Extrusion Bioprinting of Shear-Thinning Gelatin Methacryloyl Bioinks.
ABSTRACT: Bioprinting is an emerging technique for the fabrication of 3D cell-laden constructs. However, the progress for generating a 3D complex physiological microenvironment has been hampered by a lack of advanced cell-responsive bioinks that enable bioprinting with high structural fidelity, particularly in the case of extrusion-based bioprinting. Herein, this paper reports a novel strategy to directly bioprint cell-laden gelatin methacryloyl (GelMA) constructs using bioinks of GelMA physical gels (GPGs) achieved through a simple cooling process. Attributed to their shear-thinning and self-healing properties, the GPG bioinks can retain the shape and form integral structures after deposition, allowing for subsequent UV crosslinking for permanent stabilization. This paper shows the structural fidelity by bioprinting various 3D structures that are typically challenging to fabricate using conventional bioinks under extrusion modes. Moreover, the use of the GPG bioinks enables direct bioprinting of highly porous and soft constructs at relatively low concentrations (down to 3%) of GelMA. It is also demonstrated that the bioprinted constructs not only permit cell survival but also enhance cell proliferation as well as spreading at lower concentrations of the GPG bioinks. It is believed that such a strategy of bioprinting will provide many opportunities in convenient fabrication of 3D cell-laden constructs for applications in tissue engineering, regenerative medicine, and pharmaceutical screening.
Project description:Bioinks with shear-thinning/rapid solidification properties and strong mechanics are usually needed for the bioprinting of three-dimensional (3D) cell-laden constructs. As such, it remains challenging to generate soft constructs from bioinks at low concentrations that are favorable for cellular activities. Herein, we report a strategy to fabricate cell-laden constructs with tunable 3D microenvironments achieved by bioprinting of gelatin methacryloyl (GelMA)/alginate core/sheath microfibers, where the alginate sheath serves as a template to support and confine the GelMA pre-hydrogel in the core during the extrusion process, allowing for subsequent UV crosslinking. This novel strategy minimizes the bioprinting requirements for the core bioink, and facilitates the fabrication of cell-laden GelMA constructs at low concentrations. We first showed the capability of generating various alginate hollow microfibrous constructs using a coaxial nozzle setup, and verified the diffusibility and perfusability of the bioprinted hollow structures that are important for the tissue engineering applications. More importantly, the hollow alginate microfibers were then used as templates for generating cell-laden GelMA constructs with soft microenvironments, by using GelMA pre-hydrogel as the bioink for the core phase during bioprinting. As such, GelMA constructs at extremely low concentrations (<2.0%) could be extruded to effectively support cellular activities including proliferation and spreading for various cell types. We believe that our strategy is likely to provide broad opportunities in bioprinting of 3D constructs with cell-favorable microenvironments for applications in tissue engineering and pharmaceutical screening.
Project description:3D bioprinting technology provides programmable and customizable platforms to engineer cell-laden constructs mimicking human tissues for a wide range of biomedical applications. However, the encapsulated cells are often restricted in spreading and proliferation by dense biomaterial networks from gelation of bioinks. Herein, a cell-benign approach is reported to directly bioprint porous-structured hydrogel constructs by using an aqueous two-phase emulsion bioink. The bioink, which contains two immiscible aqueous phases of cell/gelatin methacryloyl (GelMA) mixture and poly(ethylene oxide) (PEO), is photocrosslinked to fabricate predesigned cell-laden hydrogel constructs by extrusion bioprinting or digital micromirror device-based stereolithographic bioprinting. The porous structure of the 3D-bioprinted hydrogel construct is formed by subsequently removing the PEO phase from the photocrosslinked GelMA hydrogel. Three different cell types (human hepatocellular carcinoma cells, human umbilical vein endothelial cells, and NIH/3T3 mouse embryonic fibroblasts) within the 3D-bioprinted porous hydrogel patterns show enhanced cell viability, spreading, and proliferation compared to the standard (i.e., nonporous) hydrogel constructs. The 3D bioprinting strategy is believed to provide a robust and versatile platform to engineer porous-structured tissue constructs and their models for a variety of applications in tissue engineering, regenerative medicine, drug development, and personalized therapeutics.
Project description:Here, we investigated the usefulness of silk fibroin nanofibers obtained via mechanical grinding of degummed silkworm silk fibers as an additive in bioinks for extrusion three-dimensional (3D) bioprinting of cell-laden constructs. The nanofibers could be sterilized by autoclaving, and addition of the nanofibers improved the shear thinning of polymeric aqueous solutions, independent of electric charge and the content of cross-linkable moieties in the polymers. The addition of nanofibers to bioinks resulted in the fabrication of hydrogel constructs with higher fidelity to blueprints. Mammalian cells in the constructs showed >85% viability independent of the presence of nanofibers. The nanofibers did not affect the morphologies of enclosed cells. These results demonstrate the great potential of silk fibroin nanofibers obtained via mechanical grinding of degummed silkworm silk fibers as an additive in bioinks for extrusion 3D bioprinting.
Project description:A stereolithography-based bioprinting platform for multimaterial fabrication of heterogeneous hydrogel constructs is presented. Dynamic patterning by a digital micromirror device, synchronized by a moving stage and a microfluidic device containing four on/off pneumatic valves, is used to create 3D constructs. The novel microfluidic device is capable of fast switching between different (cell-loaded) hydrogel bioinks, to achieve layer-by-layer multimaterial bioprinting. Compared to conventional stereolithography-based bioprinters, the system provides the unique advantage of multimaterial fabrication capability at high spatial resolution. To demonstrate the multimaterial capacity of this system, a variety of hydrogel constructs are generated, including those based on poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacryloyl (GelMA). The biocompatibility of this system is validated by introducing cell-laden GelMA into the microfluidic device and fabricating cellularized constructs. A pattern of a PEGDA frame and three different concentrations of GelMA, loaded with vascular endothelial growth factor, are further assessed for its neovascularization potential in a rat model. The proposed system provides a robust platform for bioprinting of high-fidelity multimaterial microstructures on demand for applications in tissue engineering, regenerative medicine, and biosensing, which are otherwise not readily achievable at high speed with conventional stereolithographic biofabrication platforms.
Project description:Fabrication of three dimensional (3D) organoids with controlled microarchitectures has been shown to enhance tissue functionality. Bioprinting can be used to precisely position cells and cell-laden materials to generate controlled tissue architecture. Therefore, it represents an exciting alternative for organ fabrication. Despite the rapid progress in the field, the development of printing processes that can be used to fabricate macroscale tissue constructs from ECM-derived hydrogels has remained a challenge. Here we report a strategy for bioprinting of photolabile cell-laden methacrylated gelatin (GelMA) hydrogels. We bioprinted cell-laden GelMA at concentrations ranging from 7 to 15% with varying cell densities and found a direct correlation between printability and the hydrogel mechanical properties. Furthermore, encapsulated HepG2 cells preserved cell viability for at least eight days following the bioprinting process. In summary, this work presents a strategy for direct-write bioprinting of a cell-laden photolabile ECM-derived hydrogel, which may find widespread application for tissue engineering, organ printing and the development of 3D drug discovery platforms.
Project description:Three-dimensional (3D)-bioprinting enables scientists to mimic in vivo micro-environments and to perform in vitro cell experiments under more physiological conditions than is possible with conventional two-dimensional (2D) cell culture. Cell-laden biomaterials (bioinks) are precisely processed to bioengineer tissue three-dimensionally. One primarily used matrix material is sodium alginate. This natural biopolymer provides both fine mechanical properties when gelated and high biocompatibility. Commonly, alginate is 3D bioprinted using extrusion based devices. The gelation reaction is hereby induced by a CaCl? solution in the building chamber after material extrusion. This established technique has two main disadvantages: (1) CaCl? can have toxic effects on the cell-laden hydrogels by oxygen diffusion limitation and (2) good printing resolution in the CaCl? solution is hard to achieve, since the solution needs to be removed afterwards and substituted by cell culture media. Here, we show an innovative approach of alginate bioprinting based on a CaCl? nebulizer. The device provides CaCl? mist to the building platform inducing the gelation. The necessary amount of CaCl? could be decreased as compared to previous gelation strategies and limitation of oxygen transfer during bioprinting can be reduced. The device was manufactured using the MJP-3D printing technique. Subsequently, its digital blueprint (CAD file) can be modified and additive manufactured easily and mounted in various extrusion bioprinters. With our approach, a concept for a more gentle 3D Bioprinting method could be shown. We demonstrated that the concept of an ultrasound-based nebulizer for CaCl? mist generation can be used for 3D bioprinting and that the mist-induced polymerization of alginate hydrogels of different concentrations is feasible. Furthermore, different cell-laden alginate concentrations could be used: Cell spheroids (mesenchymal stem cells) and single cells (mouse fibroblasts) were successfully 3D printed yielding viable cells and stable hydrogels after 24 h cultivation. We suggest our work to show a different and novel approach on alginate bioprinting, which could be useful in generating cell-laden hydrogel constructs for e.g., drug screening or (soft) tissue engineering applications.
Project description:A series of stable and ready-to-use bioinks have been developed based on the xeno-free and tunable hydrogel (VitroGel) system. Cell laden scaffold fabrication with optimized polysaccharide-based inks demonstrated that Ink H4 and RGD modified Ink H4-RGD had excellent rheological properties. Both bioinks were printable with 25-40 kPa extrusion pressure, showed 90% cell viability, shear-thinning and rapid shear recovery properties making them feasible for extrusion bioprinting without UV curing or temperature adjustment. Ink H4-RGD showed printability between 20 and 37 °C and the scaffolds remained stable for 15 days at temperature of 37 °C. 3D printed non-small-cell lung cancer (NSCLC) patient derived xenograft cells (PDCs) showed rapid spheroid growth of size around 500 µm in diameter and tumor microenvironment formation within 7 days. IC<sub>50</sub> values demonstrated higher resistance of 3D spheroids to docetaxel (DTX), doxorubicin (DOX) and erlotinib compared to 2D monolayers of NSCLC-PDX, wild type triple negative breast cancer (MDA-MB-231 WT) and lung adenocarcinoma (HCC-827) cells. Results of flow property, shape fidelity, scaffold stability and biocompatibility of H4-RGD suggest that this hydrogel could be considered for 3D cell bioprinting and also for in-vitro tumor microenvironment development for high throughput screening of various anti-cancer drugs.
Project description:Despite the significant technological advancement in tissue engineering, challenges still exist towards the development of complex and fully functional tissue constructs that mimic their natural counterparts. To address these challenges, bioprinting has emerged as an enabling technology to create highly organized three-dimensional (3D) vascular networks within engineered tissue constructs to promote the transport of oxygen, nutrients, and waste products, which can hardly be realized using conventional microfabrication techniques. Here, we report the development of a versatile 3D bioprinting strategy that employs biomimetic biomaterials and an advanced extrusion system to deposit perfusable vascular structures with highly ordered arrangements in a single-step process. In particular, a specially designed cell-responsive bioink consisting of gelatin methacryloyl (GelMA), sodium alginate, and 4-arm poly(ethylene glycol)-tetra-acrylate (PEGTA) was used in combination with a multilayered coaxial extrusion system to achieve direct 3D bioprinting. This blend bioink could be first ionically crosslinked by calcium ions followed by covalent photocrosslinking of GelMA and PEGTA to form stable constructs. The rheological properties of the bioink and the mechanical strengths of the resulting constructs were tuned by the introduction of PEGTA, which facilitated the precise deposition of complex multilayered 3D perfusable hollow tubes. This blend bioink also displayed favorable biological characteristics that supported the spreading and proliferation of encapsulated endothelial and stem cells in the bioprinted constructs, leading to the formation of biologically relevant, highly organized, perfusable vessels. These characteristics make this novel 3D bioprinting technique superior to conventional microfabrication or sacrificial templating approaches for fabrication of the perfusable vasculature. We envision that our advanced bioprinting technology and bioink formulation may also have significant potentials in engineering large-scale vascularized tissue constructs towards applications in organ transplantation and repair.
Project description:Cell-laden hydrogels are the primary building blocks for bioprinting, and, also termed bioinks, are the foundations for creating structures that can potentially recapitulate the architecture of articular cartilage. To be functional, hydrogel constructs need to unlock the regenerative capacity of encapsulated cells. The recent identification of multipotent articular cartilage-resident chondroprogenitor cells (ACPCs), which share important traits with adult stem cells, represents a new opportunity for cartilage regeneration. However, little is known about the suitability of ACPCs for tissue engineering, especially in combination with biomaterials. This study aimed to investigate the potential of ACPCs in hydrogels for cartilage regeneration and biofabrication, and to evaluate their ability for zone-specific matrix production. Gelatin methacryloyl (gelMA)-based hydrogels were used to culture ACPCs, bone marrow mesenchymal stromal cells (MSCs) and chondrocytes, and as bioinks for printing. Our data shows ACPCs outperformed chondrocytes in terms of neo-cartilage production and unlike MSCs, ACPCs had the lowest gene expression levels of hypertrophy marker collagen type X, and the highest expression of PRG4, a key factor in joint lubrication. Co-cultures of the cell types in multi-compartment hydrogels allowed generating constructs with a layered distribution of collagens and glycosaminoglycans. By combining ACPC- and MSC-laden bioinks, a bioprinted model of articular cartilage was generated, consisting of defined superficial and deep regions, each with distinct cellular and extracellular matrix composition. Taken together, these results provide important information for the use of ACPC-laden hydrogels in regenerative medicine, and pave the way to the biofabrication of 3D constructs with multiple cell types for cartilage regeneration or in vitro tissue models. STATEMENT OF SIGNIFICANCE:Despite its limited ability to repair, articular cartilage harbors an endogenous population of progenitor cells (ACPCs), that to date, received limited attention in biomaterials and tissue engineering applications. Harnessing the potential of these cells in 3D hydrogels can open new avenues for biomaterial-based regenerative therapies, especially with advanced biofabrication technologies (e.g. bioprinting). This study highlights the potential of ACPCs to generate neo-cartilage in a gelatin-based hydrogel and bioink. The ACPC-laden hydrogel is a suitable substrate for chondrogenesis and data shows it has a bias in directing cells towards a superficial zone phenotype. For the first time, ACPC-hydrogels are evaluated both as alternative for and in combination with chondrocytes and MSCs, using co-cultures and bioprinting for cartilage regeneration in vitro. This study provides important cues on ACPCs, indicating they represent a promising cell source for the next generation of cartilage constructs with increased biomimicry.
Project description:The development of a multimaterial extrusion bioprinting platform is reported. This platform is capable of depositing multiple coded bioinks in a continuous manner with fast and smooth switching among different reservoirs for rapid fabrication of complex constructs, through digitally controlled extrusion of bioinks from a single printhead consisting of bundled capillaries synergized with programmed movement of the motorized stage.