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.
Cell sheets have immense potential for medical and pharmaceutical applications including tissue regeneration, drug testing, and disease modelling. In this study, composite hydrogels were prepared ...from a mixture of phenolated hyaluronic acid (HA-Ph) and gelatin (Gelatin-Ph), with a controlled degree of polymer crosslinking and degradation, to fabricate muscle cell sheets from myoblasts. These hydrogels were obtained
via
hydrogen peroxide (H
2
O
2
)-mediated crosslinking catalysed by horseradish peroxidase (HRP) and peroxide-mediated cleavage of the polymer chains. The degrees of crosslinking and degradation were modulated by altering the exposure time to air containing H
2
O
2
. The results showed that exposing a solution of 2% w/v HA-Ph, 0.75% w/v Gelatin-Ph, and 1 unit mL
−1
HRP to air with 16 ppm H
2
O
2
for 60 min yielded a stiffer hydrogel (7.16 kPa Young's modulus) than exposure times of 15 min (0.46 kPa) and 120 min (3.98 kPa). Moreover, mouse myoblast C2C12 cells cultured on a stiff hydrogel and induced to undergo myogenic differentiation formed longer and higher-density myotubes than those on softer hydrogels. The cell sheets were readily detached within 5 min by immersing the HA-Ph/Gelatin-Ph hydrogels covered with a monolayer of cells in a medium containing hyaluronidase. Our findings demonstrate that composite hydrogels with properties tuned by controlling the exposure time to H
2
O
2
, show great promise as platforms for muscle cell sheet fabrication.
Hyaluronic acid/gelatin composite hydrogels with properties tuned by controlling the exposure time to H
2
O
2
show great promise as platforms for muscle cell sheet fabrication.
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.
Silk fibroin (SF) is an attractive material for composing bioinks suitable for three-dimensional (3D) bioprinting. However, the low viscosity of SF solutions obtained through common dissolution ...methods limits 3D-bioprinting applications without the addition of thickeners or partial gelation beforehand. Here, we report a method of 3D bioprinting low-viscosity SF solutions without additives. We combined a method of freeform reversible embedding of suspended hydrogels, known as the FRESH method, with horseradish peroxidase-catalyzed cross-linking. Using this method, we successfully fabricated 3D SF hydrogel constructs from low-viscosity SF ink (10% w/w, 50 mPa s at 1 s–1 shear rate), which does not yield 3D constructs when printed onto a plate in air. Studies using mouse fibroblasts confirmed that the printing process was cell-friendly. Additionally, cells enclosed in printed SF hydrogel constructs maintained > 90% viability for 11 days of culture. These results demonstrate that the 3D bioprinting technique developed in this study enables new 3D bioprinting applications using SF inks and thus has a great potential to contribute to tissue engineering and regenerative medicine.
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.
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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.
Tissue engineering has great potential to create tissue and organ constructs of clinically relevant sizes. The main obstacle to creating volumetric tissue constructs was the lack of a technique for ...fabricating dense and perfusable vascular networks in vitro within the constructs. In significant efforts to develop such a technique, hydrogels have been used as materials for templates and support architectures of vascular-like networks because of their excellent properties, such as high biocompatibility, flexibility, and the rapid diffusion of oxygen and nutrients compared with solid materials. Herein, we reviewed current hydrogel-based strategies to fabricate vascular-like networks in vitro. The first strategy was based on the ability of vascular endothelial cells to form capillary-like tubes. The second was an engineering-based strategy that can be categorized into templating, modular assembly, microfabrication, rapid prototyping technique, and their hybrid model. Finally, we discussed future directions in tissue engineering for creating transplantable and volumetric constructs.
Abstract A phenolic hydroxyl group was incorporated into gelatin, using aqueous-phase carbodiimide activation chemistry, to obtain in situ gellable and injectable protein-based materials for drug ...delivery and tissue engineering applications. By this means, gelatin derivatives that were gellable via a peroxidase-catalyzed reaction were obtained. The enzymatically cross-linked gelatin gels did not melt at 37 °C and showed tunable proteolytic degradability. The time necessary for gelation decreased with increasing content of the phenolic hydroxyl (Ph) group, peroxidase concentration and decreasing H2 O2 concentration. Resistance to gel compression also depended on the content of Ph groups, with the gel containing the lowest Ph group content showing the greatest resistance to compression. We encapsulated L929 fibroblast cells in gelatin gels under conditions that induced gelation in about 10 s. The encapsulated cells showed about 95% viability. In addition, L929 cells seeded on the gels showed the same growth profiles as those seeded on an unmodified gelatin-coated dish. Subcutaneous rodent injection experiments demonstrated successful in situ formation of gels at the injected site.
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)
) 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)
. 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.
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