Three-dimensional bioprinting uses additive manufacturing techniques for the automated fabrication of hierarchically organized living constructs. The building blocks are often hydrogel-based bioinks, which need to be printed into structures with high shape fidelity to the intended computer-aided design. For optimal cell performance, relatively soft and printable inks are preferred, although these undergo significant deformation during the printing process, which may impair shape fidelity. While the concept of good or poor printability seems rather intuitive, its quantitative definition lacks consensus and depends on multiple rheological and chemical parameters of the ink. This review discusses qualitative and quantitative methodologies to evaluate printability of bioinks for extrusion- and lithography-based bioprinting. The physicochemical parameters influencing shape fidelity are discussed, together with their importance in establishing new models, predictive tools and printing methods that are deemed instrumental for the design of next-generation bioinks, and for reproducible comparison of their structural performance.

Printability and Shape Fidelity of Bioinks in 3D Bioprinting.

Bioprinting is rapidly being adopted as a major method for fabricating tissue engineering constructs. Through the precise deposition of cell- and bioactive molecule-laden materials, bioprinting offers researchers a means to create biological constructs with enhanced spatial complexity that more closely mimics native tissue. The vast majority of materials used in bioprinting have been polymers due to their suitability toward resembling the cellular environment and the variety of methods available to process polymeric systems in ambient or relatively mild chemical and environmental conditions. In this review, we will discuss in detail the wide variety of natural and synthetic polymers that have been employed as inks in bioprinting. We will review recent bioprinting innovations, such as increasing architectural complexity and cell viability in heterogeneous tissue constructs, which allow for the investigation of biological questions that could not be addressed before. We will also survey nascent fields of study that promise to further advance the development of novel biofabrication technologies in the field, such as 4D bioprinting and the inclusion of nanomaterials. To conclude, we will examine some of the necessary steps that must take place to bring this technology to commercial markets and facilitate its use in clinical therapies.

Polymeric Systems for Bioprinting.

A major challenge in three-dimensional (3D) bioprinting is the limited number of bioinks that fulfill the physicochemical requirements of printing while also providing a desirable environment for encapsulated cells. Here, we address this limitation by temporarily stabilizing bioinks with a complementary thermo-reversible gelatin network. This strategy enables the effective printing of biomaterials that would typically not meet printing requirements, with instrument parameters and structural output largely independent of the base biomaterial. This approach is demonstrated across a library of photocrosslinkable bioinks derived from natural and synthetic polymers, including gelatin, hyaluronic acid, chondroitin sulfate, dextran, alginate, chitosan, heparin, and poly(ethylene glycol). A range of complex and heterogeneous structures are printed, including soft hydrogel constructs supporting the 3D culture of astrocytes. This highly generalizable methodology expands the palette of available bioinks, allowing the biofabrication of constructs optimized to meet the biological requirements of cell culture and tissue engineering.

Expanding and optimizing 3D bioprinting capabilities using complementary network bioinks

Bioprinting is a tool increasingly used in tissue engineering laboratories around the world As an extension to classic tissue engineering, it enables high levels of control over the spatial deposition of cells, materials, and other factors It is a field with huge promise for the production of implantable tissues and even organs, but the availability of functional bioinks is a barrier to success Extrusion bioprinting is the most commonly used technique, where high-viscosity solutions of materials and cells are required to ensure good shape fidelity of the printed tissue construct This is contradictory to hydrogels used in tissue engineering, which are generally of low viscosity prior to cross-linking to ensure cell viability, making them not directly translatable to bioprinting This review provides an overview of the important rheological parameters for bioinks and methods to assess printability, as well as the effect of bioink rheology on cell viability Developments over the last five years in bioink formulations and the use of suspended printing to overcome rheological limitations are then discussed

/pdf/the-rheology-of-direct-and-suspended-extrusion-bioprinting-1jtnd60bn4.pdf

The rheology of direct and suspended extrusion bioprinting

Sustaining the vital functions of cells outside the organism requires strictly defined parameters In order to ensure their optimal growth and development, it is necessary to provide a range of nutrients and regulators Hydrogels are excellent materials for 3D in vitro cell cultures Their ability to retain large amounts of liquid, as well as their biocompatibility, soft structures, and mechanical properties similar to these of living tissues, provide appropriate microenvironments that mimic extracellular matrix functions The wide range of natural and synthetic polymeric materials, as well as the simplicity of their physico-chemical modification, allow the mechanical properties to be adjusted for different requirements Sodium alginate-based hydrogel is a frequently used material for cell culture The lack of cell-interactive properties makes this polysaccharide the most often applied in combination with other materials, including gelatin The combination of both materials increases their biological activity and improves their material properties, making this combination a frequently used material in 3D printing technology The use of hydrogels as inks in 3D printing allows the accurate manufacturing of scaffolds with complex shapes and geometries The aim of this paper is to provide an overview of the materials used for 3D cell cultures, which are mainly alginate–gelatin hydrogels, including their properties and potential applications

https://www.mdpi.com/1996-1944/14/4/858/pdf

A Review on the Adaption of Alginate-Gelatin Hydrogels for 3D Cultures and Bioprinting.

An advantage of bioprinting is the ability to incorporate cells into the hydrogel bioink allowing for precise control over cell placement within a construct. Previous work found that the printability of collagen bioinks is highly dependent on their rheological properties. The effect of cell density on collagen rheological properties and, therefore, printability has not been assessed. Therefore, the objective of this study was to determine the effects of incorporating cells on the rheology and printability of collagen bioinks. Primary chondrocytes, at densities relevant to cartilage tissue engineering (up to 100 × 106 cells ml-1), were incorporated into 8 mg ml-1 collagen bioinks. Bioink rheological properties before, during, and after gelation as well as printability were assessed. Cell-laden printed constructs were also cultured for up to 14 d to assess longer-term cell behavior. The addition of cells resulted in an increase in the storage modulus and viscosity of the collagen before gelation. However, the storage modulus after gelation and the rate of gelation decreased with increasing cell density. Theoretical models were compared to the rheological data to suggest frameworks that could be used to predict the rheological properties of cell-laden bioinks. Printability testing showed that improved printability could be achieved with higher cell densities. Fourteen-day culture studies showed that the printing process had no adverse effects on the viability or function of printed cells. Overall, this study shows that collagen bioinks are conducive to bioprinting with a wide range of cell densities while maintaining high printability and chondrocyte viability and function.

https://iopscience.iop.org/article/10.1088/1758-5090/ab3524/pdf

High density cell seeding affects the rheology and printability of collagen bioinks

Heterogeneous matrix deposition in human tissue engineered cartilage changes the local shear modulus and resistance to local construct buckling.

The Role of Buckling Instabilities in the Global and Local Mechanical Response in Porous Collagen Scaffolds

The influence of chondrocyte source on the manufacturing reproducibility of human tissue engineered cartilage.

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Papers

High density cell seeding affects the rheology and printability of collagen bioinks

Heterogeneous matrix deposition in human tissue engineered cartilage changes the local shear modulus and resistance to local construct buckling.

The Role of Buckling Instabilities in the Global and Local Mechanical Response in Porous Collagen Scaffolds

The influence of chondrocyte source on the manufacturing reproducibility of human tissue engineered cartilage.