BioInks for Extrusion Bioprinters
Recent developments in 3D bioprinting technologies have allowed for enhanced fabrication of scaffolds and 3D tissues. These 3D printers use bioinks, a combination of biomaterials and cells, to create 3D structures that closely mimic naturally complex tissues within the human body. One of the first and most critical steps in the bioprinting process involves selecting which of these bioinks to use (Figure 1).
Figure 1: The bioprinting process. Users design constructs through CAD software, choose bioinks of cells and biomaterials then print designs. After prints, researchers analyze constructs then alter the process based on results.
Bioinks for extrusion bioprinters can be split into four general categories: matrix, curing, sacrificial and support. Each type plays a different role in the bioprinting process. Depending on the desired tissue, researchers will use bioinks from each of these categories to develop 3D tissues.
Matrix bioinks include any biomaterial used for cell encapsulation. In addition to printability, these materials must offer a compatible environment for living cells. Ideally, a matrix bioink will closely mimic the extracellular matrix, the natural environment for cells within the human body. Matrix bioinks must also shield cells from shear stresses during the printing process and offer quick, non-toxic gelation processes for optimal print resolution.
Most matrix bioinks are naturally derived hydrogels, such as collagen, hyaluronic acid or alginate (Table 1). Natural bioinks offer advantages such as excellent cell viability, but can have large batch-to-batch variability, affecting print parameters and mechanical properties. Therefore, some researchers use synthetic matrix bioinks such as poly(ethyelene glycol) diacrylate, which offer tunable mechanical properties and degradation rates as well as less variation between batches .
|Source||Gelation Time||Gelation Process||Support/Sacrificial Material needed?||References|
|Poly (ethylene glycol) diacrylate (PEGDA)||Synthetic||Minutes||Chemical||No||(1–7)|
|Methacrylated Chondroitin Sulfate||Natural||Minutes||Chemical||No||(6 8)|
|Cellulose (various modifications)||Natural||Minutes||Chemical||No||(13–16)|
|Cell- and Tissue- derived ECM(Matrigel)||Natural||Seconds||Thermal||No||(22–24)|
|Hyaluronic Acid (various modifications)||Natural||Modification Dependent||Modification Dependent||Modification Dependent||(6 8 26 29–32)|
Dextran (various modifications)
|Natural||Modification Dependent||Modification Dependent||Modification Dependent||(30 33)|
|Gelatin (various modifications)||Natural||Modification Dependent||Modification Dependent||Modification Dependent||(34–38)|
Table 1: Matrix Bioinks commonly used for bioprinting. These bioinks can be used individually or combined to create hybrid bioinks. Some bioinks such as hyaluronic acid, dextran and gelatin, can be modified for improved gelation time and printability.
Sometimes researchers want to use certain matrix bioinks such as collagen, which offer ideal environments for cells, but lack fast gelation times or the mechanical stability to develop complex geometries. In these cases, support or sacrificial bioinks may be used to provide additional support. Other matrix bioinks, such as PEGDA or modified versions of gelatin, must be combined with a curing bioink for proper gelation. These bioinks are crosslinked through a process called photopolymerization.
Figure 2: Fluorescence image of F-actin/DAPI stained lattice architecture bioprinted with matrix bioink cell-laden GelMA and curing bioink Irgacure through radical polymerization. (Adapted from 37).
Photopolymerization allows for spatial and temporal control over the gelation of a bioink (39 ).Matrix bioinks that undergo photopolymerization require curing bioinks also known as photoinitiators (Table 2). These bioinks, when exposed to light at the proper wavelength, produce free radicals. These free radicals then interact with the matrix bioink to create a solid gel (Figure 3).
Figure 3: The radical polymerization process, where R-R represents the photoinitiator and M the polymer. First, UV or blue light produces free radicals from the photoinitiator and these free radicals are added to a monomer molecule. Then, in the propagation step, this molecule continues to grow through a rapid reaction. Finally, in termination, the radical is annihilated through either combination (shown) or disproportionation.
When choosing a curing bioink, cytocompatibility must be given serious consideration. A curing bioink must also be water-soluble to mix with matrix bioinks. While Irgacure is the most commonly used photoinitiator in bioprinting, both VA-086 and BioKey, also known as LAP, have demonstrated faster and more cytocompatible crosslinking mechanisms (39, 44, 40). Eosin Y and Biokey offer the advantage of crosslinking in the visible light range, avoiding potential harmful effects of UV light exposure to cells(1,39). However, as a type-II photoinitiator, Eosin Y is less efficient than cleavable Type I initiators like BioKey and VA-086(42).
|Full Name||Distributor||Initiator Type||Wavelength for Crosslinking||References|
|VA-086||(2,2’-azobis[2-methyl-N-(2-hydroxyethyl)propionamide]||Wako Chemicals||I||365 nm (UV)||(1 40 41)|
|Irgacure||(2-hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone||Sigma||I||365 nm (UV)||(1 34 38 39 43)|
|BioKey||lithium phenyl-2,4,6-trimethylbenzoylphosphinate||BioBots||I||365 or 405 nm (UV or Blue Light)||(1 39 42)|
|Eosin Y||Eosin Y||Sigma||II||519 (Green Light)||(1 42 )|
Table 2: Curing Bioinks commonly used for bioprinting. These bioinks are used with modified matrix bioinks such as poly(ethylene glycol) diacrylate (PEDGDA) or gelatin methacrylate (GelMA).
Figure 4 (45): An engineered tissue construct of bioprinted cell-laden GelMA and sacrificial Pluronic F127. A) Design schematic of structure, in which the red filaments correspond to the sacrificial bioink. B) Composite image (top view) of the 3D printed tissue construct taken using three fluorescent channels. After the sacrificial ink is removed, the channels are endothelialized (red). C,D) Brightfield images of the construct once the sacrificial ink is removed. E) Image of the sacrificial ink evacuation.
Sacrificial bioinks can offer temporary support or can be used to create complex geometries within a structure. These materials, used in tandem with matrix biomaterials, can be washed away after printing. Ideally, a sacrificial biomaterial offers high print fidelity, cytocompatibility and ease of removal. Sacrificial bioinks are often used to develop blood vessels or vasculature within a tissue (Figure 4). Sacrificial bioinks include biomaterials such as pluronic F127, gelatin or agarose, which can be dissolved through alterations in temperature and then removed via a vacuum(45,46,47).
|Sacrificial Bioink||Source||Method for removal||References|
|Gelatin||Natural||Heat (37°C)||(48 49)|
|Pluronic F127||Synthetic||Cool (4 °C)||(38 45–46 50)|
|Agarose||Natural||Heat(40°C)||(38 46 49)|
|Carbohydrate Glass||Natural||Heat (37°C)||(46 51)|
Table 3: Sacrificial Bioinks commonly used for bioprinting. These bioinks can be used as a temporary support structure or to create intricate designs within a block of material that can be washed away after printing.
Support bioinks offer more permanent support than sacrificial bioinks. When used with matrix bioinks, support bioinks offer improved mechanical properties and structure for scaffolds. These inks are most useful when developing tissues that require higher mechanical strength, such as bone or cartilage (Figure 5).
Figure 5 (46) : 3D bioprinted ear structure with matrix and support bioinks. Blue and red correspond to cell-laden alginate matrix ink, while the white portion of the structure corresponds to a PCL support bioink.
Most support bioinks are thermoplastic polymers. Thermoplastic polymers are materials that become liquefied or molten when heated above a certain melting temperature and plastic above a glass transition temperature. While these synthetic polymers can’t encapsulate cells due to the high temperatures required for printing, they offer control over mechanical properties and biodegradability. Some common support bioinks are listed in the table below(52–54).
|Support Bioink||Source||Melting Temperature||References|
|Polylactic acid (PLA)||Synthetic||150-160°C||(55)|
|Poly(l-lactic acid) (PLLA)||Synthetic||173-178°C||(44)|
|Poly(lactic-co-glycolic acid) (PLGA)||Synthetic||-||(56)|
|Polycaprolactone (PCL)||Synthetic||80°C||(11 57)|
Table 4: Common support bioinks used in the bioprinting process. Support bioinks can be combined with matrix bioinks to offer improved control over mechanical properties and biodegradability.
Through the use of bioinks and 3D bioprinters, researchers are able to develop complex 3D tissues. As both bioprinting technologies and bioinks are developed and standardized, the process of creating these tissues will become more reproducible and less complex. Researchers aim to create these tissues as both in vitro models for disease and drug testing as well as for in vivo approaches for the regeneration or replacement of diseased tissues. These advancements will revolutionize biological research and lead to great improvements in health care.