Fluidic integrated 3D bioprinting system to sustain cell viability towards larynx fabrication.

Herein, we report the first study to create a three-dimensional (3D) bioprinted artificial larynx for whole-laryngeal replacement. Our 3D bio-printed larynx was generated using extrusion-based 3D bioprinter with rabbit's chondrocyte-laden gelatin methacryloyl (GelMA)/glycidyl-methacrylated hyaluronic acid (GMHA) hybrid bioink. We used a polycaprolactone (PCL) outer framework incorporated with pores to achieve the structural strength of printed constructs, as well as to provide a suitable microenvironment to support printed cells. Notably, we established a novel fluidics supply (FS) system that simultaneously supplies basal medium together with a 3D bioprinting process, thereby improving cell survival during the printing process. Our results showed that the FS system enhanced post-printing cell viability, which enabled the generation of a large-scale cell-laden artificial laryngeal framework. Additionally, the incorporation of the PCL outer framework with pores and inner hydrogel provides structural stability and sufficient nutrient/oxygen transport. An animal study confirmed that the transplanted 3D bio-larynx successfully maintained the airway. With further development, our new strategy holds great potential for fabricating human-scale larynxes with in vivo-like biological functions for laryngectomy patients.

5-Fluorouracil-Immobilized Hyaluronic Acid Hydrogel Arrays on an Electrospun Bilayer Membrane as a Drug Patch.

The hyaluronic acid (HA) hydrogel array was employed for immobilization of 5-fluorouracil (5-FU), and the electrospun bilayer (hydrophilic: polyurethane/pluronic F-127 and hydrophobic: polyurethane) membrane was used to support the HA hydrogel array as a patch. To visualize the drug propagating phenomenon into tissues, we experimentally investigated how FITC-BSA diffused into the tissue by applying hydrogel patches to porcine tissue samples. The diffusive phenomenon basically depends on the FITC-BSA diffusion coefficient in the hydrogel, and the degree of diffusion of FITC-BSA may be affected by the concentration of HA hydrogel, which demonstrates that the high density of HA hydrogel inhibits the diffusive FITC-BSA migration toward the low concentration region. YD-10B cells were employed to investigate the release of 5-FU from the HA array on the bilayer membrane. In the control group, YD-10B cell viability was over 98% after 3 days. However, in the 5-FU-immobilized HA hydrogel array, most of the YD-10B cells were not attached to the bilayer membrane used as a scaffold. These results suggest that 5-FU was locally released and initiated the death of the YD-10B cells. Our results show that 5-FU immobilized on HA arrays significantly reduces YD-10B cell adhesion and proliferation, affecting cells even early in the cell culture. Our results suggest that when 5-FU is immobilized in the HA hydrogel array on the bilayer membrane as a drug patch, it is possible to control the drug concentration, to release it continuously, and that the patch can be applied locally to the targeted tumor site and administer the drug in a time-stable manner. Therefore, the developed bilayer membrane-based HA hydrogel array patch can be considered for sustained release of the drug in biomedical applications.

The Effect of Bright Light Therapy on Sleep and Quality of Life in Patients With Poststroke Insomnia.

OBJECTIVE: Poststroke insomnia is common and negatively affects stroke recovery. The objective of this study was to determine the effectiveness of bright light therapy for mild-to-moderate stroke patients with insomnia. METHODS: This study was randomized, double blind, and placebo controlled. A 2-week trial was conducted on patients with mild-to-moderate stroke who had poststroke insomnia. Only patients who had experienced a first episode of stroke were enrolled in this study. Sleep parameters were measured using the Actiwatch Spectrum Pro for 7 days before and after light therapy. The instrument specifically collected data concerning sleep, mood state, fatigue, and subjective quality of life. Participants with poststroke insomnia received bright light therapy (10,000 lux) or placebo therapy for 30 minutes in the early morning. A total of 112 eligible participants entered the study, but only 56 patients were randomized to treatment (27 to bright light therapy and 29 to placebo therapy). RESULTS: Results from analysis of variance showed that the mean change of sleep latency (F(1,55) =4.793, p = .033) and sleep efficiency (F(1,55) = 5.625, p = .022) were significantly superior in bright light therapy over placebo. Bright light therapy resulted in significant improvements in daytime sleepiness, fatigue, mood, and quality of life in study participants (p < .05). CONCLUSIONS: Bright light therapy is a nonpharmacological treatment of early, poststroke insomnia in patients who had a mild to moderate stroke. In addition, bright light therapy is effective for the treatment of daytime sleepiness, fatigue, and depression and for improving quality of life in patients with poststroke insomnia. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04721574.

Locally Controlled Diffusive Release of Bone Morphogenetic Protein-2 Using Micropatterned Gelatin Methacrylate Hydrogel Carriers.

In this work, a novel and simple bone morphogenetic protein (BMP)-2 carrier is developed, which enables localized and controlled release of BMP-2 and facilitates bone regeneration. BMP-2 is localized in the gelatin methacrylate (GelMA) micropatterns on hydrophilic semi-permeable membrane (SNM), and its controlled release is regulated by the concentration of GelMA hydrogel and BMP-2. The controlled release of BMP-2 is verified using computational analysis and quantified using fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) diffusion model. The osteogenic differentiation of osteosarcoma MG-63 cells is manipulated by localized and controlled BMP-2 release. The calcium deposits are significantly higher and the actin skeletal networks are denser in MG-63 cells cultured in the BMP-2-immobilized GelMA micropattern than in the absence of BMP-2. The proposed BMP-2 carrier is expected to not only act as a barrier membrane that can prevent invasion of connective tissue during bone regeneration, but also as a carrier capable of localizing and controlling the release of BMP-2 due to GelMA micropatterning on SNM. This approach can be extensively applied to tissue engineering, including the localization and encapsulation of cells or drugs.

A novel three-dimensional magnetic particle imaging system based on the frequency mixing for the point-of-care diagnostics.

The magnetic particle imaging (MPI) is a technology that can image the concentrations of the superparamagnetic iron oxide nanoparticles (SPIONs) which can be used in biomedical diagnostics and therapeutics as non-radioactive tracers. We proposed a point-of-care testing MPI system (PoCT-MPI) that can be used for preclinical use for imaging small rodents (mice) injected with SPIONs not only in laboratories, but also at emergency sites far from laboratories. In particular, we applied a frequency mixing magnetic detection method to the PoCT-MPI, and proposed a hybrid field free line generator to reduce the power consumption, size and weight of the system. The PoCT-MPI is [Formula: see text] in size and weighs less than 100 kg. It can image a three-dimensional distribution of SPIONs injected into a biosample with less than 120 Wh of power consumption. Its detection limit is [Formula: see text], 10 mg/mL, [Formula: see text] (Fe).

Construction of 3D-rendering imaging of an ischemic rat brain model using the planar FMMD technique.

Occlusion of the major cerebral artery usually results in brain hypoxic-ischemic injury, which evokes neuroinflammation and microglial activation. Activated microglia are considered a source of multiple neurotoxic factors, such as reactive oxygen species (ROS), in the central nervous system (CNS). We herein present a 3D-rendering brain imaging technique in an experimental rodent model of cerebral ischemia based on 2D magnetic images of superparamagnetic iron oxide nanoparticles (SPIONs) using the planar frequency mixing magnetic detection (p-FMMD) technique. A rat model of cerebral ischemia was established by unilateral middle cerebral artery occlusion with reperfusion (MCAO/R) injury. 2,3,5-Triphenyltetrazolium chloride (TTC) staining was performed to demonstrate the irreversibly damaged ischemic brain tissues, and double immunofluorescent labeling of OX6 (activated microglial marker) and ethidium (ROS marker) was conducted to confirm ROS generation in the activated microglia in the infarcted brain region. The ischemic brain sections treated with OX6-conjugated SPIONs were scanned using our p-FMMD system, yielding 2D images on the basis of the nonlinear magnetic characteristics inherent in SPIONs. The p-FMMD signal images representing microglia activation show an infarct ratio of 44.6 ± 7.1% compared to the contralateral counterpart, which is smaller than observed by TTC (60.9 ± 4.9%) or magnetic resonance imaging (MRI, 65.7 ± 2.7%). Furthermore, we developed a 3D-rendering brain imaging process based on the 2D p-FMMD signal images. The 3D reconstructed model showed a decreased ratio of coincidence of the ischemic regions compared with MRI models. In this study, we successfully conducted a feasibility test on whether our p-FMMD technology, a technique for signaling and imaging based on the nonlinearity of SPIONs, can be used to visualize the ischemic brain region in real time by detecting activated microglia in an MCAO/R animal model. Therefore, our method might allow for a different approach to analyze the pathophysiology of ischemic stroke through molecular imaging. Furthermore, we propose that this magnetic particle imaging (MPI) technique that detects the nonlinear magnetization properties of SPIONs could be applied not only to a stroke model but also to various types of pathophysiological studies as a new bioimaging tool.

Magnetic immunoassay platform based on the planar frequency mixing magnetic technique.

We represent the experimental results of our planar-frequency mixing magnetic detection (p-FMMD) technique to obtain 2D superparamagnetic images for magnetic immunoassay purpose. The imaging of magnetic beads is based on the nonlinear magnetic characteristics inherent in superparamagnetic materials. The p-FMMD records the sum-frequency components originating from both a high and a low frequency magnetic field incident on the magnetically nonlinear nanoparticles. In this study, we apply the p-FMMD technique to 2D scanning imaging of superparamagnetic iron oxide nanoparticles (SPIONs) in a microfluidic platform. Our p-FMMD system enables to acquire planar images of SPIONs filled in a microchannel as narrow as 30µm in width. The minimum detectable amount is ~1.0×10(8) beads of 100nm size. The system shows a spatial resolution enabling to distinguish between two distinct channels even 2mm apart from each other. Our p-FMMD system as a magnetic immunoassaying system has permitted the detection of amyloid beta 42 (Aß42), a promising biomarker of Alzheimer's disease, at the minimum concentration of 23.8pg/ml. This may enable the identification of the Aß42 levels for the early-stage of Alzheimer's disease with the assistance of the MPI using p-FMMD technique. The results show that the deployment of the p-FMMD can be an alternative to conventional biosensing analytical methods, and can be used as a fast and portable screening method.

The effects of chest expansion resistance exercise on chest expansion and maximal respiratory pressure in elderly with inspiratory muscle weakness.

[Purpose] The aim of this study was to examine the effect of chest expansion resistance exercises (CERE) on chest expansion, maximal inspiratory pressure (MIP), and maximal expiratory pressure (MEP) in elderly people with inspiratory muscle weakness. [Subjects] Thirty elderly people with inspiratory muscle weakness (MIP < 80% of the predicted value) were randomly and equally assigned to a chest expansion resistance exercise (CERE) group, core conditioning exercise (CCE) group, and control group. [Methods] The intervention was applied to the CERE group and CCE group five times per week, 30 minutes each time, for six weeks. A tapeline was used to measure upper and lower chest expansion. MIP and MEP before and after the intervention were measured and compared. [Results] There was significant improvement in upper and lower chest expansion and MIP after the intervention in both the CERE group and the CCE group, whereas the control group did not show any significant difference. MEP did not significantly change in any of the three groups after the intervention. [Conclusion] The CERE group underwent greater changes than the CCE group, which proves that the CERE is more effective for improving elderly people's chest expansion capacity and MIP in elderly people. Therefore, application of the CERE by therapists is recommended if the environment and conditions are appropriate for enhancement of chest expansion capacity and MIP in elderly people.

Effects of trunk rotation induced treadmill gait training on gait of stroke patients: a randomized controlled trial.

[Purpose] This study was conducted to find out the effect of arm swing during treadmill training on the gait of stroke patients. [Subjects and Methods] This study subjects were 20 stroke subjects patients who were randomly assigned to either the experimental group (EG) or the control group (CG), 10 subjects in each group. Therapists induced arm swing of affected side of EG subjects using Nordic poles, while subjects in CG had the affected arm restricted to prevent arm swing. Training was performed for 30 minutes, 3 times a week for 4 weeks. The timed up and go test (TUG), the dynamic gait index (DGI) and the 6-minute walk test (6MWT) were assessed before and after the training. [Results] After the training, there were no significant differences in the TUG times of EG and CG. There were significant differences in the DGI and the 6-minute walking distance of EG, but not of CG. There were also significant differences in the improvements of the DGI and the 6-minute walking distance between the groups. [Conclusion] Arm swing training had a positive effect on patients' gait ability. Further studies are required to generalize the results of this study.

The effect of chest expansion resistance exercise in chronic stroke patients: a randomized controlled trial.

[Purpose] The aim of this study was to examine the initial effects of chest expansion resistance exercise (CERE) applied to chronic stroke patients on their pulmonary functions, chest expansion, and functional gait ability. [Subjects] Forty chronic stroke patients without any respiration-related rehabilitation program experience (21 men and 19 women; times elapsed since occurrence of stroke: 21.8 ± 5.3 months) were randomly and equally allocated to a CERE group (experimental group) and a control group. [Methods] An ordinary stroke rehabilitation program was performed on the subjects. While the experimental group received a CERE intervention, the control group performed passive range of motion exercise with automatic instruments. [Results] The CERE group's chest expansion significantly increased after the intervention, whereas the control group did not see any significant difference. As regards VC (vital capacity), FVC (forced vital capacity), and FEV1 (forced expiratory volume in one second), there were no significant changes in either the CERE or control group. In the 10MTWT (10-meter timed walking test), there were no significant changes in either group, but in the 6MWT (6-minute walk test), while there were no significant differences in the control group, the CERE group saw significant changes. [Conclusion] The results of application of CERE to chronic stroke patients demonstrated the importance of respiratory exercise in an approach to stroke rehabilitation treatment intervention and the need to add respiratory exercise to a rehabilitation intervention program.

Antibody-based magnetic nanoparticle immunoassay for quantification of Alzheimer's disease pathogenic factor.

Alzheimer's disease (AD) is a neurodegenerative disorder that leads to a decline in cognitive and intellectual abilities and an irreversible mental deterioration. Based on multidisciplinary AD research, the most universally accepted hypotheses on AD pathogenesis are the intracerebral aggregate formation of beta-amyloid (Aß) peptides. According to medical paradigmatic transition from medical treatment to early diagnostic prevention, scientists have considered physiological body fluid as a biomarker medium, in which the promising AD biomarkers could be verified. Recently, use of saliva has been considered as one of the diagnostic fluids over the past decade with meaningful diagnostic potential. We utilized saliva as a biomarker medium to correlate the salivary Aß levels to AD pathological aspects, especially to the mild cognitive impairment group among AD patients, and to verify our detecting system to be sensitive enough for an early diagnostic tool. The identification of the salivary AD biomarkers using a facile microarraying method would motivate this study with the assistance of magnetically assembled antibody-conjugated nanoparticles and a photomultiplier tube as an optical detector. This simple magnetoimmunoassay system measures the photointensity generated by fluorescence, enables the quantification of the Aß peptides from AD salivary samples, and consequently classifies the salivary Aß levels into AD pathological aspects. This method demonstrates a facile approach enabling it to simply detect salivary Aß peptides at a concentration as low as ~20 pg/ml. It is expected that our simple magnetoimmunoassay system may have a potential as a detector for low-level Aß peptides with weak-fluorescence emission.

Measurement of beta-amyloid peptides in specific cells using a photo thin-film transistor.

The existence of beta-amyloid [Aß] peptides in the brain has been regarded as the most archetypal biomarker of Alzheimer's disease [AD]. Recently, an early clinical diagnosis has been considered a great importance in identifying people who are at high risk of AD. However, no microscale electronic sensing devices for the detection of Aß peptides have been developed yet. In this study, we propose an effective method to evaluate a small quantity of Aß peptides labeled with fluorescein isothiocyanate [FITC] using a photosensitive field-effect transistor [p-FET] with an on-chip single-layer optical filter. To accurately evaluate the quantity of Aß peptides within the cells cultured on the p-FET device, we measured the photocurrents which resulted from the FITC-conjugated Aß peptides expressed from the cells and measured the number of photons of the fluorochrome in the cells using a photomultiplier tube. Thus, we evaluated the correlation between the generated photocurrents and the number of emitted photons. We also evaluated the correlation between the number of emitted photons and the amount of FITC by measuring the FITC volume using AFM. Finally, we estimated the quantity of Aß peptides of the cells placed on the p-FET sensing area on the basis of the binding ratio between FITC molecules and Aß peptides.

In situ curing of sliding SU-8 droplet over a microcontact printed pattern for tunable fabrication of a polydimethylsiloxane nanoslit.

A tunable process for polydimethylsiloxane (PDMS) nanoslit fabrication is developed for nanofluidic applications. A microcontact printing (µCP) of a laterally spreading self-assembled hexadecanethiol (HDT) layer, combined with in situ curing of a sliding SU-8 droplet, enables precise and independent tuning of a nanoslit-mold width and height using a single µCP master mold. The SU-8 nanoslit-mold is replicated using a hard-soft composite PDMS to prevent channel collapse at low (<0.2) aspect ratio (height over width). The fluidic characteristics as well as dimensions of nanoslits fabricated with various conditions are analyzed using a fluorescein sample and AFM images. Finally, concentration polarization-based sample preconcentration is successfully demonstrated at the nanoslit boundary where an electric double-layer is overlapped.

Microfluidic synthesis of pure chitosan microfibers for bio-artificial liver chip.

We developed microfluidic-based pure chitosan microfibers (approximately 1 meter long, 70-150 microm diameter) for liver tissue engineering applications. Despite the potential of the chitosan for creating bio-artificial liver chips, its major limitation is the inability to fabricate pure chitosan-based microstructures with controlled shapes because of the mechanical weakness of the pure chitosan. Previous studies have shown that chitosan micro/nanofibers can be fabricated by using chemicals and electrospinning techniques. However, there is no paper regarding pure chitosan-based microfibers in a microfluidic device. This paper suggests a unique method to fabricate pure chitosan microfibers without any chemical additive. We also analyzed the chemical, mechanical, and diffusion properties of pure chitosan microfibers. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectrometry and electron spectroscopy for chemical analysis (ESCA) were used to analyze the chemical composition of the synthesized chitosan microfibers. We measured the mechanical axial-force and diffusion coefficient in pure chitosan-based microfibers using fluorescence recovery after photobleaching (FRAP) techniques. Furthermore, to evaluate the capability of the microfibers for liver tissue formation, hepatoma HepG2 cells were seeded onto the chitosan microfibers. The functionality of these hepatic cells cultured on chitosan microfibers was analyzed by measuring albumin secretion and urea synthesis. Therefore, this pure chitosan-based microfiber chip could be a potentially useful method for liver tissue engineering applications.

Applications of micromixing technology.

This review presents an application of micromixer technologies, which have driven a number of critical research trends over the past few decades, particularly for chemical and biological fields. Micromixer technologies in this review are categorized according to their applications: (1) chemical applications, including chemical synthesis, polymerization, and extraction; (2) biological applications, including DNA analysis, biological screening enzyme assays, protein folding; and (3) detection/analysis of chemical or biochemical content combined with NMR, FTIR, or Raman spectroscopies. In the chemical application, crystallization, extraction, polymerization, and organic synthesis have been reported, not only for laboratory studies, but also for industrial applications. Microscale techniques are used in chemical synthesis to develop microreactors. In clinical medicine and biological studies, microfluidic systems have been widely applied to the identification of biochemical products, diagnosis, drug discovery, and investigation of disease symptoms. The biological and biochemical applications also include enzyme assays, biological screening assays, cell lysis, protein folding, and biological analytical assays. Nondestructive analytical/detection methods have yielded a number of benefits to chemical and biochemical processes. In this chapter, we introduce analytical methods those are frequently integrated into micromixing technologies, such as NMR, FT-IR, and Raman spectroscopies. From the study of micromixers, we discovered that the Re number and mixing time depends on the specific application, and we clustered micromixers in various applications according to the Re number and mixing performance (mixing time). We expect that this clustering will be helpful in designing of micromixers for specific applications.

Rapid photothermal lysis of the pathogenic bacteria, Escherichia coli using synthesis of gold nanorods.

A photon-to-thermal conversion nanosystem has been developed to rapidly elevate temperatures in poorly thermally conducting media using irradiation of gold nanorods. We first hypothesized that nanoparticles, especially gold nanorods, are capable of generating enough heat to lyse bacteria by heating sample solutions via laser irradiation. To test this, we synthesized Au nanorods (aspect ratio 3-4) and studied optothermal properties of these nanoparticles. The short Au nanorods were more efficient at absorbing 808 nm 450 mW laser irradiations resulting in more efficient temperature increase in glass vials compared to the long rods (aspect ratio -40). In bulk media, these nanoparticles could easily raise the temperature beyond 100 degrees C under continuous wave laser irradiation, enabling immediate cell lysis. Consequently, the pathogenic bacteria, Escherichia coli, within the sample solution are lysed by irradiating of the sample using a near infrared laser.

Role of flagellum and motility in pathogenesis of Vibrio vulnificus.

To assess the role of the flagellum which was detected by immunoscreening of surface proteins of Vibrio vulnificus, an flgE-deleted mutant was constructed and tested for its pathogenicity. The ability of this nonmotile mutant to adhere to INT-407 cells and its role in biofilm were decreased, as was its lethality to mice.