BioInks for Extrusion Bioprinters

Wikis > 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).

The bioprinting process involves selection of bioinks

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

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 .

Matrix Bioinks

Matrix Bioink

Source Gelation Time Gelation Process Support/Sacrificial Material needed? References
Poly (ethylene glycol) diacrylate (PEGDA) Synthetic Minutes Chemical No (17)
Methacrylated Chondroitin Sulfate Natural Minutes Chemical No (6 8)
Alginate Natural Seconds Chemical No (6 912)
Cellulose (various modifications) Natural Minutes Chemical No (1316)
Gellan Gum Natural Seconds Chemical No (1719)
Fibrin Natural Seconds Chemical Yes (1921)
Cell- and Tissue- derived ECM(Matrigel) Natural Seconds Thermal No (2224)
Collagen Natural 0.5-1h Thermal Yes (2527)
Spider Silk Natural Minutes Thermal No (28)
Hyaluronic Acid (various modifications) Natural Modification Dependent Modification Dependent Modification Dependent (6 8 26 2932)

Dextran (various modifications)

Natural Modification Dependent Modification Dependent Modification Dependent (30 33)
Gelatin (various modifications) Natural Modification Dependent Modification Dependent Modification Dependent (3438)

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.

Curing Bioinks

3D bioprinted structure from matrix and curing bioinks

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).

The photoinitator process
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).

Curing Bioinks

Curing Bioink

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).

Sacrificial Bioinks

Bioprint with sacrificial bioinks

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 Bioinks
Sacrificial Bioink Source Method for removal References
Gelatin Natural Heat (37°C) (48 49)
Pluronic F127 Synthetic Cool (4 °C) (38 4546 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

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).

bioprinted ear structure from curing and matrix bioinks
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(5254).

Support Bioinks
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.

References

[1]
[2]
S. Jana and A. Lerman, “Bioprinting a Cardiac Valve,” Biotech Adv, vol. 33, no. 8, pp. 1503-1521, December 2015.
[3]
R. F. Pereira and P. J. Bartolo, “3D Bioprinting of Photocrosslinkable Hydrogel Constructs,” J. Appl. Polym. Sci., vol. 132, no. 48, 2015.
[4]
C. C. Hribar, P. Soman, J. Warner, P. Chung and S. Chen, “Light-assisted Direct-Write of 3D Functional Biomaterials,” Lab Chip, vol. 14, pp. 268-275, 2014.
[5]
T. Q. Huang et al, “3D Printing of Biomimetic Microstructures for Cancer Cell Migration,Biomed Microdevices., vol. 16, no. 1, pp. 127-132, Feb 2014.
[6]
G. D. Nicodemus and S. J. Bryant, “Cell Encapsulation in Biodegradable Hydrogels for Tissue Engineering Applications,” Engineering: Part B, vol. 14, no. 2, 2008.
[7]
[8]
[9]
B. Duan et al, “3D Bioprinting of Heterogeneous Aortic Valve Conduits with Alginate/Gelatin Hydrogels,” J Biomed Mater Res A., vol. 101, no. 5, pp. 1255-1264, June 2013.
[10]
S. Khalil et al., “Bioprinting Endothelial Cells with Alginate for 3D Tissue Constructs,J Biomech Eng, vol. 131, no. 11, October 2009.
[11]
J. Kundu and e. al, “An Additive Manufacturing-Based PCL-Alginate-Chondrocyte Bioprinted Scaffold for Cartilage Tissue Engineering,Tissue Engineering: Part B, vol. 14, no. 2, 2008.
[12]
M. T. e. a. Poldervaart, “Sustained Release of BMP-2 in Bioprinted Alginate for Osteogenicity in Mice and Rats,Plos one, vol. 8, no. 8, August 2013.
[13]
Q. Zhang et. al, “Review on Biomedical and Bioengineering Applications of Cellulose Sulfate,” Carbohydrate Polymers., pp. 311-322, November 2015.
[14]
Mohite et al. “A Novel Biomaterial: Bacterial Cellulose and its New Era Applications,Biotechnology and Applied Biochemistry, vol. 61, no. 2, pp. 101-110, 2014.
[15]
[16]
[17]
R. Levato et al, “Biofabrication of Tissue Constructs by 3D Printing of Cell-laden Microcarriers,Biofabrication, vol. 6, 2014.
[18]
J. Visser et. al, “Biofabrication of Multi-material Anatomically Shaped Tissue Constructs,Biofabrication, vol. 5, 2013.
[19]
D. Kirchmajer et. al, “An Overview of the Suitability of Hydrogel-Forming Polymers for Extrusion-Based 3D-Printing,J. Mater. Chem. B., vol. 3, pp. 4105-4117, 2015.
[20]
Y.-B. Lee, “Bioprinting of collagen and VEGF-releasing Fibrin Gel Scaffolds for Neural Stem Cell Culture,” Experimental Neurology, vol. 223, pp. 645-652, 2010.
[21]
T. A. Ahmed et al, “Fibrin: A Versatile Scaffold for TIssue Engineering Applications,” Tissue Engineering: Part B, vol. 14, no. 2, 2008.
[22]
S. Hong et al, “Cellular Behavior in Micropatterned Hydrogels by Bioprinting System Depended on the Cell Types and Cellular Interaction,” Journal of Bioscience and Bioengineering, vol. 116, no. 2, pp. 224-230, August 2013.
[23]
L. Horvarth et al, “Engineering an in vitro Air-blood Barrier by 3D Bioprinting,” Nature Scientific Reports, vol. 5, 2015.
[24]
 D. W. G. Astashkina et al, “Critical Analysis of 3-D Organoid in vitro Cell Culture Models for High-Throughput Drug Candidate Toxicity Assessments,” Advanced Drug Delivery Reviews, vol. 69, pp. 1-18, 2014.
[25]
A. D. Nocera et al., “Printing Collagen 3D Structures,” in VI Latin American Congress on Biomedical Engineering CLAIB 2014 (Vol 49), Parana, Argentina, IFMBE Proceedings, 2014, pp. 136-139.
[26]
[27]
V. Lee et al, “Design and Fabrication of Human Skin by Three-Dimensional Bioprinting,” Tissue Engineering: Part C, vol. 20, no. 6, 2014.
[28]
K. Schacht et al, “Biofabrication of Cell-Loaded 3D Spider Silk Constructs,Angew. Cehm. Int. Ed., vol. 54, pp. 1-6, 2015.
[29]
C. B. Highley et al, “Direct 3D Printing of Shear-Thinning Hydrogels into Self-Healing Hydrogels,Adv. Mater., vol. 27, 2015.
[30]
L. Pescosolido et al, “Hyaluronic Acid and Dextran-Based Semi-IPN Hydorgels as Biomaterials for Bioprinting,” Biomacromolecules, vol. 12, no. 5, pp. 1831-1838, 2011.
[31]
C. B. Rodell, J. Mealy and J. A. Burdick, “Supramolecular Guest-Host Interactions for the Preparation of Biomedical Materials,” Bioconjugate Chem, 2015.
[32]
J. A. Burdick et al, “Controlled Degradation and Mechanical Behavior of Photopolymerized Hyaluronic Acid Networks,Biomacromolecules, vol. 6, no. 1, pp. 386-391, 2005.
[33]
S. Levesque et. al., “Macroporous interconnected Dextran Scaffolds of Controlled Pororsity for Tissue-Engineering Applications,” Biomaterials, vol. 26, pp. 7436-46, 2005.
[34]
H. Aubin et al, “Directed 3D Cell Alignment and Elongation in Microengineered Hydrogels,Biomaterials, vol. 31, no. 27, pp. 6941-6951, September 2010.
[35]
J. W. Nichol and e. al, “Cell-laden Microengineered Gelatin Methacrylate Hydrogels,Biomaterials, vol. 31, pp. 5536-5544, 2010.
[36]
M. Nikkhah et al, “Directed Endothelial Cell Morphogenesis in Micropatterned Gelatin Methacrylate Hydrogels,” Biomaterials, vol. 33, no. 35, pp. 9009-9018, December 2012.
[37]
[38]
[39]
[40]
[41]
[42]
X.-H. Qin et al, “Additive Manufacturing of Photosensitive Hydrogels for Tissue Engineering Applications,” BioNanoMat, vol. 15, no. 3-4, pp. 49-70, 2014.
[43]
S. SJ et al, “Sodium Alginate Hydrogel-Based Bioprinting Using a Novel Multinozzle Bioprinting System,” Artif Organs, vol. 35, no. 11, pp. 1132-6, 2011.
[44]
J. K. e. a. Carrow, “Polymers for Bioprinting,” in Essentials of 3D Biofabrication and Translation, Elsevier Inc, January 2015, pp. 229-248.
[45]
D. B. Kolesky et al, “3D Bioprinting of Vascularized, Heterogeneous Cell-laden Tissue Constructs,” Adv. Mater., vol. 26, pp. 3124-3130, 2014.
[46]
Z. Wang et al, “3D Bioprinting for Engineering Complex Tissues,” Biotechnology Advances, 2015.
[47]
R. L. e. a. Reis, “Natural Polymers for the Microencapsulation of Cells.,J.R.Soc. Interface, vol. 11, 2014.
[48]
W. Lee et al, “On-Demand Three-Dimensional Freeform Fabrication of Multi-Layered Hydrogel Scaffold with Fluidic Channels,” Biotechnology and Bioengineering, vol. 105, no. 6, pp. 1178-1186, 2010.
[49]
[50]
W. Wu et al, “Omnidirectional Printing of 3D Microvascular Networks,” Adv Mater, vol. 23, pp. H178-H183, 2011.
[51]
J. S. Miller et al., “Rapid Casting of Patterned Vascular Networks for Perfusable Engineered 3D Tissues,” Nature Materials, vol. 11, no. 9, pp. 768-74, June 2012.
[52]
[53]
J. Malda et al, “25th Anniversary Article: Engineering Hydrogels for Biofabrication,Adv. Mater., vol. 25, pp. 5011-5028, 2013.
[54]
C. M. Piard et al, “Cell-laden 3D Printed Scaffolds for Bone Tissue Engineering,Clinic Rev Bone Miner Metab, vol. 13, pp. 245-255, 2015.
[55]
A. Bandyopadhyay et al, “3D Printing of Biomaterials,” MRS Bulletin, vol. 40, 2015.
[56]
Sawkins et al, “Bioprinting as a Tool for Osteochondral Tissue Engineering,” European Cells & Materials, vol. 22, no. 3, December 2010.
[57]
[58]
L. P. Schuurman W et al., “Gelatin-methacrylamide hydrogels as potential biomaterials for fabrication of tissue-engineered cartilage constructs,” Macromol Biosci, vol. 13, pp. 551-61, 2013.
[59]
J. Liu et al, “Hydrogels for Engineering of Perfusable Vascular Networks,” Int. J. Mol. Sci., vol. 16, no. 7, pp. 15997-16016, 2015.
[60]
H. Kim and e. al, “Patterning Methods for Polymers in Cell and Tissue Engineering,” Annals of Biomedical Engineering, vol. 40, no. 6, pp. 1339-1355, June 2012.
[61]
[62]
M. Kesti, “Development of 3D Bioprinting Inks Based on Tandem Crosslinked Hydrogels,” Tampere University of Technology, Zurich, 2013.
Category: Tags: