not fully understood. To address this fundamental gap, we investigated the dynamics of Leidenfrost droplets restricted by metal disks. The disk alters the accumulation and release of the vapour generated by the droplet, and substantially changes its dynamic characteristics. Our experiments reveal the formation of oscillating multi-lobed structures when restricting the droplet within a disk. In comparison, patterning offset radial grooves on the surface of the disk rectifies the vapour flow and facilitates the self-propulsion of the droplet along the edge of the disk. Our work offers opportunities for developing soft and short-living actuators, which can operate at high temperatures.

Studying the Response of Aortic Endothelial Cells under Pulsatile Flow Using a Compact Microfluidic System.

We describe a piezoelectric pumping system for studying the mechanobiology of human aortic endothelial cells (HAECs) under pulsatile flow in microfluidic structures. The system takes advantage of commercially available components, including pumps, flow sensors, and microfluidic channels, which can be easily integrated, programmed, and operated by cellular biologists. Proof-of-concept experiments were performed to elucidate the complex mechanotransduction processes of endothelial cells to pulsatile flow. In particular, we investigated the effect of atheroprone and atheroprotective pulsatile shear stress on endothelial cytoskeleton remodeling and distribution of ß-catenin, as well as nuclear shape and size. The system is simple to operate, relatively inexpensive, portable, and controllable, providing opportunities for studying the mechanobiology of endothelial cells using microfluidic technologies.

Reconfigurable, Self-Sufficient Convective Heat Exchanger for Temperature Control of Microfluidic Systems.

Here, we demonstrate a modular, reconfigurable, and self-sufficient convective heat exchanger for regulation of temperature in microfluidic systems. The heat exchanger consists of polymer tubes wrapped around a plastic pole and fully embedded in an elastomer block, which can be easily mounted onto the microfluidic structure. It is compatible with various microfluidic geometries and materials. Miniaturized, battery-powered piezoelectric pumps are utilized to drive the heat carrying liquid through the heat exchanger at desired flow rates and temperatures. Customized temperature profiles can be generated by changing the configuration of the heat exchanger with respect to the microfluidic structure. Tailored dynamic temperature profiles can be generated by changing the temperature of the heat carrying liquid in successive cycles. This feature is used to study the calcium signaling of endothelial cells under successive temperature cycles of 24 to 37 °C. The versatility, simplicity, and self-sufficiency of the heat exchanger makes it suitable for various microfluidic based cellular assays.

Evaluation of the use of therapeutic peptides for cancer treatment.

Cancer along with cardiovascular disease are the main causes of death in the industrialised countries around the World. Conventional cancer treatments are losing their therapeutic uses due to drug resistance, lack of tumour selectivity and solubility and as such there is a need to develop new therapeutic agents. Therapeutic peptides are a promising and a novel approach to treat many diseases including cancer. They have several advantages over proteins or antibodies: as they are (a) easy to synthesise, (b) have a high target specificity and selectivity and (c) have low toxicity. Therapeutic peptides do have some significant drawbacks related to their stability and short half-life. In this review, strategies used to overcome peptide limitations and to enhance their therapeutic effect will be compared. The use of short cell permeable peptides that interfere and inhibit protein-protein interactions will also be evaluated.

NEST3D printed bone-mimicking scaffolds: assessment of the effect of geometrical design on stiffness and angiogenic potential.

Tissue-engineered implants for bone regeneration require consideration regarding their mineralization and vascularization capacity. Different geometries, such as biomimetic designs and lattices, can influence the mechanical properties and the vascularization capacity of bone-mimicking implants. Negative Embodied Sacrificial Template 3D (NEST3D) printing is a versatile technique across a wide range of materials that enables the production of bone-mimicking scaffolds. In this study, different scaffold motifs (logpile, Voronoi, and trabecular bone) were fabricated via NEST3D printing in polycaprolactone to determine the effect of geometrical design on stiffness (10.44 ± 6.71, 12.61 ± 5.71, and 25.93 ± 4.16 MPa, respectively) and vascularization. The same designs, in a polycaprolactone scaffold only, or when combined with gelatin methacryloyl, were then assessed for their ability to allow the infiltration of blood vessels in a chick chorioallantoic membrane (CAM) assay, a cost-effective and time-efficient in ovo assay to assess vascularization. Our findings showed that gelatin methacrylolyl alone did not allow new chorioallantoic membrane tissue or blood vessels to infiltrate within its structure. However, polycaprolactone on its own or when combined with gelatin methacrylolyl allowed tissue and vessel infiltration in all scaffold designs. The trabecular bone design showed the greatest mineralized matrix production over the three designs tested. This reinforces our hypothesis that both biomaterial choice and scaffold motifs are crucial components for a bone-mimicking scaffold.

Negative Printing for the Reinforcement of In Situ Tissue-Engineered Cartilage.

In the realm of in situ cartilage engineering, the targeted delivery of both cells and hydrogel materials to the site of a defect serves to directly stimulate chondral repair. Although the in situ application of stem cell-laden soft hydrogels to tissue defects holds great promise for cartilage regeneration, a significant challenge lies in overcoming the inherent limitation of these soft hydrogels, which must attain mechanical properties akin to the native tissue to withstand physiological loading. We therefore developed a system where a gelatin methacryloyl hydrogel laden with human adipose-derived mesenchymal stem cells is combined with a secondary structure to provide bulk mechanical reinforcement. In this study, we used the negative embodied sacrificial template 3D printing technique to generate eight different lattice-based reinforcement structures made of polycaprolactone, which ranged in porosity from 80% to 90% with stiffnesses from 28 ± 5 kPa to 2853 ± 236 kPa. The most promising of these designs, the hex prism edge, was combined with the cellular hydrogel and retained a stable stiffness over 41 days of chondrogenic differentiation. There was no significant difference between the hydrogel-only and hydrogel scaffold group in the sulfated glycosaminoglycan production (340.46 ± 13.32 µg and 338.92 ± 47.33 µg, respectively) or Type II Collagen gene expression. As such, the use of negative printing represents a promising solution for the integration of bulk reinforcement without losing the ability to produce new chondrogenic matrix.

Engineering interfacial tissues: The myotendinous junction.

The myotendinous junction (MTJ) is the interface connecting skeletal muscle and tendon tissues. This specialized region represents the bridge that facilitates the transmission of contractile forces from muscle to tendon, and ultimately the skeletal system for the creation of movement. MTJs are, therefore, subject to high stress concentrations, rendering them susceptible to severe, life-altering injuries. Despite the scarcity of knowledge obtained from MTJ formation during embryogenesis, several attempts have been made to engineer this complex interfacial tissue. These attempts, however, fail to achieve the level of maturity and mechanical complexity required for in vivo transplantation. This review summarizes the strategies taken to engineer the MTJ, with an emphasis on how transitioning from static to mechanically inducive dynamic cultures may assist in achieving myotendinous maturity.

Virtual Reality Assisted Motor Imagery for Early Post-Stroke Recovery: A Review.

Stroke is a serious neurological disease that may lead to long-term disabilities and even death for stroke patients worldwide. The acute period, ( ≤ 1 mo post-stroke), is crucial for rehabilitation but the current standard clinical practice may be ineffective for patients with severe motor impairment, since most rehabilitation programs involve physical movement. Imagined movement - the so-called motor imagery (MI) - has been shown to activate motor areas of the brain without physical movement. MI therefore offers an opportunity for early rehabilitation of stroke patients. MI, however, is not widely employed in clinical practice due to a lack of evidence-based research. Here, we review MI-based approaches to rehabilitation of stroke patients and immersive virtual reality (VR) technologies to potentially assist MI and thus, promote recovery of motor function.

Advanced flexible transvaginal mesh with high visibility under computerized tomography (CT) scan.

Pelvic floor disorders, including pelvic organ prolapse (POP) and stress urinary incontinence (SUI), are serious and very common. Surgery is commonly undertaken to restore the strength of the vaginal wall using transvaginal surgical mesh (TVM). However, up to 15% of TVM implants result in long-term complications, including pain, recurrent symptoms, and infection.Clinical Relevance- In this study, a new bioengineered TVM has been developed to address these issues. The TVM is visible using noninvasive imaging techniques such as computed tomography (CT); it has a highly similar structural profile to human tissue and potential to reduce pain and inflammation. These combined technological advances have the potential to revolutionize women's health.

Layer-by-Layer Analysis of In Vitro Skin Models.

In vitro human skin models are evolving into versatile platforms for the study of skin biology and disorders. These models have many potential applications in the fields of drug testing and safety assessment, as well as cosmetic and new treatment development. The development of in vitro skin models that accurately mimic native human skin can reduce reliance on animal models and also allow for more precise, clinically relevant testing. Recent advances in biofabrication techniques and biomaterials have led to the creation of increasingly complex, multilayered skin models that incorporate important functional components of skin, such as the skin barrier, mechanical properties, pigmentation, vasculature, hair follicles, glands, and subcutaneous layer. This improved ability to recapitulate the functional aspects of native skin enhances the ability to model the behavior and response of native human skin, as the complex interplay of cell-to-cell and cell-to-material interactions are incorporated. In this review, we summarize the recent developments in in vitro skin models, with a focus on their applications, limitations, and future directions.

Studying the Synergistic Effect of Substrate Stiffness and Cyclic Stretch Level on Endothelial Cells Using an Elastomeric Cell Culture Chamber.

Endothelial cells lining blood vessels are continuously exposed to biophysical cues that regulate their function in health and disease. As we age, blood vessels lose their elasticity and become stiffer. Vessel stiffness alters the mechanical forces that endothelial cells experience. Despite ample evidence on the contribution of endothelial cells to vessel stiffness, less is known about how vessel stiffness affects endothelial cells. In this study, we developed a versatile model to study the cooperative effect of substrate stiffness and cyclic stretch on human aortic endothelial cells. We cultured endothelial cells on elastomeric wells covered with fibronectin-coated polyacrylamide gel. Varying the concentrations of acrylamide and bis-acrylamide enabled us to produce soft and stiff substrates with elastic modules of 40 and 200 kPa, respectively. Using a customized three-dimensional (3D) printed cam-driven system, the cells were exposed to 5 and 10% cyclic stretch levels. This enabled us to mimic the stiffness and stretch levels that endothelial cells experience in young and aged arteries. Using this model, we found that endothelial cells cultured on a soft substrate had minimal cytoskeletal alignment to the direction of the stretch compared to the ones cultured on the stiff substrate. We also observed an increase in the cellular area and aspect ratio in cells cultured on the stiff substrate, both of which are positively regulated by cyclic stretch. However, neither cyclic stretch nor substrate stiffness significantly affected the nuclear circularity. Additionally, we found that the accumulation of NF-κB in the nucleus, endothelial proliferation, tube formation, and expression of IL1ß depends on the stretch level and substrate stiffness. Our model can be further used to investigate the complex signaling pathways associated with vessel stiffening that govern the endothelial responses to mechanical forces.

A bioactive peptide analogue for myxoma virus protein with a targeted cytotoxicity for human skin cancer in vitro.

BACKGROUND: Cancer is an international health problem, and the search for effective treatments is still in progress. Peptide therapy is focused on the development of short peptides with strong tumoricidal activity and low toxicity. In this study, we investigated the efficacy of a myxoma virus peptide analogue (RRM-MV) as a candidate for skin cancer therapy. RRM-MV was designed using the Resonant Recognition Model (RRM) and its effect was examined on human skin cancer and normal human skin cells in vitro. METHODS: Cell cultures were treated with various concentrations of the peptides at different incubation intervals. Cellular morphological changes (apoptosis and necrosis) were evaluated using confocal laser scanning microscopy. The cytotoxic effects of RRM-MV on human skin cancer and normal human skin cells were quantitatively determined by cytotoxicity and cell viability assays. The effect on human erythrocytes was also determined using quantitative hemolysis assay. DNA fragmentation assay was performed to detect early apoptotic events in treated cancer cells. Furthermore, to investigate the possible cell signalling pathway targeted by the peptides treatment, the levels of p-Akt expression in skin cancer and normal cells were detected by immunoblotting. RESULTS: Our results indicate that RRM-MV has a dose-dependent toxic effect on cancer cells only up to 18 h. The immunoblotting results indicated that the RRM-MV slightly increased p-Akt expression in melanoma and carcinoma cells, but did not seem to affect p-Akt expression in normal skin cells. CONCLUSIONS: RRM-MV targets and lethally harms cancer cells and leaves normal cells unharmed. It is able to reduce the cancer cell viability, disrupting the LDH activity in cancer cells and can significantly affect cancer progression. Further investigation into other cell signalling pathways is needed in the process leading to the in vivo testing of this peptide to prove its safety as a possible effective treatment for skin cancer.

Generation of dynamic vortices in a microfluidic system incorporating stenosis barrier by tube oscillation.

Microfluidic systems incorporating sudden expansions are widely used for generation of vortex flow patterns. However, the formation of vortices requires high flow rates to induce inertial effects. Here, we introduce a new method for generating dynamic vortices in microfluidics at low static flow rates. Human blood is driven through a microfluidic channel incorporating a semi-circular stenosis barrier. The inlet tube of the channel is axially oscillated using a computer-controlled audio-speaker. The tube oscillation induces high transient flow rates in the channel, which generates dynamic vortices across the stenosis barrier. The size of the vortices can be modulated by varying the frequency and amplitude of tube oscillation. Various vortex flow patterns can be generated by varying the flow rate. The formation and size of the vortices can be predicted using the Reynolds number of the oscillating tube. We demonstrate the potential application of the system for investigating the adhesion and phagocytosis of circulating immune cells under pathologically high shear rates induced at the stenosis. This approach facilitates the development of versatile and controllable inertial microfluidic systems for performing various cellular assays while operating at low static flow rates and low sample volumes.

Investigating the effects of low intensity visible light on human keratinocytes using a customized LED exposure system.

Photobiomodulation (PBM) refers to the use of light to modulate cellular processes, and has demonstrated utility in improving wound healing outcomes, and reducing pain and inflammation. Despite the potential benefits of PBM, the precise molecular mechanisms through which it influences cell behavior are not yet well understood. Inconsistent reporting of key light parameters has created uncertainty around optimal exposure profiles. In addition, very low intensities of light, < 0.1 J/cm2, have not been thoroughly examined for their use in PBM. Here, we present a custom-made compact, and modular LED-based exposure system for studying the effects of very low-intensity visible light (cell proliferation, migration, ROS production, and mitochondrial membrane potential) of three different wavelengths in a parallel manner. The device allows for six repeats of three different exposure conditions plus a non-irradiated control on a single 24-well plate. The immortalised human keratinocyte cell line, HaCaT, was selected as a major cellular component of the skin epidermal barrier. Furthermore, an in vitro wound model was developed by allowing the HaCaT to form a confluent monolayer, then scratching the cells with a pipette tip to form a wound. Cells were exposed to yellow (585 nm, 0.09 mW, ~ 3.7 mJ/cm2), orange (610 nm, 0.8 mW, ~ 31 mJ/cm2), and red (660 nm, 0.8 mW, ~ 31 mJ/cm2) light for 10 min. 48 h post-irradiation, immunohistochemistry was performed to evaluate cell viability, proliferation, ROS production, and mitochondrial membrane potential. The results demonstrate increased proliferation and decreased scratch area for all exposure conditions, however only red light increased the mitochondrial activity. Oxidative stress levels did not increase for any of the exposures. The present exposure system provides opportunities to better understand the complex cellular mechanisms driven by the irradiation of skin cells with visible light.