Gelatin/Hyaluronic Acid Content in Hydrogels Obtained through Blue Light-Induced Gelation Affects Hydrogel Properties and Adipose Stem Cell Behaviors.

Composite hydrogels of hyaluronic acid and gelatin attract great attention in biomedical fields. In particular, the composite hydrogels obtained through processes that are mild for cells are useful in tissue engineering. In this study, hyaluronic acid/gelatin composite hydrogels obtained through a blue light-induced gelation that is mild for mammalian cells were studied for the effect of the content of each polymer in the precursor solution on gelation, properties of resultant hydrogels, and behaviors of human adipose stem cells laden in the hydrogels. Control of the content enabled gelation in less than 20 s, and also enabled hydrogels to be obtained with 0.5-1.2 kPa Young's modulus. Human adipose stem cells were more elongated in hydrogels with a higher rather than lower content of hyaluronic acid. Stem cell marker genes, Nanog, Oct4, and Sox2, were expressed more in the cells in the composite hydrogels with a higher content of hyaluronic acid compared with those in the hydrogel composed of gelatin alone and on tissue culture dishes. These results are useful for designing conditions for using gelatin/hyaluronic acid composite hydrogels obtained through blue light-induced gelation suitable for tissue engineering applications.

Inkjet micropatterning through horseradish peroxidase-mediated hydrogelation for controlled cell immobilization and microtissue fabrication.

A simple fabrication method for cell micropatterns on hydrogel substrates was developed using an inkjet printing system that induced hydrogel micropatterns. The hydrogel micropatterns were created from inks resulting in cell-adhesive and non-cell-adhesive printed regions by horseradish peroxidase-catalyzed reaction onto non-cell-adhesive and cell-adhesive hydrogel substrates, respectively, to obtain the cell micropatterns. Cell-adhesive and non-cell-adhesive regions were obtained from gelatin and alginate derivatives, respectively. The cells on the cell-adhesive regions were successfully aligned, resulting in recognizable patterns. Furthermore, the proposed system permits the patterning of multiple cell types by switching the non-cell-adhesive region to the cell-adhesive region in the presence of growing cells. Also, we could fabricate disc- and filament-shaped small tissues by degrading the non-cell-adhesive substrates having dot- and line-shaped cell-adhesive micropatterns using alginate-lyase. These results indicate that our system is useful for fabrication of tailor-made cell patterns and microtissues with the shape defined by the micropattern, and will be conducive to a diverse range of biological applications.

Horseradish peroxidase-catalyzed hydrogelation consuming enzyme-produced hydrogen peroxide in the presence of reducing sugars.

In the present work, three kinds of reducing sugars: glucose, galactose, and mannose, are applied to horseradish peroxidase (HRP)-catalyzed hydrogelation of an aqueous solution containing natural polymers modified with phenolic hydroxyl moieties. In this system, HRP consumes hydrogen peroxide that was generated from the oxidation of thiol groups in HRP in the presence of reducing sugars. Herein, we highlight the versatility of applicable sugar types and the controllable hydrogel properties. The mechanical properties and microstructures of the resultant hydrogels can be well controlled by varying the concentration and the reducing power of sugars. Moreover, reducing sugar-independent cytocompatibility of the hydrogels was confirmed by the growth of cells on them. The wide selection of sugar types provides a better understanding of the reaction mechanism and enables the characterization of hydrogels with well-controlled properties.

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.

Inducing flocculation of non-floc-forming Escherichia coli cells.

The present article reviews several approaches for inducing flocculation of Escherichia coli cells. The common industrially used bacterium E. coli does not naturally have floc-forming ability. However, there are several approaches to induce flocculation of E. coli cells. One is induction by flocculants-polyvalent inorganic salts, synthetic polymeric flocculants, or bio-based polymeric materials, including polysaccharide derivatives. Another method is the induction of spontaneous flocculation by changing the phenotypes of E. coli cells; several studies have shown that physical treatment or gene modification can endow E. coli cells with floc-forming ability. Coculturing E. coli with other microbes is another approach to induce E. coli flocculation. These approaches have particular advantages and disadvantages, and remain open to clarification of the flocculation mechanisms and improvement of the induction processes. In this review, several approaches to the induction of E. coli flocculation are summarized and discussed. This review will be a useful guide for the future development of methods for the flocculation of non-floc-forming microorganisms.

Peroxidase-catalyzed microextrusion bioprinting of cell-laden hydrogel constructs in vaporized ppm-level hydrogen peroxide.

Hydrogels were prepared by contacting air containing 10-50 ppm H2O2 with an aqueous solution containing polymer(s) possessing phenolic hydroxyl (Ph) moieties (polymer-Ph) and horseradish peroxidase (HRP). In this system, HRP catalyzes cross-linking of the Ph moieties by consuming H2O2 diffused from the air. The hydrogelation rate and mechanical properties of the resultant hydrogels can be tuned by controlling the H2O2 concentration in air, the exposure time of the air containing H2O2 to the solution containing polymer-Phs and HRP, and the HRP concentration. The shortest hydrogelation time of the solution stirred in air containing 16 ppm H2O2 was 6 s. Based on these findings, this hydrogelation system was applied to microextrusion bioprinting, in which bioink containing polymer-Phs, HRP, and cells were extruded into air containing H2O2. The superior cytocompatibility of the bioprinting method was confirmed by more than 90% viability, migration, and the spreading of mouse fibroblast 10T1/2 cells enclosed in the bioprinted hydrogels composed of derivatives of hyaluronic acid and gelatin, both possessing Ph moieties. These results demonstrate the great potency of HRP-catalyzed hydrogelation consuming H2O2 supplied in surrounding air for various biomedical applications, especially bioprinting.

Visible Light-Induced Hydrogelation of an Alginate Derivative and Application to Stereolithographic Bioprinting Using a Visible Light Projector and Acid Red.

Visible light-induced hydrogelation is attractive for various biomedical applications. In this study, hydrogels of alginate with phenolic hydroxyl groups (Alg-Ph) were obtained by irradiating a solution containing the polymer, ruthenium II trisbipyridyl chloride ([Ru(bpy)3]2+) and sodium persulfate (SPS), with visible light. The hydrogelation kinetics and the mechanical properties of the resultant hydrogels were tunable by controlling the intensity of the light and the concentrations of [Ru(bpy)3]2+ and SPS. With appropriate concentrations of [Ru(bpy)3]2+ and SPS, the hydrogel could be obtained following approximately 10 s of irradiation using a normal desktop lamp. The hydrogelation process and the resultant hydrogel were cytocompatible; mouse fibroblast cells enclosed in the Alg-Ph hydrogel maintained more than 90% viability for 1 week. The solution containing Alg-Ph, [Ru(bpy)3]2+ and SPS was useful as a bioink for stereolithographic bioprinting. Cell-laden hydrogel constructs could be printed using the bioprinting system equipped with a visible light projector without a significant decrease in cell viability in the presence of photoabsorbent Acid Red 18. The hydrogel construct including a perfusable helical lumen of 1 mm in diameter could be fabricated using the printing system. These results demonstrate the significant potential of this visible light-induced hydrogelation system and the stereolithographic bioprinting using the hydrogelation system for tissue engineering and regenerative medicine.

Displaying a recombinant protein on flocs self-produced by Escherichia coli through fused expression with elongation factor Ts.

The utility of engineering flocculation is wildly recognized in applied and environmental microbiology. We previously reported self-produced flocculation of Escherichia coli cells by overexpressing the native bcsB gene that encodes a component of the cellulose synthesis pathway. Further experiments clarified that the spontaneous E. coli flocs were proteinous, and elongation factor Ts (Tsf) was the main component. In this study, we demonstrated successful expression of a fusion protein consisting of Tsf and green fluorescence protein (GFP) on E. coli flocs. Interestingly, the percentage of Tsf-GFP in total floc protein reached approximately 15% (w/w). The proposed design of a fusion protein with Tsf enables displaying a recombinant target protein on the floc structure.

Drop-On-Drop Multimaterial 3D Bioprinting Realized by Peroxidase-Mediated Cross-Linking.

A cytocompatible inkjet bioprinting approach that enables the use of a variety of bioinks to produce hydrogels with a wide range of characteristics is developed. Stabilization of bioinks is caused by horseradish peroxidase (HRP)-catalyzed cross-linking consuming hydrogen peroxide (H2 O2 ). 3D cell-laden hydrogels are fabricated by the sequential dropping of a bioink containing polymer(s) cross-linkable through the enzymatic reaction and H2 O2 onto droplets of another bioink containing the polymer, HRP, and cells. The ≈95% viability of enclosed mouse fibroblasts and subsequent elongation of the cells in a bioprinted hydrogel consisting of gelatin and hyaluronic acid derivatives suggest the high cytocompatibility of the developed printing approach. The existence of numerous polymers, including derivatives of polysaccharides, proteins, and synthetic polymers, cross-linkable through the HRP-catalyzed reaction, means the current approach shows great promise for biofabrication of functional and structurally complex tissues.

Differentiation potential of human adipose stem cells bioprinted with hyaluronic acid/gelatin-based bioink through microextrusion and visible light-initiated crosslinking.

Bioprinting has a great potential to fabricate three-dimensional (3D) functional tissues and organs. In particular, the technique enables fabrication of 3D constructs containing stem cells while maintaining cell proliferation and differentiation abilities, which is believed to be promising in the fields of tissue engineering and regenerative medicine. We aimed to demonstrate the utility of the bioprinting technique to create hydrogel constructs consisting of hyaluronic acid (HA) and gelatin derivatives through irradiation by visible light to fabricate 3D constructs containing human adipose stem cells (hADSCs). The hydrogel was obtained from a solution of HA and gelatin derivatives possessing phenolic hydroxyl moieties in the presence of ruthenium(II) tris-bipyridyl dication and sodium ammonium persulfate. hADSCs enclosed in the bioprinted hydrogel construct elongated and proliferated in the hydrogel. In addition, their differentiation potential was confirmed by examining the expression of pluripotency marker genes and cell surface marker proteins, and differentiation to adipocytes in adipogenic differentiation medium. Our results demonstrate the great potential of the bioprinting method and the resultant hADSC-laden HA/gelatin constructs for applications in tissue engineering and regenerative medicine.

Quantitative Evaluation of Recombinant Protein Packaged into Outer Membrane Vesicles of Escherichia coli Cells.

Outer membrane vesicles (OMVs) are spherical bilayered proteolipids released from the cell surfaces of bacteria, which have gained traction in the biotechnology fields. Bacterial cellular machinery can be genetically engineered to produce and package heterologous enzymes into OMVs, producing nanocarriers and nanoparticle catalysts. However, the productivity or efficiency of packaging the target protein into OMVs has not been quantitatively evaluated. In this study, we packaged green fluorescence protein (GFP) into the OMVs of Escherichia coli through N-terminal fused expression to outer membrane protein W (OmpW). The OMV productivity and amount of OmpW-GFP packaged in the OMVs were quantitatively compared between two hypervesiculating mutant strains ΔnlpI and ΔdegP. Both strains increased the OMV production, but the ΔnlpI strain additionally enhanced the packaging of OmpW-GFP into OMVs. It was further confirmed that Spr, a peptidoglycan endopeptidase, plays an important role in the enhanced packaging of OmpW-GFP into OMVs through the increased OmpW-GFP expression on the ΔnlpI cells. Finally, the amount of OmpW-GFP released in the OMV fraction of both mutants was determined in terms of the OMV productivity and the packaging efficiency of OmpW-GFP into OMVs. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:51-57, 2018.

Cell-laden microgel prepared using a biocompatible aqueous two-phase strategy.

Microfluidic methods are frequently used to produce cell-laden microgels for various biomedical purposes. Such microfluidic methods generally employ oil-water systems. The poor distribution of crosslinking reagents in the oil phase limits the available gelation strategies. Extracting the microgel from the oil-phase also reduces its production efficiency. In this study, an aqueous two-phase system (ATPS) involving dextran (DEX) and polyethylene glycol (PEG) was used to prepare cell-laden microgel. This avoided the problems associated with an oil phase. The microgel precursor polymers and crosslinking reagents were dispersed in the DEX and PEG phases, respectively. The ultra-low interfacial tension of the ATPS hindered droplet formation. A co-flow microfluidic device was fabricated to overcome this problem. The device incorporated a square-wave-changing injection force, to improve the efficiency of droplet formation. The microgel precursor (including alginate and carboxymethyl cellulose derivatives possessing phenolic hydroxyl moieties) could be dispersed in the DEX solution at various concentrations. Uniform droplets were formed with controllable diameters, and were sequentially converted to microgel by horseradish peroxidase-catalyzed crosslinking. Cells were dispersed in the DEX phase with the microgel precursor polymer, and retained their high viability and proliferation in the resulting microgel. The solubility of gelatin derivatives in the DEX phase was low, but was sufficient to impart cell adhesion properties on the microgel.

Fabrication of single and bundled filament-like tissues using biodegradable hyaluronic acid-based hollow hydrogel fibers.

Hydrogel fibers with biodegradable and biocompatible features are useful for the fabrication of filament-like tissues. We developed cell-laden hyaluronic acid (HA)-based hollow hydrogel fibers to create single and bundled filament-like tissues. The cell-laden fibers were fabricated by crosslinking phenolic-substituted hyaluronic acid (HA-Ph) in an aqueous solution containing cells through a horseradish peroxidase (HRP)-catalyzed reaction in the presence of catalase by extruding the solution in ambient flow of an aqueous solution containing H2O2. The encapsulated cells proliferated and grew within the hollow core, and the cells formed filament-like constructs in both single and bundled fibers, which were obtained by collection on a rotating cylindrical tube. Single and bundled filament-like tissues covered with an additional heterogeneous cell layer were obtained by degrading the fiber membrane using hyaluronidase after covering the fiber surface with heterogeneous cells. Cellular viability was preserved during HA-Ph hydrogel fiber fabrication and filament-like tissue formation. These results demonstrate the feasibility of HA-based hollow hydrogel fibers obtained through HRP- and catalase-mediated reactions to engineer filament-like tissues.

Impact of immobilizing of low molecular weight hyaluronic acid within gelatin-based hydrogel through enzymatic reaction on behavior of enclosed endothelial cells.

The hydrogels having the ability to promote migration and morphogenesis of endothelial cells (ECs) are useful for fabricating vascularized dense tissues in vitro. The present study explores the immobilization of low molecular weight hyaluronic acid (LMWHA) derivative within gelatin-based hydrogel to stimulate migration of ECs. The LMWHA derivative possessing phenolic hydroxyl moieties (LMWHA-Ph) was bound to gelatin-based derivative hydrogel through the horseradish peroxidase-catalyzed reaction. The motility of ECs was analyzed by scratch migration assay and microparticle-based cell migration assay. The incorporated LMWHA-Ph molecules within hydrogel was found to be preserved stably through covalent bonds during incubation. The free and immobilized LMWHA-Ph did not lose an inherent stimulatory effect on human umbilical vein endothelial cells (HUVECs). The immobilized LMWHA-Ph within gelatin-based hydrogel induced the high motility of HUVECs, accompanied by robust cytoskeleton extension, and cell subpopulation expressing CD44 cell receptor. In the presence of immobilized LMWHA-Ph, the migration distance and the number of existing HUVECs were demonstrated to be encouraged in dose-dependent and time-dependent manners. Based on the results obtained in this work, it was concluded that the enzymatic immobilization of LMWHA-Ph within gelatin-based hydrogel represents a promising approach to promote ECs' motility and further exploitation for vascular tissue engineering applications.

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.

Inkjetting Plus Peroxidase-Mediated Hydrogelation Produces Cell-Laden, Cell-Sized Particles with Suitable Characters for Individual Applications.

The authors report a method to prepare cell-laden, cell-sized microparticles from various materials suitable for individual applications. The method includes a piezoelectric inkjetting technology and a horseradish peroxidase (HRP)-catalyzed crosslinking reaction. The piezoelectric inkjetting technology enables production of cell-laden, cell-sized (20-60 µm) droplets from a polymer aqueous solution. The HRP-catalyzed crosslinking of the polymer in the ejected solution enables production of spherical microparticles from various materials. Superior cytocompatibility of the microencapsulation method is confirmed from the viability and growth profiles of normal murine mammary gland epithelial cells.

5,5-Dithiobis(2-nitrobenzoic acid) pyrene derivative-carbon nanotube electrodes for NADH electrooxidation and oriented immobilization of multicopper oxidases for the development of glucose/O2 biofuel cells.

We report the functionalization of multi-walled carbon nanotubes (MWCNTs) electrodes by a bifunctional nitroaromatic molecule accomplished via π-π interactions of a pyrene derivative. DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) has the particularity to possess both electroactivable nitro groups and negatively charged carboxylic groups. The integration of the DTNB-modified MWCNTs was evaluated for different bioelectrocatalytic systems. The immobilized DTNB-based electrodes showed electrocatalytic activity toward the oxidation of the reduced form of nicotinamide adenine dinucleotide (NADH) with low overpotential of -0.09V vs Ag/AgCl at neutral pH. Glucose dehydrogenase was successfully immobilized at the surface of DTNB-based electrodes and, in the presence of NAD+, the resulting bioelectrode achieved efficient glucose oxidation with high current densities of 2.03mAcm-2. On the other hand, the aromatic structure and the negatively charged nature of the DTNB provoked orientation of both laccase and bilirubin oxidase onto the electrode, which enhanced their ability to undergo a direct electron transfer for oxygen reduction. Due to the proper orientation, low overpotentials were obtained (ca. 0.6V vs Ag/AgCl) and high electrocatalytic currents of about 3.5mAcm-2 were recorded at neutral pH in O2 saturated conditions for bilirubin oxidase electrodes. The combination of these bioanodes and bilirubin oxidase biocathodes provided glucose/O2 enzymatic biofuel cells (EBFC) exhibiting an open-circuit potential of 0.640V, with an associated maximum current density of 2.10mAcm-2. Moreover, the fuel cell delivered a maximum power density of 0.50mWcm-2 at 0.36 V.

Deletion of degQ gene enhances outer membrane vesicle production of Shewanella oneidensis cells.

Shewanella oneidensis is a Gram-negative facultative anaerobe that can use a wide variety of terminal electron acceptors for anaerobic respiration. In this study, S. oneidensis degQ gene, encoding a putative periplasmic serine protease, was cloned and expressed. The activity of purified DegQ was inhibited by diisopropyl fluorophosphate, a typical serine protease-specific inhibitor, indicating that DegQ is a serine protease. In-frame deletion and subsequent complementation of the degQ were carried out to examine the effect of envelope stress on the production of outer membrane vesicles (OMVs). Analysis of periplasmic proteins from the resulting S. oneidensis strain showed that deletion of degQ induced protein accumulation and resulted in a significant decrease in protease activity within the periplasmic space. OMVs from the wild-type and mutant strains were purified and observed by transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the OMVs showed a prominent band at ~37 kDa. Nanoliquid chromatography-tandem mass spectrometry analysis identified three outer membrane porins (SO3896, SO1821, and SO3545) as dominant components of the band, suggesting that these proteins could be used as indices for comparing OMV production by S. oneidensis strains. Quantitative evaluation showed that degQ-deficient cells had a fivefold increase in OMV production compared with wild-type cells. Thus, the increased OMV production following the deletion of DegQ in S. oneidensis may be responsible for the increase in envelope stress.

Antibiofilm effect of warfarin on biofilm formation of Escherichia coli promoted by antimicrobial treatment.

Enhancement of microbial biofilm formation by low antimicrobial doses is a critical problem in the medical field. The objective of this study was to propose a new drug candidate against the biofilm formation promoted by subinhibitory dose of antimicrobials. To determine the effect on biofilm formation of Escherichia coli, a subinhibitory concentration of lactoferrin (LF), a milk protein involved in a broad range of biological properties including antimicrobial action, or ampicillin (AMP), a typical antibiotic, was added to an E. coli cell culture in a 96-well microtiter plate. On the other hand, warfarin (WARF), an oral anticoagulant, or polymyxin B (PMB), a strong antibiotic for biofilm treatment, was added as an antagonist against the biofilm promoted by LF or AMP. The amount of biofilm formed at 100µg/mL LF in lysogeny broth medium was four times higher than in the absence of LF. Meanwhile, it was found that WARF suppressed the LF-promoted biofilm formation to a level comparable with the LF-free condition. WARF worked in a similar manner to PMB, which is known as an antibiofilm agent. Furthermore, WARF could also suppress the biofilm promoted by AMP. In conclusion, this study suggests that WARF can work as an antibiofilm agent against the biofilm formation promoted by subinhibitory dose of antimicrobials.

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.