Hydrogel Constructs Research Articles

Optimization of methacrylated gelatin /layered double hydroxides nanocomposite cell-laden hydrogel bioinks with high printability for 3D extrusion bioprinting.

Layered double hydroxides (LDHs) offer unique source of inspiration for design of bone mimetic biomaterials due to their superior mechanical properties, drug delivery capability and regulation cellular behaviors, particularly by divalent metal cations in their structure. Three-dimensional (3D) bioprinting of LDHs holds great promise as a novel strategy thanks to highly tunable physiochemical properties and shear-thinning ability of LDHs, which allow shape fidelity after deposition. Herein, we introduce a straightforward strategy for extrusion bioprinting of cell laden nanocomposite hydrogel bioink of gelatin methacryloyl (GelMA) biopolymer and LDHs nanoparticles. First, we synthesized LDHs by co-precipitation process and systematically examined the effect of LDHs addition on printing parameters such as printing pressure, extrusion rate, printing speed, and finally bioink printability in creating grid-like constructs. The developed hydrogel bioinks provided precise control over extrudability, extrusion uniformity, and structural integrity after deposition. Based on the printability and rheological analysis, the printability could be altered by controlling the concentration of LDHs, and printability was found to be ideal with the addition of 3 wt % LDHs. The addition of LDHs resulted in remarkably enhanced compressive strength from 652 kPa (G-LDH0) to 1168 kPa (G-LDH3). It was shown that the printed nanocomposite hydrogel scaffolds were able to support encapsulated osteoblast survival, spreading, and proliferation in the absence of any osteoinductive factors taking advantage of LDHs. In addition, cells encapsulated in G-LDH3 had a larger cell spreading area and higher cell aspect ratio than those encapsulated in G-LDH0. Altogether, the results demonstrated that the developed GelMA/LDHs nanocomposite hydrogel bioink revealed a high potential for extrusion bioprinting with high structural fidelity to fabricate implantable 3D hydrogel constructs for repair of bone defects.

Filamented Light (FLight) Biofabrication of Highly Aligned Tissue-Engineered Constructs.

Cell-laden hydrogels used in tissue engineering generally lack sufficient 3Dtopographical guidance for cells to mature into aligned tissues. A new strategy called filamented light (FLight) biofabrication rapidly creates hydrogels composed of unidirectional microfilament networks, with diameters on the length scale of single cells. Due to optical modulation instability, a light beam is divided optically into FLight beams. Local polymerization of a photoactive resin is triggered, leading to local increase in refractive index, which itself creates self-focusing waveguides and further polymerization of photoresin into long hydrogel microfilaments. Diameter and spacing of the microfilaments can be tuned from 2 to 30µm by changing the coherence length of the light beam. Microfilaments show outstanding cell instructive properties with fibroblasts, tenocytes, endothelial cells, and myoblasts, influencing cell alignment, nuclear deformation, and extracellular matrix deposition. FLight is compatible with multiple types of photoresins and allows for biofabrication of centimeter-scale hydrogel constructs with excellent cell viability within seconds (<10 s per construct). Multidirectional microfilaments are achievable within a single hydrogel construct by changing the direction of FLight projection, and complex multimaterial/multicellular tissue-engineered constructs are possible by sequentially exchanging the cell-laden photoresin. FLight offers a transformational approach to developing anisotropic tissues using photo-crosslinkable biomaterials.

Highly bioactive cell-laden hydrogel constructs bioprinted using an emulsion bioink for tissue engineering applications

The insufficient pore structure of cell-laden hydrogel scaffolds has limited their application in various tissue regeneration applications owing to low cell-to-cell/matrix interactions and low transfer of nutrients and metabolic wastes. Herein, we designed a highly porous cell-laden hydrogel scaffold fabricated using an emulsion bioink consisting of methacrylated collagen (CMA), mineral oil (MO), and human adipose stem cells (hASCs) to induce efficient cell infiltration and cellular activities. By selecting the most appropriate concentration of CMA and MO, the emulsion bioink can be successfully formulated with proper yield stress and printability. The cell-laden scaffold exhibited significantly greater cell growth and cytoskeletal reorganization than the normally printed cell-laden CMA scaffold. Furthermore, two bioactive components (kartogenin and bone morphogenetic protein-2) were physically encapsulated in the oil droplets of the cell construct, and the molecules in the cell constructs enhanced chondrogenic or osteogenic differentiation of hASCs in the printed structure. Based on these results, the cell-printed structure using an emulsion bioink can not only provide a good cellular microenvironment but also be a new potential method to accelerate stem cell differentiation by combining bioactive molecules and cell-laden scaffolds.

Dot extrusion bioprinting of spatially controlled heterogenous tumor models

Bioprinting of cell-laden hydrogel constructs providing three-dimensional (3D) spatial pattern capacity and suitable cellular microenvironment have become essential tools in the field of tissue engineering. For heterogeneous tissue development, the printing approaches permitting controllable deposition of multicellular components in a flexible manner are urgently needed. Here, a facile dot-extrusion printing (DEP) system for one-step generation of cell-laden gelatin methacrylate (GelMa) hydrogel beads (GHBs) is developed, which allows size-tunable GHBs programmable positioned into complex 3D constructs. The GelMa is in situ semi-gelled at the printhead, thus enabling one-step generation of GHBs onto the platform, as a result of improved printing simplicity, fidelity and flexibility. The size and spatial position of the GHBs are adjustable by programming G-code parameters. Further, by integrating multiple printheads, GHBs encapsulating different cellular components can be printed to fabricate heterogeneous tissue constructs, and maintain the post-printed cell viability over 95%. As an example of application, two different tumor-stroma spatial phases were developed through spatial regionally printing tumor cells and normal fibroblasts into juxtapositional or overlapping microcapsule structures, giving an access to study the complex tumor-stroma interactions in different microenvironments. The developed DEP system thus holds promise for creation of complex and heterogeneous tissues toward various biology studies.

Hydrogel Injection Molding to Generate Complex Cell Encapsulation Geometries.

Biofabrication methods capable of generating complex, three-dimensional, cell-laden hydrogel geometries are often challenging technologies to implement in the clinic and scaled manufacturing processes. Hydrogel injection molding capitalizes on the reproducibility, efficiency, and scalability of the injection molding process, and we adapt this technique to biofabrication using a library of natural and synthetic hydrogels with varied crosslinking chemistries and kinetics. We use computational modeling to evaluate hydrogel library fluid dynamics within the injection molds in order to predict molding feasibility and cytocompatibility. We evaluate the reproducibility of hydrogel construct molding and extraction and establish criteria for the selection of hydrogels suitable for injection molding. We demonstrate that hydrogel injection molding is capable of generating complex three-dimensional cell-laden construct geometries using diverse hydrogel materials and that this platform is compatible with primary human islet encapsulation. These results highlight the versatility and feasibility of hydrogel injection molding as a biofabrication technique with potential applications in the clinic and biomanufacturing.

Chiral fiber supramolecular hydrogels for tissue engineering.

Tissue engineering (TE), as a new interdisciplinary discipline, aims to develop biological substitutes for repairing damaged tissues and organs. For the success of tissue regeneration, such biomaterials need to support the physiological activities of cells and allow the growth and maturation of tissues. Naturally, this regulation is achieved through the dynamic remodeling of the extracellular matrix (ECM) of cells. In recent years, chiral supramolecular hydrogels have shown higher application potential in the TE field than traditional polymer hydrogels due to their dynamic noncovalent interactions, adjustable self-assembly structure, and good biocompatibility. These advantages make it possible to construct hydrogels under physiological conditions with structure and function similar to those of the natural ECM. Meanwhile, the chiral characteristics of hydrogels play an important role in regulating cellular activities such as differentiation, adhesion, and proliferation, which is beneficial for tissue formation. In this review, a brief introduction is presented to highlight the importance of chiral fiber supramolecular hydrogels for TE at first. Afterward, the considerations for chiral supramolecular hydrogel design, as well as the influence of external stimuli on chiral hydrogel construction, are discussed. Finally, the potential application prospects of these materials in TE and the significant contribution made by our group in this field are summarized. This review not only helps to reveal the importance of chiral properties in TE but also provides new strategies for TE research based on chiral bionic microenvironments. This article is categorized under: Implantable Materials and Surgical Technologies > Nanotechnology in Tissue Repair and Replacement Biology-Inspired Nanomaterials > Peptide-Based Structures Nanotechnology Approaches to Biology > Cells at the Nanoscale Therapeutic Approaches and Drug Discovery > Emerging Technologies.

3D printing sacrificial templates for manufacturing hydrogel constructs with channel networks

In hydrogel-based tissue engineering, channel network is an efficient structure for transporting nutrients/oxygen to support cell survival and construct living tissues in vitro. 3D printing can create complex hydrogel-based tissue constructs, however, due to the weak mechanical properties of hydrogel bioinks, cell-laden hydrogel constructs with effective channel networks are difficult to be directly printed. Here, an easy sacrificial 3D printing method based on commercial desktop 3D printer and water-soluble polyvinyl alcohol (PVA) to construct effective hydrogel channel networks is introduced. Specifically, i) commercial PVA consumables are printed to be sacrificial templates; ii) gelatin methacryloyl (GelMA) solution is cast to encapsulate the sacrificial template; iii) the sacrificial template is dissolved to form channel networks. PVA is a water-soluble sacrificial material with sufficient dissolution time and high mechanical strength, which can avoid the disadvantages of classic sacrificial materials (sugar and Pluronic F127). High roundness of printed PVA filaments results in high roundness of the channel networks. The interconnected channel networks accelerate the supply of oxygen/nutrients and promote cell growth. And with a period of culture, the higher the channel networks density, the better the cell growth. Additionally, PVA sacrificial templates have high stability and long-term preservation, facilitating transportation, circulation and use, which is conducive to the manufacturing and promotion of hydrogel-based constructs with channel networks. Taken together, our easy strategy of manufacturing hydrogel constructs with channel networks has broad application prospects, such as 3D cell culture, construction of functional tissue in vitro or tissue repair in vivo etc.

Advanced Hydrogel systems for mandibular reconstruction

Mandibular defect becomes a prevalent maxillofacial disease resulting in mandibular dysfunctions and huge psychological burdens to the patients. Considering the routine presence of oral contaminations and aesthetic restoration of facial structures, the current clinical treatments are however limited, incapable to reconstruct the structural integrity and regeneration, spurring the need for cost-effective mandibular tissue engineering. Hydrogel systems possess great merit for mandibular reconstruction with precise involvement of cells and bioactive factors. In this review, current clinical treatments and distinct mode(s) of mandible formation and pathological resorption are summarized, followed by a review of hydrogel-related mandibular tissue engineering, and an update on the advanced fabrication of hydrogels with improved mechanical property, antibacterial ability, injectable form, and 3D bioprinted hydrogel constructs. The exploration of advanced hydrogel systems will lay down a solid foundation for a bright future with more biocompatible, effective, and personalized treatment in mandibular reconstruction.

3D Bioprinted GelMA-Gelatin-Hydroxyapatite Osteoblast Laden Composite Hydrogels for Bone Tissue Engineering.

Introduction 3D bioprinting offers a novel solution to critical sized bone defects by allowing the placement of osteogenic cells, additive biomaterials and bioactive signaling that mimic native tissue. We describe the suitability of an extrusion-based 3D bioink composed of gelatin methacryloyl (GelMA), gelatin, hydroxyapatite (HA), and osteoblasts for bone tissue engineering. Methods A mouse calvarial osteoblast-laden GelMA-gelatin-LAP bioink consisting of various concentrations of HA was 3D-bioprinted into a porous hydrogel construct. After days 1, 14, and 28, five constructs from each HA concentration were analyzed. The water weight percent differences of the hydrogels and the degradation behavior in enzyme solution were characterized. An ALP assay and histological analysis were performed. Cell survivability was determined using a LIVE/DEAD Viability/Cytoxicity Kit and AlamarBlue Cell Viability reagent. Real-time polymerase chain reaction (RT-qPCR) was performed to measure expression levels of bone morphogenetic protein-7 (BMP-7) and Osteocalcin (BGLAP). Results The addition of 5, 10, and 20 mg/ml of HA significantly reduced hydrogel swelling (p ≤ 0.01) from baseline GelMA-Gelatin hydrogels (Figure 3A). HA significantly decreased hydrogel breakdown in a concentration dependent manner (Figure 3B, p ≤ 0.001). A significant increase in cell proliferation at day 28 was noted in all groups (Figure 3D). ALP activity (Figure 3E) significantly increased with the addition of 5mg/ml and 20mg/ml of HA at days 7 and 28 (p ≤ 0.05). Live/dead staining at 1, 14, and 28 days showed high chondrocyte viability. The addition of 20mg/ml of HA demonstrated significantly greater BMP7 and BGLAP gene expression at both 14 and 28 days over the hydrogels without HA (Figure 5, p ≤ 0.05). Conclusion The addition of HA to GelMA-gelatin hydrogels significantly decreased hydrogel swelling, improved the ability of the hydrogel to resist enzymatic degradation, increased osteoblastic differentiation and mineralization, and increased osteogenic gene expression while maintaining equal cell viability and proliferation to non-HA hydrogels.

Studies on oxygen availability and the creation of natural and artificial oxygen gradients in gelatin-methacryloyl hydrogel 3D cell culture.

Three-dimensional (3D) cultivation platforms allow the creation of cell models, which more closely resemble in vivo-like cell behavior. Therefore, 3D cell culture platforms have started to replace conventional two-dimensional (2D) cultivation techniques in many fields. Besides the advantages of 3D culture, there are also some challenges: cultivation in 3D often results in an inhomogeneous microenvironment and therefore unique cultivation conditions for each cell inside the construct. As a result, the analysis and precise control over the singular cell state is limited in 3D. In this work, we address these challenges by exploring ways to monitor oxygen concentrations in gelatin methacryloyl (GelMA) 3D hydrogel culture at the cellular level using hypoxia reporter cells and deep within the construct using a non-invasive optical oxygen sensing spot. We could show that the appearance of oxygen limitations is more prominent in softer GelMA-hydrogels, which enable better cell spreading. Beyond demonstrating novel or space-resolved techniques of visualizing oxygen availability in hydrogel constructs, we also describe a method to create a stable and controlled oxygen gradient throughout the construct using a 3D printed flow-through chamber.

Extrusion 3D printing of keratin protein hydrogels free of exogenous chemical agents

Keratins are a class of intermediate filament proteins that can be obtained from numerous sources including human hair. Materials fabricated from keratins offer desirable characteristics as scaffolds for tissue engineering, including intrinsic cell adhesion sequences and tunable degradation kinetics. The capacity to create 3D printed constructs from keratin-based bio-inks generates unique opportunities for spatial control of scaffold physicochemical properties to direct scaffold functions in ways not readily achieved through other means. The aim of this study was to leverage the controllable rheological properties of keratin hydrogels to create a strategy for extrusion 3D printing of keratin bio-inks without the use of exogenous rheological modifiers, crosslinking agents, or photocurable resins. The rheological properties of keratin hydrogels were tuned by varying two parameters: (a) the ratio of keratose (obtained by oxidative extraction of keratin) to kerateine (obtained by reductive extraction of keratin); and (b) the weight percentage of total keratin protein in the gel. A computational model of the dispensing nozzle for a commercially available extrusion 3D printer was developed to calculate the needed pneumatic printing pressures based on the known rheological properties of the gels. Keratin hydrogel constructs, of varying keratose/kerateine ratios and total keratin weight percentages, were 3D printed in cylindrical geometries via extrusion 3D printing. Rheology and degradation studies showed that gels with greater relative kerateine content exhibited greater flow resistance and slower degradation kinetics when submerged in phosphate buffered saline solution at 37 °C, owing to the presence of cysteine residues in kerateine and the capability of forming disulfide bonds. Total keratin weight percentage was found to influence gel yield stress, with possible implications for tuning filament fidelity. Findings from this work support the use of keratose/kerateine ratio and total keratin weight percentage as handles for modulating rheological characteristics of keratin hydrogels to enhance printability and control scaffold properties.

Porous hydrogel constructs based on methacrylated gelatin/polyethylene oxide for corneal stromal regeneration

The lack of donor sources for corneal transplantation leads to the development of alternative materials for corneal regeneration and repair. As the stroma occupies the thickest layer of the cornea, biomimetic corneal stromal constructs were widely observed by tissue engineering. In this study, a light-curing porous hydrogel based on the two-phase aqueous emulsion was prepared to induce the regeneration of corneal stroma. Methacrylated gelatin (GelMA) solution containing photoinitiator was mixed with aqueous polyethylene oxide (PEO) solution to form an immiscible two-phase emulsion. We characterized physical and chemical properties, containing the pore size, mechanical properties, degradation properties, and swelling properties, to choose the preparation process of the porous hydrogel. The in vitro studies performed in this study suggested that the porous hydrogels had good biocompatibility and the specific matrix secreted by corneal stromal cells gradually increased, so the porous hydrogels promoted the secretion of extracellular matrix. The hydrogels were inserted in a mid-stromal pocket created by minor surgery to confirm their histocompatibility and application in corneal regeneration and repair.

A 3D bioprinted nano-laponite hydrogel construct promotes osteogenesis by activating PI3K/AKT signaling pathway.

Development of nano-laponite as bioinks based on cell-loaded hydrogels has recently attracted significant attention for promoting bone defect repairs and regeneration. However, the underlying mechanisms of the positive function of laponite in hydrogel was not fully explored. In this study, the effect of 3D bioprinted nano-laponite hydrogel construct on bone regeneration and the potential mechanism was explored in vitro and in vivo. In vitro analyses showed that the 3D construct protected encapsulated cells from shear stresses during bioprinting, promoted cell growth and cell spreading, and BMSCs at a density of 107/mL exhibited an optimal osteogenesis potential. Osteogenic differentiation and ectopic bone formation of BMSCs encapsulated inside the 3D construct were explored by determination of calcium deposition and x-ray, micro-CT analysis, respectively. RNA sequencing revealed that activation of PI3K/AKT signaling pathway of BMSCs inside the laponite hydrogel significantly upregulated expression of osteogenic related proteins. Expression of osteogenic proteins was significantly downregulated when the PI3K/AKT pathway was inhibited. The 3D bioprinted nano-laponite hydrogel construct exhibited a superior ability for bone regeneration in rat bones with defects compared with groups without laponite as shown by micro-CT and histological examination, while the osteogenesis activity was weakened by applications of a PI3K inhibitor. In summary, the 3D bioprinted nano-laponite hydrogel construct promoted bone osteogenesis by promoting cell proliferation, differentiation through activation of the PI3K/AKT signaling pathway.

Ultrasound-Responsive Hydrogels for On-Demand Protein Release.

The development of tunable, ultrasound-responsive hydrogels that can deliver protein payload on-demand when exposed to focused ultrasound is described in this study. Reversible Diels-Alder linkers, which undergo a retro reaction when stimulated with ultrasound, were used to cross-link chitosan hydrogels with entrapped FITC-BSA as a model protein therapeutic payload. Two Diels-Alder linkage compositions with large differences in the reverse reaction energy barriers were compared to explore the influence of linker composition on ultrasound response. Selected physicochemical properties of the hydrogel construct, its basic degradation kinetics, and its cytocompatibility were measured with respect to Diels-Alder linkage composition. Focused ultrasound initiated the retro Diels-Alder reaction, controlling the release of the entrapped payload while also allowing for real-time visualization of the ongoing process. Additionally, increasing the focused ultrasound amplitude and time correlated with an increased rate of protein release, indicating stimuli responsive control.

Inhibition Effect of Ti3C2Tx MXene on Ice Crystals Combined with Laser-Mediated Heating Facilitates High-Performance Cryopreservation

The phenomena of ice formation and growth are of great importance for climate science, regenerative medicine, cryobiology, and food science. Hence, how to control ice formation and growth remains a challenge in these fields and attracts great interest from widespread researchers. Herein, the ice regulation ability of the two-dimensional MXene Ti3C2Tx in both the cooling and thawing processes is explored. Molecularly speaking, the ice growth inhibition mechanism of Ti3C2Tx MXene is ascribed to the formation of hydrogen bonds between functional groups of -O-, -OH, and -F distributed on the surface of Ti3C2Tx and ice/water molecules, which was elucidated by the molecular dynamics simulation method. In the cooling process, Ti3C2Tx can decrease the supercooling degree and inhibit the sharp edge morphology of ice crystals. Moreover, taking advantage of the outstanding photothermal conversion property of Ti3C2Tx, rapid ice melting can be achieved, thus reducing the phenomena of devitrification and ice recrystallization. Based on the ice restriction performance of Ti3C2Tx mentioned above, Ti3C2Tx is applied for cryopreservation of stem-cell-laden hydrogel constructs. The results show that Ti3C2Tx can reduce cryodamage to stem cells induced by ice injury in both the cooling and thawing processes and finally increase the cell viability from 38.4% to 80.9%. In addition, Ti3C2Tx also shows synergetic antibacterial activity under laser irradiation, thus realizing sterile cryopreservation of stem cells. Overall, this work explores the ice inhibition performance of Ti3C2Tx, elucidates the physical mechanism, and further achieves application of Ti3C2Tx in the field of cell cryopreservation.

A transcriptome sequencing study on the effect of macro-pores in hydrogel scaffolds on global gene expression of laden human cartilage chondrocytes

The macro-porous hydrogel scaffolds can not only enhance the proliferation of laden chondrocytes but also favor the deposition of hyaline cartilaginous extracellular matrix, however, the underlying molecular mechanism is still unclear. Herein, the global gene expression of human cartilage chondrocytes (HCCs) encapsulated in traditional hydrogel (Gel) constructs and micro-cavitary gel (MCG) constructs are investigated by using high-throughput RNA sequencing (RNA-seq). The differentially expressed genes (DEGs) between the HCCs cultured in Gel and MCG constructs have been identified via bioinformatics analysis. Significantly, the DEGs that promote cell proliferation (e.g. POSTN, MKI67, KIF20A) or neo-cartilage formation (e.g. COL2, ASPN, COMP, FMOD, FN1), are more highly expressed in MCG constructs than in Gel constructs, while the expressions of the DEGs associated with chondrocyte hypertrophy (e.g. EGR1, IBSP) are upregulated in Gel constructs. The expression of representative DEGs is verified at both mRNA and protein levels. Besides, cellular viability and morphology as well as the enriched signaling pathway of DEGs are studied in detail. These results of this work may provide data for functional tissue engineering of cartilage.

Conductive hydrogel constructs with three-dimensionally connected graphene networks for biomedical applications

Conductive hydrogels afford efficient electrical communication with biological systems while providing soft and hydrated interfaces. However, facile fabrication of the conductive hydrogels of high electrical properties with minimal incorporation of conductive components is still challenge. We developed a conductive hydrogel composed of three-dimensionally connected reduced graphene oxide (rGO) networks using graphene oxide (GO)-coated agarose microbeads and thermal annealing. Self-assembly of GO-coated agarose microbeads to granulate hydrogels and subsequent mild heating allowed the production of conductive hydrogels (named thermally annealed graphene-channeled agarose hydrogel (TAGAH)) containing a three-dimensionally connected rGO network. TAGAH exhibited high electrical conductivity with a small amount of graphene, low impedance, and soft tissue-like elasticity. Various conductive constructs could be easily fabricated by molding and 3D printing. In vitro and in vivo studies revealed their excellent biocompatibility. Moreover, potential biomedical applications of TAGAH-based materials were successfully demonstrated as soft bioelectrodes, pressure sensors, strain sensors, and conductive tissue scaffolds.

Nanoparticle‐Stabilized Emulsion Bioink for Digital Light Processing Based 3D Bioprinting of Porous Tissue Constructs (Adv. Healthcare Mater. 12/2022)

Emulsion Bioinks In article number 2102810 by Rui Liu and co-workers, a nanoparticle-stabilized emulsion bioink via integration of two immiscible aqueous phases is developed to directly bioprint macroporous hydrogel constructs. The bioprinted macroporous hydrogel constructs do excellent service to the proliferation, spreading, and maturation of encapsulated cells in vitro and in vivo.

Acute Response of Engineered Cardiac Tissue to Pressure and Stretch

The heart is a dynamic organ, and the cardiac tissue experiences changes in pressure and stretch during the cardiac cycle. Existing cell culture and animal models are limited in their capacity to decouple and tune specific hemodynamic stresses implicated in the development of physiological and pathophysiological cardiac tissue remodeling. This study focused on creating a system to subject engineered cardiac tissue to either pressure or stretch stimuli in isolation and the subsequent evaluation of acute tissue remodeling. We developed a cardiac tissue chip containing three-dimensional (3-D) cell-laden hydrogel constructs and cultured them within systems where we could expose them to either pressure changes or volume changes as seen in the left ventricle. Acute cellular remodeling with each condition was qualitatively and quantitatively assessed using histology, immunohistochemistry, gene expression studies, and soluble factor analysis. Using our unique model system, we isolated the effects of pressure and stretch on engineered cardiac tissue. Our results confirm that both pressure and stretch mediate acute stress responses in the engineered cardiac tissue. However, both experimental conditions elicited a similar acute phase injury response within this timeframe. This study demonstrates our ability to subject engineered cardiac tissue to either pressure or stretch stimuli in isolation, both of which elicited acute tissue remodeling responses.

One-Step FRESH Bioprinting of Low-Viscosity Silk Fibroin Inks.

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.