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.

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.