Characterization of Chitosan Hydrogels Obtained through Phenol and Tripolyphosphate Anionic Crosslinking.

Chitosan is a deacetylated polymer of chitin that is extracted mainly from the exoskeleton of crustaceans and is the second-most abundant polymer in nature. Chitosan hydrogels are preferred for a variety of applications in bio-related fields due to their functional properties, such as antimicrobial activity and wound healing effects; however, the existing hydrogelation methods require toxic reagents and exhibit slow gelation times, which limit their application in biological fields. Therefore, a mild and rapid gelation method is necessary. We previously demonstrated that the visible light-induced gelation of chitosan obtained through phenol crosslinking (ChPh) is a rapid gelation method. To further advance this method (<10 s), we propose a dual-crosslinked chitosan hydrogel obtained by crosslinking phenol groups and crosslinking sodium tripolyphosphate (TPP) and the amino groups of chitosan. The chitosan hydrogel was prepared by immersing the ChPh hydrogel in a TPP solution after phenol crosslinking via exposure to visible light. The physicochemical properties of the dual-crosslinked hydrogels, including Young's moduli and water retentions, were subsequently investigated. Young's moduli of the dual-crosslinked hydrogels were 20 times higher than those of the hydrogels without TPP ion crosslinking. The stiffness could be manipulated by varying the immersion time, and the water retention properties of the ChPh hydrogel were improved by TPP crosslinking. Ion crosslinking could be reversed using an iron chloride solution. This method facilitates chitosan hydrogel use for various applications, particularly tissue engineering and drug delivery.

Magnetic Microrobots Fabricated by Photopolymerization and Assembly.

Magnetic soft microrobots have great potential to access narrow spaces and conduct multiple tasks in the biomedical field. Until now, drug delivery, microsurgery, disease diagnosis, and dredging the blocked blood vessel have been realized by magnetic soft microrobots in vivo or in vitro. However, as the tasks become more and more complex, more functional units have been embedded in the body of the developed magnetic microrobots. These magnetic soft microrobots with complex designed geometries, mechanisms, and magnetic orientation are now greatly challenging the fabrication of the magnetic microrobots. In this paper, we propose a new method combining photopolymerization and assembly for the fabrication of magnetic soft microrobots. Utilizing the micro-hand assembly system, magnetic modules with different shapes and materials are firstly arrayed with precise position and orientation control. Then, the developed photopolymerization system is employed to fix and link these modules with soft materials. Based on the proposed fabrication method, 3 kinds of soft magnetic microrobots were fabricated, and the fundamental locomotion was presented. We believe that the presented fabrication strategy could help accelerate the clinical application of magnetic microrobots.

Randomized phase III study of high-dose methotrexate and whole-brain radiotherapy with/without temozolomide for newly diagnosed primary CNS lymphoma: JCOG1114C.

BACKGROUND: The goal was to determine whether the addition of temozolomide (TMZ) to the standard treatment of high-dose methotrexate (HD-MTX) and whole-brain radiotherapy (WBRT) for primary central nervous system lymphoma (PCNSL) improves survival. METHODS: An open-label, randomized, phase III trial was conducted in Japan, enrolling immunocompetent patients aged 20-70 years with histologically confirmed, newly diagnosed PCNSL. After administration of HD-MTX, patients were randomly assigned to receive WBRT (30 Gy) ±â€…10 Gy boost (arm A) or WBRT ±â€…boost with concomitant and maintenance TMZ for 2 years (arm B). The primary endpoint was overall survival (OS). RESULTS: Between September 29, 2014 and October 15, 2018, 134 patients were enrolled, of whom 122 were randomly assigned and analyzed. At the planned interim analysis, 2-year OS was 86.8% (95% confidence interval [CI]: 72.5-94.0%) in arm A and 71.4% (56.0-82.2%) in arm B. The hazard ratio was 2.18 (95% CI: 0.95-4.98), with the predicted probability of showing the superiority of arm B at the final analysis estimated to be 1.3%. The study was terminated early due to futility. O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status was measured in 115 tumors, and it was neither prognostic nor predictive of TMZ response. CONCLUSIONS: This study failed to demonstrate the benefit of concomitant and maintenance TMZ in newly diagnosed PCNSL.

Development of non-adherent cell-enclosing domes with enzymatically cross-linked hydrogel shell.

Non-adherent cells, such as hematopoietic cells and lymphocytes, are important research subjects in medical and biological fields. Therefore, a system that enables the handling of non-adherent cells in solutions in the same manner as that of adhering cells during medium exchange, exposure to chemicals, washing, and staining in imaging applications would be useful. Here, we report a 'Cell Dome' platform in which non-adherent cells can be enclosed and grown in the cavities of about 1 mm diameter and 270µm height. The domes consist of an alginate-based hydrogel shell of 90µm thickness. Cell Domes were formed on glass plates by horseradish peroxidase-mediated cross-linking. Human leukaemia cell line K562 cells enclosed in Cell Domes were stable for 29 days with every 2-3 days of medium change. The enclosed cells grew in the cavities and were stained and differentiated with reagents supplied from the surrounding medium. Additionally, K562 cells that filled the cavities (a 3D microenvironment) were more hypoxic and highly resistant to mitomycin C than those cultured in 2D. These findings demonstrate that the 'Cell Dome' may be a promising tool for conveniently culturing and evaluating non-adherent cells.

Nematode surface functionalization with hydrogel sheaths tailored in situ.

Engineering the surfaces of biological organisms allows the introduction of novel functions and enhances their native functions. However, studies on surface engineering remained limited to unicellular organisms. Herein, nematode surfaces are engineered through in situ hydrogelation mediated by horseradish peroxidase (HRP) anchored to nematode cuticles. With this method, hydrogel sheaths of approximately 10-µm thickness are fabricated from a variety of polysaccharides, proteins, and synthetic polymers. Caenorhabditis elegans and Anisakis simplex coated with a hydrogel sheath showed a negligible decrease in viability, chemotaxis and locomotion. Hydrogel sheaths containing UV-absorbable groups and catalase functioned as shields to protect nematodes from UV and hydrogen peroxide, respectively. The results also showed that hydrogel sheaths containing glucose oxidase have the potential to be used as living drug delivery systems for cancer therapy. The nematode functionalization method developed in this study has the potential to impact a wide range of fields from agriculture to medicine.

A Bio-synthetic Hybrid Hydrogel Formed under Physiological Conditions Consisting of Mucin and a Synthetic Polymer Carrying Boronic Acid.

Mucin-containing bio-synthetic hybrid hydrogel is successfully formed under physiological conditions upon mixing aqueous solutions of native mucin and synthetic polymers carrying boronic acids. The mechanical properties and stability of the hydrogel in physiological solutions, e.g., cell culture media, are tunable depending on the boronic acid content of polymers. The hydrogel dissolved in the physiological solutions releases native mucin and boronic acid-containing polymer, which can control the adhesion of mammalian cells to the surface.

Modulation of Cell-Cycle Progression by Hydrogen Peroxide-Mediated Cross-Linking and Degradation of Cell-Adhesive Hydrogels.

The cell cycle is known to be regulated by features such as the mechanical properties of the surrounding environment and interaction of cells with the adhering substrates. Here, we investigated the possibility of regulating cell-cycle progression of the cells on gelatin/hyaluronic acid composite hydrogels obtained through hydrogen peroxide (H2O2)-mediated cross-linking and degradation of the polymers by varying the exposure time to H2O2 contained in the air. The stiffness of the hydrogel varied with the exposure time. Human cervical cancer cells (HeLa) and mouse mammary gland epithelial cells (NMuMG) expressing cell-cycle reporter Fucci2 showed the exposure-time-dependent different cell-cycle progressions on the hydrogels. Although HeLa/Fucci2 cells cultured on the soft hydrogel (Young's modulus: 0.20 and 0.40 kPa) obtained through 15 min and 120 min of the H2O2 exposure showed a G2/M-phase arrest, NMuMG cells showed a G1-phase arrest. Additionally, the cell-cycle progression of NMuMG cells was not only governed by the hydrogel stiffness, but also by the low-molecular-weight HA resulting from H2O2-mediated degradation. These results indicate that H2O2-mediated cross-linking and degradation of gelatin/hyaluronic acid composite hydrogel could be used to control the cell adhesion and cell-cycle progression.

Development of phenol-grafted polyglucuronic acid and its application to extrusion-based bioprinting inks.

In this present work, we developed a phenol grafted polyglucuronic acid (PGU) and investigated the usefulness in tissue engineering field by using this derivative as a bioink component allowing gelation in extrusion-based 3D bioprinting. The PGU derivative was obtained by conjugating with tyramine, and the aqueous solution of the derivative was curable through a horseradish peroxidase (HRP)-catalyzed reaction. From 2.0 w/v% solution of the derivative containing 5 U/mL HRP, hydrogel constructs were successfully obtained with a good shape fidelity to blueprints. Mouse fibroblasts and human hepatoma cells enclosed in the printed constructs showed about 95% viability the day after printing and survived for 11 days of study without a remarkable decrease in viability. These results demonstrate the great potential of the PGU derivative in tissue engineering field especially as an ink component of extrusion-based 3D bioprinting.

A tetrahedral DNA nanorobot with conformational change in response to molecular trigger.

Dynamic DNA origami nanostructures that respond to external stimuli are promising platforms for cargo delivery and nanoscale sensing. However, the low stability of such nanostructures under physiological conditions presents a major obstacle for their use in biomedical applications. This article describes a stable tetrahedral DNA nanorobot (TDN) programmed to undergo a controlled conformational change in response to epithelial cell adhesion molecule (EpCAM), a molecular biomarker specifically expressed on the circulating tumor cells. Multiresolution molecular dynamics simulations verified the overall stability of the folded TDN design and characterized local distortions in the folded structure. Atomic force microscopy and gel electrophoresis results showed that tetragonal structures are more stable than unfolded DNA origami sheets. Live cell experiments demonstrated the low cytotoxicity and target specificity of TDN. In summary, the proposed TDN can not only effectively resist nuclease catalysis but also has the potential to monitor EpCAM-positive cells precisely.

Bio-assembling and Bioprinting for Engineering Microvessels from the Bottom Up.

Blood vessels are essential in transporting nutrients, oxygen, metabolic wastes, and maintaining the homeostasis of the whole human body. Mass of engineered microvessels is required to deliver nutrients to the cells included in the constructed large three-dimensional (3D) functional tissues by diffusion. It is a formidable challenge to regenerate microvessels and build a microvascular network, mimicking the cellular viabilities and activities in the engineered organs with traditional or existing manufacturing techniques. Modular tissue engineering adopting the "bottom-up" approach builds one-dimensional (1D) or two-dimensional (2D) modular tissues in micro scale first and then uses these modules as building blocks to generate large tissues and organs with complex but indispensable microstructural features. Building the microvascular network utilizing this approach could be appropriate and adequate. In this review, we introduced existing methods using the "bottom-up" concept developed to fabricate microvessels including bio-assembling powered by different micromanipulation techniques and bioprinting utilizing varied solidification mechanisms. We compared and discussed the features of the artificial microvessels engineered by these two strategies from multiple aspects. Regarding the future development of engineering the microvessels from the bottom up, potential directions were also concluded.

Automated Cell Mechanical Characterization by On-Chip Sequential Squeezing: From Static to Dynamic.

The mechanical properties of cells are harmless biomarkers for cell identification and disease diagnosis. Although many systems have been developed to evaluate the static mechanical properties of cells for biomedical research, their robustness, effectiveness, and cost do not meet clinical requirements or the experiments with a large number of cell samples. In this paper, we propose an approach for on-chip cell mechanical characterization by analyzing the dynamic behavior of cells as they pass through multiple constrictions. The proposed serpentine microfluidic channel consisted of 20 constrictions connected in series and divided into five rows for tracking cell dynamic behavior. Assisted by computer vision, the squeezing time of each cell through five rows of constrictions was automatically collected and filtered to evaluate the cell's mechanical deformability. We observed a decreasing passage time and increasing dynamic deformability of the cells as they passed through the multiple constrictions. The deformability increase rate of the HeLa cells was eight times greater than that of MEF cells. Moreover, the weak correlation between the deformability increase rate and the cell size indicated that cell recognition based on measuring the deformability increase rate could hardly be affected by the cell size variation. These findings showed that the deformability increase rate of the cell under on-chip sequential squeezing as a new index has great potential in cancer cell recognition.

Magnetic Driven Two-Finger Micro-Hand with Soft Magnetic End-Effector for Force-Controlled Stable Manipulation in Microscale.

In recent years, micromanipulators have provided the ability to interact with micro-objects in industrial and biomedical fields. However, traditional manipulators still encounter challenges in gaining the force feedback at the micro-scale. In this paper, we present a micronewton force-controlled two-finger microhand with a soft magnetic end-effector for stable grasping. In this system, a homemade electromagnet was used as the driving device to execute micro-objects manipulation. There were two soft end-effectors with diameters of 300 µm. One was a fixed end-effector that was only made of hydrogel, and the other one was a magnetic end-effector that contained a uniform mixture of polydimethylsiloxane (PDMS) and paramagnetic particles. The magnetic force on the soft magnetic end-effector was calibrated using an atomic force microscopy (AFM) probe. The performance tests demonstrated that the magnetically driven soft microhand had a grasping range of 0-260 µm, which allowed a clamping force with a resolution of 0.48 µN. The stable grasping capability of the magnetically driven soft microhand was validated by grasping different sized microbeads, transport under different velocities, and assembly of microbeads. The proposed system enables force-controlled manipulation, and we believe it has great potential in biological and industrial micromanipulation.

Visible Light-Curable Chitosan Ink for Extrusion-Based and Vat Polymerization-Based 3D Bioprintings.

Three-dimensional bioprinting has attracted much attention for biomedical applications, including wound dressing and tissue regeneration. The development of functional and easy-to-handle inks is expected to expand the applications of this technology. In this study, aqueous solutions of chitosan derivatives containing sodium persulfate (SPS) and Tris(2,2'-bipyridyl) ruthenium(II) chloride (Ru(bpy)3) were applied as inks for both extrusion-based and vat polymerization-based bioprinting. In both the printing systems, the curation of ink was caused by visible light irradiation. The gelation time of the solution and the mechanical properties of the resultant hydrogels could be altered by changing the concentrations of SPS and Ru(bpy)3. The 3D hydrogel constructs with a good shape fidelity were obtained from the chitosan inks with a composition that formed gel within 10 s. In addition, we confirmed that the chitosan hydrogels have biodegradability and antimicrobial activity. These results demonstrate the significant potential of using the visible light-curable inks containing a chitosan derivative for extrusion and vat polymerization-based bioprinting toward biomedical applications.

A Wearable Navigation Device for Visually Impaired People Based on the Real-Time Semantic Visual SLAM System.

Wearable auxiliary devices for visually impaired people are highly attractive research topics. Although many proposed wearable navigation devices can assist visually impaired people in obstacle avoidance and navigation, these devices cannot feedback detailed information about the obstacles or help the visually impaired understand the environment. In this paper, we proposed a wearable navigation device for the visually impaired by integrating the semantic visual SLAM (Simultaneous Localization And Mapping) and the newly launched powerful mobile computing platform. This system uses an Image-Depth (RGB-D) camera based on structured light as the sensor, as the control center. We also focused on the technology that combines SLAM technology with the extraction of semantic information from the environment. It ensures that the computing platform understands the surrounding environment in real-time and can feed it back to the visually impaired in the form of voice broadcast. Finally, we tested the performance of the proposed semantic visual SLAM system on this device. The results indicate that the system can run in real-time on a wearable navigation device with sufficient accuracy.

On-Chip Cell-Cell Interaction Monitoring at Single-Cell Level by Efficient Immobilization of Multiple Cells in Adjustable Quantities.

In recent decades, cell immobilization using microfluidic chips has facilitated significant advancements in biological analyses at the single-cell level. However, the efficient capture of multiple cells as a cluster in adjustable quantities for cell-cell interaction has not been achieved. In this paper, aiming to monitor the cell-cell interaction at the single-cell level, we proposed a novel method for the efficient immobilization of adjustable quantities of cells on the basis of passive hydrodynamics so that different cell-cell interaction patterns could be generated. Experiments were conducted to characterize the key geometric parameters of the chip to optimize the efficiency of trapping different quantities of cells. In the microfluidic chips optimized for immobilizing one to five cells, the trapping success rates (TSRs) were up to 97%, 87%, 84%, 58%, and 54%, respectively. Furthermore, the throughput was over 200 cells min-1 with a minimum cell density of 350 cells mm-2. Finally, in the experiments of applying the proposed multicell immobilization chips to cell-cell interaction monitoring, calcein-AM transfer between multiple cells under different patterns has been studied through quantifying the local fluorescent intensity. The results demonstrated that the proposed method could be a promising opportunity in the widening field of biological research at the single-cell level.

Extrusion-Based Bioprinting through Glucose-Mediated Enzymatic Hydrogelation.

We report an extrusion-based bioprinting approach, in which stabilization of extruded bioink is achieved through horseradish peroxidase (HRP)-catalyzed cross-linking consuming hydrogen peroxide (H2O2) supplied from HRP and glucose. The bioinks containing living cells, HRP, glucose, alginate possessing phenolic hydroxyl (Ph) groups, and cellulose nanofiber were extruded to fabricate 3D hydrogel constructs. Lattice- and human nose-shaped 3D constructs were successfully printed and showed good stability in cell culture medium for over a week. Mouse 10T1/2 fibroblasts enclosed in the printed constructs remained viable after 7 days of culture. It was also able to switch a non-cell-adhesive surface of the printed construct to cell-adhesive surface for culturing cells on it through a subsequent cross-linking of gelatin possessing Ph moieties. These results demonstrate the possibility of utilizing the presented cross-linking method for 3D bioprinting.

High-Speed Manipulation of Microobjects Using an Automated Two-Fingered Microhand for 3D Microassembly.

To assemble microobjects including biological cells quickly and precisely, a fully automated pick-and-place operation is applied. In micromanipulation in liquid, the challenges include strong adhesion forces and high dynamic viscosity. To solve these problems, a reliable manipulation system and special releasing techniques are indispensable. A microhand having dexterous motion is utilized to grasp an object stably, and an automated stage transports the object quickly. To detach the object adhered to one of the end effectors, two releasing methods-local stream and a dynamic releasing-are utilized. A system using vision-based techniques for the recognition of two fingertips and an object, as well automated releasing methods, can increase the manipulation speed to faster than 800 ms/sphere with a 100% success rate (N = 100). To extend this manipulation technique, 2D and 3D assembly that manipulates several objects is attained by compensating the positional error. Finally, we succeed in assembling 80-120 µm of microbeads and spheroids integrated by NIH3T3 cells.

An extranodal histopathological analysis of idiopathic multicentric Castleman disease with and without TAFRO syndrome.

Thrombocytopenia, anasarca, fever, renal failure or reticulin fibrosis, and organomegaly (TAFRO) syndrome, a poor prognostic clinical condition showing similar histopathological findings to idiopathic multicentric Castleman disease (iMCD), has been reported in Japan. In our previous report, a clinicopathological analysis was performed on 70 nodal cases of iMCD with/without TAFRO. iMCD is classified into three types based on histopathology: (i) plasmacytic (PC), (ii) mixed, and (iii) hypervascular (hyperV). In this report, extranodal histopathological changes of iMCD with/without TAFRO were analyzed. Regarding the kidney pathology, we observed the proliferation of mesangial cells with positive staining of interleukin-6 (IL-6), consistent with membranoproliferative glomerulonephritis, in two cases of iMCD with TAFRO. The number of megakaryocytes per high-powered fields was not significantly different between iMCD cases with and without TAFRO. In conclusion, extranodal lesions of iMCD with/without TAFRO showed various interesting histopathological findings. These lesions may therefore be related to the clinical condition of TAFRO. Obtaining further knowledge about TAFRO will require the observation of nodal as well as extranodal lesions.

Prognostic impact of trisomy 21 in follicular lymphoma.

The chromosomal abnormalities associated with follicular lymphoma (FL) prognosis are not fully elucidated. Here, we evaluated the pattern of chromosomal abnormalities in FL, and clarified the correlations between the cytogenetic features and clinical outcome. Cytogenetic analysis was performed using standard methods of Giemsa-banding at diagnosis for 201 FL patients admitted to our hospitals between 2001 and 2013. The identified chromosomal abnormalities were: t(14;18)(q32;q21) (59·2%), +X (17·9%), del(6)(q)/-6 (16·9%), +7 (14·4%), abnormality of 1q12-21/1q (12·9%), del(13)(q)/-13 (11·9%), abnormality of 3q27 (10·4%), abnormality of 10q22-24 (10·0%), +12/dup(12)(q) (10·0%), abnormality of 1p21-22/1p (9·0%), +18 (9·0%), del(17)(p)/-17 (5·0%), and a complex karyotype (54·7%). Patients with trisomy 21 had a significantly shorter progression-free survival (P = 0·00171) and overall survival (OS) (P < 0·001) than those without trisomy 21; additionally, patients with trisomy 21 in the rituximab-treated cohort also had a significantly shorter OS (P = 0·000428). Multivariate analysis identified trisomy 21 as an independent risk factor in our cohorts with or without t(14;18) (P = 0·015). In conclusion, the presence of trisomy 21 was an independent risk factor for in FL. Chromosomal analysis of FL patients at diagnosis can provide useful information about their expected survival.