Cell Cycle Modulation through Physical Confinement in Micrometer-Thick Hydrogel Sheaths.

Recently, surface engineering of the cell membrane with biomaterials has attracted great attention for various biomedical applications. In this study, we investigated the possibility of modulating cell cycle progression using alginate and gelatin-based hydrogel sheaths with a thickness of ∼1 µm. The hydrogel sheath was formed on cell surfaces through cross-linking catalyzed by horseradish peroxidase immobilized on the cell surface. The hydrogel sheath did not decrease the viability (>95% during 2 days of culture) of the human cervical carcinoma cell line (HeLa) expressing the fluorescent ubiquitination-based cell cycle indicator 2 (HeLa/Fucci2). Coating the HeLa/Fucci2 cells with the hydrogel sheath resulted in a cell cycle arrest in the G2/M phase, which can be caused by the reduced F-actin formation. As a result of this cell cycle arrest, an inhibition of cell growth was observed in the HeLa/Fucci2 cells. Taken together, our results demonstrate that the hydrogel sheath coating on the cell surface is a feasible approach to modulating cell cycle progression.

3D Bioprinting of Sugar Beet Pectin through Horseradish Peroxidase-Catalyzed Cross-Linking.

Horseradish peroxidase (HRP)-mediated hydrogelation, caused by the cross-linking of phenolic groups in polymers in the presence of hydrogen peroxide (H2O2), is an effective route for bioink solidification in 3D bioprinting. Sugar beet pectin (SBP) naturally has cross-linkable phenols through the enzymatic reaction. Therefore, chemical modifications are not required, unlike the various polymers that have been used in the enzymatic cross-linking system. In this study, we report the application of SBP in extrusion-based bioprinting including HRP-mediated bioink solidification. In this system, H2O2 necessary for the solidification of inks is supplied in the gas phase. Cell-laden liver lobule-like constructs could be fabricated using bioinks consisting of 10 U/mL HRP, 4.0 and 6.0 w/v% SBP, and 6.0 × 106 cells/mL human hepatoblastoma (HepG2) cells exposed to air containing 16 ppm of H2O2 concurrently during printing and 10 min postprinting. The HepG2 cells enclosed in the printed constructs maintained their viability, metabolic activity, and hepatic functions from day 1 to day 7 of the culture, which indicates the cytocompatibility of this system. Taken together, this result demonstrates the potential of SBP and HRP cross-linking systems for 3D bioprinting, which can be applied in tissue engineering applications.

Transduction and Genome Editing of the Heart with Adeno-Associated Viral Vectors Loaded onto Electrospun Polydioxanone Nonwoven Fabrics.

In this study, we introduce electrospun polydioxanone (PDO) nonwoven fabrics as a platform for the delivery of adeno-associated virus (AAV) vectors for transduction and genome editing by adhering them to organ surfaces, including the heart. AAV vectors were loaded onto the PDO fabrics by soaking the fabrics in a solution containing AAV vectors. In vitro, the amount of AAV vectors loaded onto the fabrics could be adjusted by changing their concentration in the solution, and the number of cells expressing the green fluorescent protein (GFP) encoded by the AAV vectors increased in correlation with the increasing amount of loaded AAV vectors. In vivo, both transduction and genome editing resulted in the observation of GFP expression around AAV vector-loaded PDO fabrics attached to the surfaces of mouse hearts, indicating effective transduction and expression at the target site. These results demonstrate the great potential of electrospun PDO nonwoven fabrics carrying therapeutic AAV vectors for gene therapy.

Effect of cell adhesiveness of Cell Dome shell on enclosed HeLa cells.

The Cell Dome is a dome-shaped structure (diameter: 1 mm, height: 270 µm) with cells enclosed within a cavity, covered by a hemispherical hydrogel shell, and immobilized on a glass plate. Given that the cells within Cell Dome are in contact with the inner walls of the hydrogel shell, the properties of the shell are anticipated to influence cell behavior. To date, the impact of the hydrogel shell properties on the enclosed cells has not been investigated. In this study, we explored the effects of the cell adhesiveness of hydrogel shell on the behavior of enclosed cancer cells. Hydrogel shells with varying degrees of cell adhesiveness were fabricated using aqueous solutions containing either an alginate derivative with phenolic hydroxyl moieties exclusively or a mixture of alginate and gelatin derivatives with phenolic hydroxyl moieties. Hydrogel formation was mediated by horseradish peroxidase. We used the HeLa human cervical cancer cell line, which expresses fucci2, a cell cycle marker, to observe cell behavior. Cells cultured in hydrogel shells with cell adhesiveness proliferated along the inner wall of the hydrogel shell. Conversely, cells in hydrogel shells without cell adhesiveness grew uniformly at the bottom of the cavities. Furthermore, cells in non-adhesive hydrogel shells had a higher percentage of cells in the G1/G0 phase compared to those in adhesive shells and exhibited increased resistance to mitomycin hydrochloride when the cavities became filled with cells. These results highlight the need to consider the cell adhesiveness of the hydrogel shell when selecting materials for constructing Cell Dome.

Local delivery of adeno-associated viral vectors with electrospun gelatin nanofiber mats.

Adeno-associated viral (AAV) vectors play a significant role in gene therapy, yet the typical delivery methods, like systemic and local AAV injections, often lead to unintended off-target distribution and tissue damage due to injection. In this study, we propose a localized delivery approach for AAV vectors utilizing electrospun gelatin nanofiber mats, which are cross-linked with glutaraldehyde. The AAV vectors, which encoded a green fluorescent protein (GFP), were loaded onto the mats by immersing them in a solution containing the vectors. The amount of AAV vector loaded onto the mats increased as the vector concentration in the solution increased. The loaded AAV vector was steadily released into the cell culture medium over 3 days. The mats incubated for 3 days also showed the ability to transduce into the cells cultured on them. We evaluated the effectiveness of this delivery system by attaching the mats to mouse livers. GFP expression was visible on the surface of the liver beneath the attached mats, but not in areas in direct contact with the mats. These findings suggest that the attachment of AAV vector-loaded electrospun gelatin nanofiber mats to a target site present a promising solution for localized gene delivery while reducing off-target distribution.

Horseradish Peroxidase-Mediated Bioprinting via Bioink Gelation by Alternately Extruded Support Material.

Horseradish peroxidase (HRP)-mediated extrusion bioprinting has a significant potential in tissue engineering and regenerative medicine. However, they often face challenges in terms of printing fidelity and structural integrity when using low-viscosity inks. To address this issue, a method that alternately extrudes bioinks and support material was developed in this study. The bioinks consisting of cells, HRP, and phenolated polymers, and the support material contained hydrogen peroxide (H2O2). The support material not only prevented the collapse of the constructs but also supplied H2O2 to facilitate the enzymatic reaction. 3D constructs with tall and complex shapes were successfully printed from a low-viscosity ink containing 10 U/mL HRP and 1.0% w/v phenolated hyaluronic acid (HA-Ph), with a support material containing 10 mM H2O2. Over 90% viability of mouse fibroblasts (10T1/2) was achieved following the printing process, along with a morphology and proliferation rate similar to that of nontreated cells. Furthermore, human hepatoblastoma (HepG2) cells showed an increased spheroid size over 14 days in the printed constructs. The 10T1/2 cells adhered and proliferated on the constructs printed from inks containing both phenolated gelatin and HA-Ph. These results demonstrate the great potential of this HRP-mediated extrusion bioprinting technique for tissue engineering applications.

Visible light photocrosslinking of sugar beet pectin for 3D bioprinting applications.

Herein, we report the hydrogelation of sugar beet pectin (SBP) via visible light-mediated photocrosslinking and its applications in extrusion-based 3D bioprinting. Rapid hydrogelation (<15 s) was achieved by applying 405 nm visible light to an SBP solution in the presence of tris(bipyridine)ruthenium(II) chloride hexahydrate ([Ru(bpy)3]2+) and sodium persulfate (SPS). The mechanical properties of the hydrogel could be tuned by controlling the visible light irradiation time and concentrations of SBP, [Ru(bpy)3]2+, and SPS. High-fidelity 3D hydrogel constructs were fabricated by extruding inks containing 3.0 wt% SBP, 1.0 mM [Ru(bpy)3]2+, and 1.0 mM SPS. Human hepatoblastoma (HepG2) cells encapsulated in SBP hydrogels remained viable and metabolically active after 14 d of culture. Overall, this study demonstrates the feasibility of applying SBP and a visible light-mediated photocrosslinking system to the 3D bioprinting of cell-laden constructs for tissue engineering applications.

Fabrication of cell-laden microbeads and microcapsules composed of bacterial polyglucuronic acid.

In the past decades, the microencapsulation of mammalian cells into microparticles has been extensively studied for various in vitro and in vivo applications. The aim of this study was to demonstrate the viability of bacterial polyglucuronic acid (PGU), an exopolysaccharide derived from bacteria and composed of glucuronic acid units, as an effective material for cell microencapsulation. Using the method of dropping an aqueous solution of PGU-containing cells into a Ca2+-loaded solution, we produced spherical PGU microbeads with >93 % viability of the encapsulated human hepatoma HepG2 cells. Hollow-core microcapsules were formed via polyelectrolyte complex layer formation of PGU and poly-l-lysine, after which Ca2+, a cross-linker of PGU, was chelated, and this was accomplished by sequential immersion of microbeads in aqueous solutions of poly-l-lysine and sodium citrate. The encapsulated HepG2 cells proliferated and formed cell aggregates within the microparticles over a 14-day culture, with significantly larger aggregates forming within the microcapsules. Our results provide evidence for the viability of PGU for cell microencapsulation for the first time, thereby contributing to advancements in tissue engineering.

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.

Cell Dome as an Evaluation Platform for Organized HepG2 Cells.

Human-hepatoblastoma-derived cell line, HepG2, has been widely used in liver and liver cancer studies. HepG2 spheroids produced in a three-dimensional (3D) culture system provide a better biological model than cells cultured in a two-dimensional (2D) culture system. Since cells at the center of spheroids exhibit specific behaviors attributed to hypoxic conditions, a 3D cell culture system that allows the observation of such cells using conventional optical or fluorescence microscopes would be useful. In this study, HepG2 cells were cultured in "Cell Dome", a micro-dome in which cells are enclosed in a cavity consisting of a hemispherical hydrogel shell. HepG2 cells formed hemispherical cell aggregates which filled the cavity of Cell Domes on 18 days of culture and the cells could continue to be cultured for 29 days. The cells at the center of hemispherical cell aggregates were observed using a fluorescence microscope. The cells grew in Cell Domes for 18 days exhibited higher Pi-class Glutathione S-Transferase enzymatic activity, hypoxia inducible factor-1α gene expression, and higher tolerance to mitomycin C than those cultured in 2D on tissue culture dishes (* p < 0.05). These results indicate that the center of the glass adhesive surface of hemispherical cell aggregates which is expected to have the similar environment as the center of the spheroids can be directly observed through glass plates. In conclusion, Cell Dome would be useful as an evaluation platform for organized HepG2 cells.

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.

Successful introduction of robotic-assisted percutaneous coronary intervention system into Japanese clinical practice: a first-year survey at single center.

In Japan, a robotic-assisted PCI (R-PCI) system, the CorPath GRX System (Corindus Inc.), has been approved for clinical use in 2018, which is the first introduction of R-PCI into Japan. In this study, the clinical performance of the R-PCI system in the initial year at Kurume University Hospital was evaluated comparing with conventional manual PCI (M-PCI). A total of 30 R-PCI and 77 M-PCI procedures performed between April 2019 and March 2020, were retrospectively included. The primary outcome was the rate of clinical success defined as < 30% residual stenosis without in-hospital major adverse cardiovascular events (MACE). The secondary outcomes were fluoroscopy time, dose area product (DAP), amount of radiation exposure to operators and assistants, procedural time, and contrast volume. Propensity-matching technique was used to match each R-PCI lesion to the nearest M-PCI lesion without replacement. After propensity score matching, 30 R-PCI procedures in 28 patients and 37 M-PCI procedures in 35 patients were analyzed. Clinical success rate with R-PCI was favorable and comparable to M-PCI (93.3 vs. 94.6%, p = 0.97), without any in-hospital MACE. The operator radiation exposure was significantly lower in R-PCI (0 vs. 24.5 µSV, p < 0.0001). Radiation exposure to the patients was tended to be reduced by R-PCI (DAP: 77.6 vs. 100.2 Gycm2, p = 0.07). There were no statistically significant differences in radiation exposure to the assistant, fluoroscopy time, procedural time and contrast volume between the two groups (radiation exposure to the assistant: 10.5 vs. 10.0 µSV, p = 0.64, fluoroscopy time: 27.5 vs. 30.1 min, p = 0.55, procedural time: 72.4 vs. 61.6 min, p = 0.23, and contrast volume: 93.2 vs. 102.0 ml, p = 0.36). R-PCI in selected patients demonstrated favorable clinical outcomes with dramatical reduction of radiation exposure to operators.

Characterization of encapsulated cells within hyaluronic acid and alginate microcapsules produced via horseradish peroxidase-catalyzed crosslinking.

Hydrogel microcapsules having the ability to promote cell adhesion and proliferation are a useful tool for fabricating tissue in vitro. The present study explored the effects of two anionic polysaccharide hydrogel membranes which have an impact on the adhesiveness, morphology and growth of cells. Microcapsules were made by coating a cell-laden gelatin microparticle with a hydrogel membrane produced from modified hyaluronic acid or alginate possessing phenolic hydroxyl moieties (HA-Ph or Alg-Ph respectively) via a horseradish peroxidase-catalyzed crosslinking reaction. Some gelatin was retained within the microcapsules to support the attachment and growth of encapsulated cells. The morphological and functional characteristics of encapsulated HeLa and 10T1/2 cells were evaluated. The HA-Ph hydrogel, which exhibited greater retention of gelatin, showed a higher degree of cytocompatibility with respect to cell adhesion, spreading and proliferation compared with the Alg-Ph hydrogel membrane. These findings indicate that HA-Ph microcapsules synthesized around a temporary gelatin microparticles are a promising cell vehicle for tissue engineering applications.

Renal histopathological findings of retinal vasculopathy with cerebral leukodystrophy.

Retinal vasculopathy with cerebral leukodystrophy (RVCL) is a rare autosomal dominant systemic microvascular disease. Neurological disorders and visual disturbance are highlighted as manifestations of RVCL; however, there are few reports focused on nephropathy. Herein, we describe detailed renal histopathological findings in a daughter and father with RVCL, proven by TREX1 genetic analysis. A kidney biopsy of the daughter, 35-year-old with asymptomatic proteinuria, revealed unique and various glomerular changes. Atypical double contour (not tram track-like) of the capillary wall was widely found, an apparent characteristic finding. Glomerular findings were varied due to a combination of new and old segmental mesangial proliferative changes, mesangiolysis, and segmental glomerulosclerosis-like lesions; these changes may be related to endothelial cell damage. Collapsed tufts were also found and thought to be the result of ischemia due to arterial changes. Glomerular findings in a kidney biopsy of the father revealed similarity to the daughter's glomerulus at a relatively advanced stage, but the degree of variety in the glomerular findings was much less. Kidney biopsy findings suggesting endothelial cell damage of unknown etiology need to be considered for possible RVCL.

Cytocompatible Enzymatic Hydrogelation Mediated by Glucose and Cysteine Residues.

Hydrogels were obtained from aqueous solution containing polymer(s) possessing phenolic hydroxyl moieties through horseradish peroxidase (HRP)-catalyzed reaction without direct addition of H2O2. In this hydrogelation process, H2O2 was generated from HRP and glucose contained in the aqueous solution, that is, HRP functioned not only as a catalyst, but also as a source of H2O2. The gelation time and mechanical properties of the resultant hydrogel could be altered by changing the concentrations of HRP and glucose. Cytocompatibility of the hydrogelation process was confirmed from cell studies using mouse 10T1/2 fibroblast cells.

Enzymatically-gellable galactosylated chitosan: Hydrogel characteristics and hepatic cell behavior.

The influence of contents of galactose and phenolic hydroxyl (Ph) groups incorporated into chitosan was investigated on characteristics of the chitosan derivatives and the resultant gels as well as HepG2 cell attachment and growth behaviors. Introduction of galactose groups increased the solubility of the chitosan derivatives. The gelation time decreased with increasing content of Ph groups in the chitosan derivatives. The increase of galactose groups incorporated at a fixed content of Ph groups improved mechanical properties of the resultant gels. In vitro degradation rate of the resultant gels decreased by increasing Ph groups and decreasing galactose groups incorporated into the chitosan derivatives. The HepG2 cells formed dense spheroid cell clusters when the galactose groups were absent or incorporated at high level into chitosan (13.8mol%). However, the cells exhibited spreading morphology with spheroid formation on the gels containing 1.1 and 5.2mol% galactose groups. The albumin secretion level on a cellular basis also increased considerably when the galactose groups increased to 13.8mol%. The results demonstrated the potential of the chitosan derivative hydrogels for liver tissue engineering applications.

Anchoring PEG-oleate to cell membranes stimulates reactive oxygen species production.

Polyethylene glycol (PEG) derivatives possessing oleyl and reactive groups for conjugating functional substrates, such as proteins and quantum dots, are useful materials for cell-surface engineering and cell immobilization onto substrates. The reagent is known as a biocompatible anchor for cell membranes (BAM). Here, BAM-anchoring on cell membranes is reported to stimulate reactive oxygen species (ROS) production in those cells. Significant increases in ROS production and release to the surrounding environment were detected in mouse fibroblast cell line 10T1/2 when soaked in a solution containing BAM conjugated with 1/10mol/mol bovine serum albumin at 1.5µM-protein. ROS production stimulation was confirmed to be independent of the protein crosslinked with BAM and of cell type. Similar stimulation was detected for BAMs conjugated with ovalbumin and casein, in human hepatoma cell line HepG2, and human umbilical vein endothelial cells. Considering the effects of ROS on a variety of cellular processes, these results demonstrated the necessity for focusing attention on the effects of generated and released ROS on the behaviors of cells in the studies applying BAM to cells.