Matrix Bioinks

 

Just as a 2D printer has a variety of color inks to create an assortment of images, bioprinters utilize a variety of bioinks to create an assortment of viable 3D environments.  One of the first steps in the bioprinting process involves choosing what inks to print.

 
  Figure 1: Many different types of bioinks are used to develop viable 3D tissues with complex geometries. The main categories include matrix, sacrificial, and support bioinks.

Figure 1: Many different types of bioinks are used to develop viable 3D tissues with complex geometries. The main categories include matrix, sacrificial, and support bioinks.

 

Bioinks for extrusion bioprinters can be split into three general categories: matrix, sacrificial and support. Matrix bioinks include any biomaterial used for cell encapsulation. In addition to printability, these materials must offer a compatible environment for living cells. Most matrix bioinks are naturally derived hydrogels, such as collagen, hyaluronic acid or alginate [Table 1]. A hydrogel is a very hydrated material of hydrophilic polymers that crosslink to form 3D networks[1]. These materials offer the necessary permeability for nutrient and waste transport as well as a non-toxic gelation process.

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.
Matrix Bioink Source Gelation Time Gelation Process Support/Sacrificial Needed? Sources
Poly(ethylene glycol) diacrylate (PEGDA) Synthetic Minutes Chemical No (1-8)
Methacrylate Chondroitin Sulfate Natural Minutes Chemical No (6,9)
Sodium Alginate Natural Seconds Chemical Yes (6,10-14)
Cellulose (various modifications) Natural Minutes Chemical No (15-18)
Gellan Gum Natural Seconds Chemical No (19-21)
Fibrin Natural Seconds Chemical Yes (21-25)
Cell- and Tissue- derived ECM (ie, Matrigel) Natural Minutes Thermal No (26-29)
Collagen Natural 0.5 - 1h Thermal Yes (29-31)
Spider Silk Nautral Modification Dependent Modification Dependent Modification Dependent (32)
Hyaluronic Acid (various modifications) Natural Modification Dependent Modification Dependent Modification Dependent (6,9,30,33-36)
Dextran (various modifications) Natural Modification Dependent Modification Dependent Modification Dependent (34,37)
Gelatin (various modifications) Natural Modification Dependent Modification Dependent Modification Dependent (24-25, 38-42)

 

If matrix materials have too long of a gelation time or lack the mechanical stability for printing on their own, support or sacrificial bioinks may be used to provide the necessary structure needed to create complex geometries. Some common matrix bioinks are listed in Table 1. These bioinks can be used on their own or combined together to create hybrid inks.

Important Bioink Properties

With so many options, choosing the best bioink for specific applications can be daunting. While different applications and cell types will require different bioinks, there are a few general properties that are important to consider when choosing a matrix bioink, including viscosity, gelation process, biological interaction and general material properties.

 
  Figure 2: The above matrix properties (viscosity, gelation process, material properties and biological interactions) all play a role in the effectiveness of the bioink for creating viable 3D tissues with complex geometries.

Figure 2: The above matrix properties (viscosity, gelation process, material properties and biological interactions) all play a role in the effectiveness of the bioink for creating viable 3D tissues with complex geometries.

 

Viscosity

In general, the rheological properties and polymer structure are very important when considering any bioink to ensure smooth, consistent extrusion. Specifically, matrix bioinks must work in multiple phases, first as a fluid during cell encapsulation, then as a solid once dispensed out of the printer. This transition generally works best with shear thinning materials.

A shear-thinning material is a non-newtonian material that exhibits a decrease in viscosity as shear rate increases. This phenomenon is caused from the reorganization of polymer chains due to the shear rate, which leads to decreased entanglement and thus lower viscosity [43]. This property minimizes shear stress on cells and promotes physical gelation as viscosity increases sharply right after extrusion [43,44].

Gelation Process

The gelation process, or how a matrix bioink forms a solid structure after extrusion, can affect both viability and print resolution. Ideally, the gelation process for matrix bioinks should be fast as well as non-toxic to cells. Bioinks undergo either a physical or chemical gelation process, and these processes may be reversible or irreversible [44]. Some common gelation processes are described in more detail below. Researchers will use one of these individual methods or, especially when working with hybrid bioinks, may combine gelation methods together [24,25].

Table 2: Various mechanisms used for gelation of matrix bioinks.
Mechanism Advantages Disadvantages Examples Sources
Ionic Constant viscosity during print due to reversible interactions Post-process crosslinking needed with additional crosslinking agent
Mechanically weak construct
Alginate (6,10-14,17)
Stereocomplex Constant viscosity during print due to reversible interactions High viscosity of building blocks, needs post-process crosslinking, mechanically weak constructs Stereocomplexed PEG-PLA block copolymers, Dextran stereocomplexed hydrogels (43,45)
Thermal Gelation process simple and highly compatible with biological systems Long gelation time Matrigel, Collagen, Agarose (26-29)
Photocrosslinking Photopolymerization after extrusion does not affect viscosity during extrusion;
Post-process crosslinking for tunable mechanical properties
Exposure to free radicals may affect viability Gelatin Methacrylate;Poly(ethylene glycol) diacrylate; Collagen Methacrylate (1-9,36,41)
Enzymatic Highly compatible with cells Gelation can be too fast, need special extruder or post-crosslinking to avoid gelation prior to extrusion; low mechanical properties Fibrin (21-25)
Click Chemistry (Michael-type Addition) Tunable mechanical properties Long gelation time Modified multi-arm poly(ethyelene glycol) (46-47)

Ionic Crosslinking

Ionic crosslinking creates physical crosslinks through cation solutions. Sodium alginate, a common matrix bioink, gels with Ca2+  ions. This process is compatible with cells and degree of gelation can be tuned through the adjustment of CaCl2 concentration in solution [43–44]. While it offers constant viscosity during prints due to reversible interactions, the drawbacks include the need for post-process crosslinking with an additional crosslinking agent and mechanically weak constructs [44].

Stereocomplex Crosslinking

Stereocomplex crosslinking involves the coupling of lactic acid oligomers of opposing chirality [43]. These oligomers can be coupled with water-soluble polymers such as dextran or polyethylene glycol to form solid hydrogels [43,45]. Additional forms of complexation mechanisms include formation of inclusion complexes such as cyclodextrins. In these cases, hydrophilic polymers are derivatized with cyclodextrin units and combined with guest molecule-derivatized polymers to create a hydrogel [43]. This mechanism offers constant viscosity during printing due to reversible interactions but requires post-process crosslinking and leads to mechanically weak constructs [43].

Thermal Crosslinking

Thermal crosslinking involves materials that gel from temperature changes. For matrix bioinks, thermal crosslinking must occur at room or body temperature (25 – 37 °C). While this process requires longer gelation times, the simple mechanism for gelation is highly compatible with biological systems.

Photocrosslinking

Photocrosslinking gelation occurs through the process of free radical polymerization. First, matrix bioinks such as gelatin, poly(ethylene glycol) and collagen are modified with acrylate groups. Then, UV, blue or visible wavelength light excites free radicals from a curing bioink that serves as the photoinitiator, which interact with a matrix bioink to create a solid gel through covalent bonds. As photopolymerization involves exposure to free radicals and sometimes harmful wavelengths, length of crosslinking time must be minimized to avoid effects on viability. This polymerization process, which occurs after extrusion, does not affect viscosity during extrusion and allows for tunable mechanical properties through post-process crosslinking.

Enzymatic Gelation

Another method of gelation involves enzymatic crosslinking, such as the gelation of fibrin. This method encompasses materials that are gelled through an enzymatic reaction, such as the interaction of thrombin with fibrinogen to create fibrin gels. This process is highly compatible with cells, but special extruders or a post-crosslinking process is needed to avoid gelation prior to extrusion.

Click Chemisty (Michael-type Addition)

Through click chemistry, Michael-type addition reactions create a hydrogel through di-thiolated oligopeptides and vinyl sulfone groups on PEG[46]. This process offers tunable mechanical properties but has a long gelation time [47].

Support Material needed?

In cases where a matrix bioink takes a long time to gel or lacks in mechanical properties, support or sacrificial bioinks may be used in combination to enhance the final structure. Print methods, such as the FRESH method, can also help improve printability with matrix bioinks [48].

Biological Interactions

Physical properties of bioinks, such as hydrophilicity and surface energy, can affect cell behavior, while materials with cell adhesive sites can enhance survival and proliferation of various cell types [44]. These biological interactions of materials can affect cell behavior and final outcome of a construct, so it is important to keep these properties in mind when choosing a matrix bioink [44]. Depending on specific application and cell type, different biological interactions may be desired [44].

The choice between natural or synthetic bioinks can have a significant effect on biological interactions. To mimic native environments, many focus on natural polymers [44].  These materials often also circumvent harsh fabrication conditions such as extreme temperature and organic solvents. However, naturally derived bioinks cause challenges such as batch-to-batch variability, affecting rheological properties and reproducibility in prints, and poor mechanical properties [44]. Synthetic polymers solve the challenges of batch-to-batch variability and allow for control over degradation rates and functionalization, but do not offer the natural cell adhesive sites in many natural materials [44].

Material Properties

Other important material properties to keep in mind include mechanical properties, degradation, permeability and hydration of a material. Different mechanical properties are desired for different cell and tissue types and can have a significant effect on cell growth and differentiation. Degradation properties are important to consider for biocompatibility and rate. Hydration and permeability of a material can affect material viscosity, oxygen and nutrient transport to cells, mechanical strength and the swelling or shrinking of a construct post-print [44].

As the direct materials used to encapsulate cells,  matrix bioinks are one of the most crucial pieces of the bioprinting process. Each matrix bioink currently available offers both advantages and disadvantages. As bioprinting technologies continue to grow and become more relevant, more complex and specialized matrix bioinks will be developed. As opposed to one comprehensive bioink to solve current limitations, these future bioinks will cater to specific cells and tissues, allowing for the development of more accurate tissues and 3D environments.

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