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Bioink Selection for 3D Bioprinting

What Is 3D Bioprinting?

3D bioprinting enables the generation of precisely controlled 3D cell models and tissue constructs, by engineering anatomically-shaped substrates with tissue-like complexity. Due to the high degree of control on structure and composition, 3D bioprinting has the potential to solve many critical unmet needs in medical research, including applications in cosmetics testing, drug discovery, regenerative medicine, and functional organ replacement.Personalized models of disease can be created using patient-derived stem cells, such as induced pluripotent stem cells (iPS cells) or mesenchymal stem cells. Depending on the application, a range of materials, methods, and cells can be used to yield the desired tissue construct (Figure 1). For more in-depth information, including expert review articles on 3D Bioprinting, protocols, and related products, please explore our 3D Bioprinting Handbook.

The process of 3D bioprinting of tissue and organs. The image is divided into three sections: “Extrusion-based bioprinting” with a syringe extruding bioink, “Inkjet-based bioprinting” with an inkjet printer head releasing polymer-based bioink, and “Laser-assisted bioprinting” using a laser pulse to deposit material. Below these sections, diagrams depict the composition of bioinks and their applications in tissue engineering, drug screening, and in vitro disease modeling.

Figure 1.3D Bioprinting of tissue and organs. Bioinks are created by combining cultured cells and various biocompatable materials. Bioinks can then be 3D bioprinted into functional tissue constructs for drug screening, disease modeling, and in vitro transplantation.

What Are Bioinks?

Bioinks contain living cells and biomaterials that mimic the extracellular matrix environment, supporting cell adhesion, proliferation, and differentiation after printing. In contrast to tradtional 3D printing materials, bioinks must have:

  • Print temperatures that do not exceed physiological temperatures
  • Mild cross-linking or gelation conditions
  • Bioative components that are non-toxic and able to be modified by the cells after printing

Bioinks for Extrusion-based Printing

Cell-encapsulating hydrogels are used in 3D bioprinting to create living tissue structures by forming multicellular bioprinting building blocks. Cell encapsulation allows for precise control over cell attachment and the spatial distribution of the cells and biomolecules within the scaffold, in comparison to other methods and materials.1 Combining multiple cell types and growth factors in a prescribed pattern allows for the generation of highly-complex tissue constructs.3 In addition to biocompatibility, bioprinting materials used  for cellular encapsulation must feaure high water content and porosity, allowing encapsulated cells to receive nutrients and remove waste.1 As water-swollen, porous networks, hydrogels are ideal materials for cell-encapsulation, tissue engineering, and 3D bioprinting applications. Hydrogels for 3D bioprinting must also feature tunable substrate stiffness and allow for network remodeling post-printing, so cells can spread, migrate, proliferate, and interact.9 While a wide variety of materials are used for bioinks, the most popular materials include gelatin methacrylol (GelMA), collagen, poly(ethylene glycol) (PEG), Pluronic®, alginate, and decellularized extracellular matrix (ECM)-based materials (Table 1).

Featured Bioink Material

Gelatin MethacryloylGelatin methacryloyl (GelMA) can be used to form crosslinked hydrogels for tissue engineering and 3D printing. GelMA-based bioinks feature excellent cytocompatibility, tunable substrate stiffness, improved printability, and rapid crosslinking with exposure to UV or visible light (depending on the identity of the photoinitiator)11. GelMA has been used in endothelial cell morphogenesis, cardiomyocytes, epidermal tissue, injectable tissue constructs, bone differentiation, and cartilage regeneration. Gelatin methacryloyl has also been used in microspheres and hydrogels for drug delivery applications.

A structural chemical formula of Gelatin Methacryloyl (GelMA), showing a complex arrangement of carbon rings, chains, nitrogen bases, oxygen, and hydrogen atoms. The structure includes repeating units indicated by “(N)” to denote polymerization, with various functional groups such as hydroxyls, ketones, and amides. GelMA is a modified gelatin used in biomedicine and tissue engineering for creating hydrogels.

Figure 2.Gelatin Methacryloyl

Acellular Materials

In addition to bioinks, acellular materials are also used in 3D bioprinted structures.2 Acellular materials typically provide structural support for tissue constructs and when utilized with bioinks, can generate functional, bioprinted tissues. Acellular materials are porous structures that recapitulate both mechanical and biochemical properties of the native extracellular matrix (ECM)4. Porosity enables cell migration, tissue growth, vascular formation, and cell viability within these structural constructs.6  In addition, acellular materials must also have the necessary surface chemistry for cell attachment, proliferation, and differentiation.5 Popular acellular materials include: collagen, fibrin, chitosan, nanocellulose, poly(lactic acid) (PLA), polycaprolactone (PCL), hydroxyapatite (HA), and β-tricalcium phosphate (β-TCP) (Table 1).

Bioink Material Building Blocks

Bioink MaterialOverviewAdvantageDisadvantage
Agarose
Polysaccharide extracted from seaweed
Non-toxic crosslinking
High stability
Not degradable;
Poor cell adhesion
AlginateNaturally-derived biopolymer from brown algaeMild crosslinking conditions (Ca2+)
Rapid gelation
High biocompatibility
Slow degradation kinetics;
Poor cell adhesion

ChitosanPolysaccharide obtained from the outer skeleton of shellfish (e.g. shrimp). Non-animal derived chitosan can be obtained from fungal fermentation.High biocompatibility
Antibacterial properties
Slow gelation rate
CollagenPrimary structural protein found in skin and other connective tissuesHigh biological relevanceAcid-soluble
Decellularized ECMIsolated extracellular matrix of a tissue from inhabiting native cellsHigh biological relevance
Tissue-specific
High cell survival
Undefined and inconsistent;
Loss of native ECM organization;
Low stability
Fibrin/FibrinogenInsoluble protein formed during blood clottingHigh biological relevance
Rapid gelation
Limited printability
GelatinProtein substance derived from partial hydrolysis of collagenHigh biocompatibility
High water solubility
Thermally reversible gelation
Poor shape fidelity;
Limited rigidity
GrapheneCarbon-based material that can be viewed as a one atom thick sheet of graphiteFlexible
Electrically-conductive
Low biological relevance
Hyaluronic Acid (HA)Non-sulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues.Fast gelation
Promotes cell proliferation
Poor stability
HydroxyapatiteNaturally-occurring mineral form of calcium apatite found in teeth and bonesHigh strength and rigidityLow printability;
Limited tissue specificity
PCL/PLA/PLGABiodegradable, thermoplastic polymers and/or copolymersHigh strength and rigidityLow cell adhesion and proliferation
Pluronic® F127Poly(ethylene oxide) and poly(propylene oxide) block copolymerPrintable at room temperatures
Shear thinning material
Not suitable for long-term cell culture
Table 1Biomaterials commonly used in 3D bioprinting.

What 3D Bioprinting Method Should Be Used?

Depending on the type of ink (bioink or acellular materials) selected and complexity of the final tissue construct, different 3D printing methods can be used (Figure 1). Advantages and disadvantages of common methods can be found in the table below (Table 2).

Printing methodAdvantagesDisadvantagesCell compatible?
Extrusion-based
  • Printing speed and structures can be highly controlled
  • Shear stress can impact cell viability
Yes
Inkjet-based
  • Fast printing speed
  • Compatible with biological components
  • Low cost
  • Requires low viscosity materials
Yes
Stereolithography (SLA)
  • High resolution
  • Requires large amounts of material
  • Long processing time
  • Long printing times can decrease cell viability
Yes
Laser-based
  • Can be used with viscous materials
  • Highly accurate
  • Heat generated by laser can impact cells
Yes
Fused-deposition modeling (FDM)
  • Yields highly porous structures
  • Materials must exhibit a molten phase
  • Heat used to melt materials not compatible with cells
  • Difficult to make complex geometries
No
Selective laser sintering (SLS)
  • Capable of making complex structures
  • Yields better bonding between each printed layer
  • Heat generated by laser not compatible with cells
No
Table 2.Summary of 3D bioprinting methods.

In addition to ink type, the bioprinting method can also be dictated by the end application of the printed construct (Table 3).

Tissue Engineering Applications

Tissue ModelCells UsedBioprinter UsedBioink Material UsedReference
CartilageMesenchymal Stem CellsHP® Deskjet 500 printerPEG diacrylateInkjet‐bioprinted acrylated peptides and PEG hydrogel with human mesenchymal stem cells promote robust bone and cartilage formation with minimal printhead clogging
 iPS Cells/ Chondrocytes3DDiscovery™Alginate/
Nanocellulose
Cartilage tissue engineering by the 3D bioprinting of iPS cells in a nanocellulose/alginate bioink
 ChondrocytesITOPPCL/Pluronic®A 3D bioprinting system to produce human-scale tissue constructs with structural integrity
BoneMC3T3-E1In-HouseAlginate/
Polyvinyl alcohol/ Hydroxyapatite
Development of a novel alginate-polyvinyl alcohol-hydroxyapatite hydrogel for 3D bioprinting bone tissue engineered scaffolds
 Mesenchymal
Stem Cells
HP® Deskjet 500 printerGelMAImproved properties of bone and cartilage tissue from 3D inkjet-printed human mesenchymal stem cells by simultaneous deposition and photocrosslinking in PEG-GelMA
SkinMesenchymal Stem CellsIn-HouseFibrin/Collagen IBioprinted amniotic fluid‐derived stem cells accelerate healing of large skin wounds
 Keratinocytes/ FibroblastsIn-HouseCollagen IDesign and fabrication of human skin by three-dimensional bioprinting
Blood VesselHUVEC/HUVSMC/ FibroblastsIn-HouseAgaroseScaffold-free vascular tissue engineering using bioprinting
MuscleMuscle Derived Stem CellsIn-HouseFibrinMicroenvironments engineered by inkjet bioprinting spatially direct adult stem cells toward muscle‐ and bone‐like subpopulations
BrainNeural Stem CellIn-HousePolyurethane3D bioprinting of neural stem cell-laden thermoresponsive biodegradable polyurethane hydrogel and potential in central nervous system repair
LiveriPS CellsNanolitre Dispensing SystemAlginate-RGDBioprinting of human pluripotent stem cells and their directed differentiation into hepatocyte-like cells for the generation of mini-livers in 3D
LungA549/HUVECBioFactory™ECM GelEngineering an in vitro air-blood barrier by 3D bioprinting
KidneyImmortalized PTECs/Primary RPTECsIn-HouseGelatin/FibrinBioprinting of 3D convoluted renal proximal tubules on perfusable chips
HeartHUVEC/Neonatal CardiomyocytesNovoGen MMXAlginate/GelMABioprinting 3D microfibrous scaffolds for engineering endothelialized myocardium and heart-on-a-chip
 Mesenchymal Stem Cells/iPSC Derived Neurons3D-BioPlotter®Graphene/ PLGAThree-Dimensional printing of high-content graphene scaffolds for electronic and biomedical applications
Table 3.3D Bioprinting of tissue constructs.

Conclusion

3D bioprinting allows for the spatially-controlled placement of cells in a defined 3D microenvironment. Bioinks are formed by combining cells and various biocompatible materials, which are subsequently printed in specific shapes to generate tissue-like, 3D structures. Combing our expertise in materials science and cell biology, we offer a variety of solutions to simplify the 3D bioprinting workflow.

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