Injectable Gelatin Microgel-Based Composite Ink for 3D Bioprinting in Air.

Injectable hydrogels have attracted much attention in tissue engineering and regenerative medicine for their capability to replace implantation surgeries with a minimally invasive injection procedure and ability to fill irregular defects. The proposed composite ink is a gelatin microgel-based yield-stress and shear-thinning composite material that is injectable and solidifies quickly after injection at room temperature, which can be utilized for the creation of three-dimensional parts in air directly. The gelatin composite ink consists of a microgel solid phase (gelled gelatin microgels) and a cross-linkable solution phase (gelatin solution-based acellular or cellular suspension). The gelatin composite ink can be injected or printed directly in air and solidifies as physical cross-linking to hold printed structures at room temperature. The fabricated part further undergoes a chemical cross-linking process when immersed in a transglutaminase solution to enzymatically gel the gelatin solution, making a physiologically stable construct as needed. Lattice, tube-shaped, cup-shaped, and human anatomical (ear and nose) structures are printed to demonstrate the feasibility of the proposed composite ink for printing applications. The morphology and metabolic activity of cells cultured in the gelatin composite ink are further analyzed to confirm the suitability of the proposed composite ink to provide a beneficial physiological environment for bioprinting needs.

Cross-Linkable Microgel Composite Matrix Bath for Embedded Bioprinting of Perfusable Tissue Constructs and Sculpting of Solid Objects.

Tissue engineering is a rapidly growing field, which requires advanced fabrication technologies to generate cell-laden tissue analogues with a wide range of internal and external physical features including perfusable channels, cavities, custom shapes, and spatially varying material and/or cell compositions. A versatile embedded printing methodology is proposed in this work for creating custom biomedical acellular and cell-laden hydrogel constructs by utilizing a biocompatible microgel composite matrix bath. A sacrificial material is patterned within a biocompatible hydrogel precursor matrix bath using extrusion printing to create three-dimensional features; after printing, the matrix bath is cross-linked, and the sacrificial material is flushed away to create perfusable channels within the bulk composite hydrogel matrix. The composite matrix bath material consists of jammed cross-linked hydrogel microparticles (microgels) to control rheology during fabrication along with a fluid hydrogel precursor, which is cross-linked after fabrication to form the continuous phase of the composite hydrogel. For demonstration, gellan or enzymatically cross-linked gelatin microgels are utilized with a continuous gelatin hydrogel precursor solution to make the composite matrix bath herein; the composite hydrogel matrix is formed by cross-linking the continuous gelatin phase enzymatically after printing. A variety of features including discrete channels, junctions, networks, and external contours are fabricated in the proposed composite matrix bath using embedded printing. Cell-laden constructs with printed features are also evaluated; the microgel composite hydrogel matrices support cell activity, and printed channels enhance proliferation compared to solid constructs even in static culture. The proposed method can be expanded as a solid object sculpting method to sculpt external contours by printing a shell of sacrificial ink and further discarding excess composite hydrogel matrix after printing and cross-linking. While aqueous alginate solution is used as a sacrificial ink, more advanced sacrificial materials can be utilized for better printing resolution.

Gellan Fluid Gel as a Versatile Support Bath Material for Fluid Extrusion Bioprinting.

Biomedical applications of three-dimensional (3D) printing demand complex hydrogel-based constructs laden with living cells. Advanced support materials facilitate the fabrication of such constructs. This work demonstrates the versatility and utility of a gellan fluid gel as a support bath material for fabricating freeform 3D hydrogel constructs from a variety of materials. Notably, the gellan fluid gel support bath can supply sensitive biological cross-linking agents such as enzymes to printed fluid hydrogel precursors for mild covalent hydrogel cross-linking. This mild fabrication approach is suitable for fabricating cell-laden gelatin-based constructs in which mammalian cells can form intercellular contacts within hours of fabrication; cellular activity is observed over several days within printed constructs. In addition, gellan is compatible with a wide range of ionic and thermal conditions, which makes it a suitable support material for ionically cross-linked structures generated by printing alginate-based ink formulations as well as thermosensitive hydrogel constructs formed from gelatin. Ultraviolet irradiation of printed structures within the support bath is also demonstrated for photoinitiated cross-linking of acrylated ink materials. Furthermore, gellan support material performance in terms of printed filament stability and residual support material on constructs is found to be comparable and superior, respectively, to previously reported support materials.

Self-Supporting Nanoclay as Internal Scaffold Material for Direct Printing of Soft Hydrogel Composite Structures in Air.

Three dimensional (3D) bioprinting technology enables the freeform fabrication of complex constructs from various hydrogels and is receiving increasing attention in tissue engineering. The objective of this study is to develop a novel self-supporting direct hydrogel printing approach to extrude complex 3D hydrogel composite structures in air without the help of a support bath. Laponite, a member of the smectite mineral family, is investigated to serve as an internal scaffold material for the direct printing of hydrogel composite structures in air. In the proposed printing approach, due to its yield-stress property, Laponite nanoclay can be easily extruded through a nozzle as a liquid and self-supported after extrusion as a solid. Its unique crystal structure with positive and negative charges enables it to be mixed with many chemically and physically cross-linked hydrogels, which makes it an ideal internal scaffold material for the fabrication of various hydrogel structures. By mixing Laponite nanoclay with various hydrogel precursors, the hydrogel composites retain their self-supporting capacity and can be printed into 3D structures directly in air and retain their shapes before cross-linking. Then, the whole structures are solidified in situ by applying suitable cross-linking stimuli. The addition of Laponite nanoclay can effectively improve the mechanical and biological properties of hydrogel composites. Specifically, the addition of Laponite nanoclay results in a significant increase in the Young's modulus of each hydrogel-Laponite composite: 1.9-fold increase for the poly(ethylene glycol) diacrylate (PEGDA)-Laponite composite, 7.4-fold increase for the alginate-Laponite composite, and 3.3-fold increase for the gelatin-Laponite composite.

Functional Nanoclay Suspension for Printing-Then-Solidification of Liquid Materials.

Additive manufacturing (AM) enables the freeform fabrication of complex structures from various build materials. The objective of this study is to develop a novel Laponite nanoclay-enabled "printing-then-solidification" additive manufacturing approach to extrude complex three-dimensional (3D) structures made of various liquid build materials. Laponite, a member of the smectite mineral family, is investigated to serve as a yield-stress support bath material for the extrusion printing of liquid build materials. Using the printing-then-solidification approach, the printed structure remains liquid and retains its shape with the help of the Laponite support bath. Then the completed liquid structures are solidified in situ by applying suitable cross-linking mechanisms. Finally, the solidified structures are harvested from the Laponite nanoclay support bath for any further processing as needed. Due to its chemical and physical stability, liquid build materials with different solidification/curing/gelation mechanisms can be fabricated in the Laponite bath using the printing-then-solidification approach. The feasibility of the proposed Laponite-enabled printing-then-solidification approach is demonstrated by fabricating several complicated structures made of various liquid build materials, including alginate with ionic cross-linking, gelatin with thermal cross-linking, and SU-8 with photo-cross-linking. During gelatin structure printing, living cells are included and the postfabrication cell viability is above 90%.

Inkjet Bioprinting of 3D Silk Fibroin Cellular Constructs Using Sacrificial Alginate.

Silk fibroin is a natural protein which has shown great promise for tissue engineering but is not printable due to slow gelation or harsh gelation conditions which are not cell-friendly. In this study, a two-step gelation process is proposed for the printing of silk fibroin, which utilizes alginate as a sacrificial hydrogel during an inkjetting-based process. A cell-laden blend of alginate with silk fibroin is utilized to achieve rapid gelation by calcium alginate formation during printing; it is followed by horseradish peroxidase (HRP) catalyzed covalent cross-linking of the fibroin protein at tyrosine residues after printing. This two-step gelation process successfully enables 3D bioprinting of well-defined cell-laden silk fibroin constructs suitable for long-term culture. The constructs remain intact after calcium chelation to liquefy the alginate component, demonstrating the formation of silk fibroin hydrogel. NIH 3T3 fibroblasts proliferate and spread through the hydrogel after printing. Increasing metabolic activity is observed for 5 weeks after printing, and histology shows dense cell populations in cultured constructs. The proposed two-step gelation technique is expected to enable 3D silk fibroin printing for various applications.

In Situ Printing-then-Mixing for Biological Structure Fabrication Using Intersecting Jets.

Although traditional three-dimensional bioprinting technology is suitable for many tissue engineering applications, various biomaterials and constructs call for bioprinting innovations. There is a need for the fabrication of complex structures from reactive biomaterials as well as heterogeneous structures with controlled material compositions. In particular, during reactive material printing, reactive solutions/suspensions that undergo changes in rheological properties or cytocompatibility are not printable using traditional bioprinting approaches that require all components of bioinks to be mixed before deposition. The objective of this study is to develop and implement an intersecting jets-based inkjet bioprinting approach, which enables voxel-resolution printing-then-mixing for the fabrication of biological structures using reactive materials as well as structures having a compositional gradient. Inkjetting is implemented herein as a versatile technique to simultaneously deposit droplets of disparate materials at controlled locations where active collision, mixing, and coalescence occur. For reactive material printing, neural stem cell (NSC) spheres are fabricated from reactive PuraMatrix hydrogel solution and physiological cell suspension, and cell-laden alginate structures are also printed in air directly from reactive sodium alginate and calcium chloride solutions. For heterogeneous structure printing, collagen sheets with a hydroxyapatite (HAP) content gradient are fabricated to demonstrate the unique online control of material composition throughout a structure. It is demonstrated that the proposed bioprinting approach is feasible for applications that utilize reactive materials or require heterogeneous compositions.

Granular gel support-enabled extrusion of three-dimensional alginate and cellular structures.

Freeform fabrication of soft structures has been of great interest in recent years. In particular, it is viewed as a critical step toward the grand vision of organ printing--the on-demand design and fabrication of three-dimensional (3D) human organ constructs for implantation and regenerative medicine. The objective of this study is to develop a novel granular gel support material-enabled, two-step gelation-based 'printing-then-gelation' approach to fabricate 3D alginate structures using filament extrusion. Specifically, a granular Carbopol microgel bath holds the ungelled alginate structure being extruded, avoiding the instantaneous gelation of each printed layer as well as resultant surface tension-induced nozzle clogging. Since Carbopol microgels react with multivalent cations, which are needed for alginate crosslinking, gelatin is introduced as a sacrificial material to make an alginate and gelatin bioink for extrusion, which gels thermally (step-one gelation) to initially stabilize the printed structure for removal from Carbopol. Then gelatin is melted and diffused away while alginate is ionically crosslinked in a 37 °C calcium chloride bath (step-two gelation), resulting in an alginate structure. The proposed 'printing-then-gelation' approach works for alginate structure fabrication, and it is also applicable for the printing of cellular constructs and other similar homogeneous soft structures using a two-step or even multi-step approach. The main conclusions are: (1) 0.8% (w/v) Carbopol bath with a neutral pH value may be most suitable for soft structure printing; (2) it is most effective to use a 0.9% (w/v) NaCl solution to facilitate the removal of residual Carbopol; and (3) alginate structures fabricated using the proposed approach demonstrate better mechanical properties than those fabricated using the conventional 'gelation-while-printing' approach.