Collagen Bioinks for Bioprinting: A Systematic Review of Hydrogel Properties, Bioprinting Parameters, Protocols, and Bioprinted Structure Characteristics

Abstract

Bioprinting is a modern tool suitable for creating cell scaffolds and tissue or organ carriers from polymers that mimic tissue properties and create a natural environment for cell development. A wide range of polymers, both natural and synthetic, are used, including extracellular matrix and collagen-based polymers. Bioprinting technologies, based on syringe deposition or laser technologies, are optimal tools for creating precise constructs precisely from the combination of collagen hydrogel and cells. This review describes the different stages of bioprinting, from the extraction of collagen hydrogels and bioink preparation, over the parameters of the printing itself, to the final testing of the constructs. This study mainly focuses on the use of physically crosslinked high-concentrated collagen hydrogels, which represents the optimal way to create a biocompatible 3D construct with sufficient stiffness. The cell viability in these gels is mainly influenced by the composition of the bioink and the parameters of the bioprinting process itself (temperature, pressure, cell density, etc.). In addition, a detailed table is included that lists the bioprinting parameters and composition of custom bioinks from current studies focusing on printing collagen gels without the addition of other polymers. Last but not least, our work also tries to refute the often-mentioned fact that highly concentrated collagen hydrogel is not suitable for 3D bioprinting and cell growth and development.

Keywords: bioink; bioprinting; bioprinting parameters; collagen; hydrogel; hydrogel properties.

Figure 1

Design and phases of the bioprinting process.

Figure 2

The Prisma workflow diagram of the article search.

Figure 3

Collagen and its non-enzymatic (A) and enzymatic divalent (B) and trivalent (C) nature crosslinks.

Figure 4

Formation of hydrogel from the same source of collagen however isolated by different way. (A) phosphate buffer pre-treatment and extraction by 0.1 M acetic acid; collagen gelled only locally (B) ethanol pre-treatment and extraction by 0.02 M acetic acid followed by neutralization by 0.1 M NaOH to pH = 7, collagen precipitate is centrifugated and dissolved again in 0.02 M acetic acid; collagen gelled completely.

Figure 5

Porcine stromal cells in 3D printed collagen hydrogel (15 mg/mL), substrate coverslip glass, red F-actin, blue cell nuclei. (A) 3D image from confocal microscopy. (B) side projection from confocal microscopy. (C) printing process on substrate fixed using heated vacuum bed. The microscopy image was acquired on Andor Dragonfly spinning disk confocal system with high-speed CMOS camera Zyla 4.2 (Andor Technology Ltd., Belfast, UK) mounted on Leica DMi8 inversed fully mechanized microscope (Leica, Wetzlar, Germany) using 20× lens (NA = 0.75) with water immersion. The wavelengths of the excitation lasers were 405 nm and 561 nm and the transmission characteristics of the emission filters were 450/50 nm and 600/50 nm. Used disk pinhole diameter was 40 µm. The hydrogel samples were photographed in PBS buffer in 35 mm Petri dish with #1.5 glass bottom (0.17 mm thickness, Cellvis, Sunnyvale, CA, USA).

Figure 6

Porcine stromal cells in 3D printed collagen hydrogel with concentrations 10, 20 and 30 mg/mL substrate coverslip glass, red F-actin, blue cell nuclei. Top row shows maximum intensity projection and bottom row show side projection from confocal microscopy. The microscopy images were acquired on Andor Dragonfly spinning disk confocal system with high-speed CMOS camera Zyla 4.2 (Andor Technology Ltd., Belfast, UK) mounted on Leica DMi8 inversed fully mechanized microscope (Leica, Wetzlar, Germany) using 20× lens (NA = 0.75) with water immersion. The wavelengths of the excitation lasers were 405 nm and 561 nm and the transmission characteristics of the emission filters were 450/50 nm and 600/50 nm. Used disk pinhole diameter was 40 µm. The hydrogel samples were photographed in PBS buffer in 35 mm Petri dish with #1.5 glass bottom (0.17 mm thickness, Cellvis, Sunnyvale, CA, USA).

Figure 7

Main additive manufacturing technologies for collagen bioprinting with a description of the main parts of the assemblies.

Figure 8

Modification of amino groups in gelatine/collagen with methacrylic anhydride.