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Introduction to Bioprinting
2-26-2018
Biofabrication Workshop
Biomaterials Lab and Center for Engineering Complex
Tissues
Anthony J. Melchiorri, Ph.D.
Associate Director, Biomaterials Lab
Rice University
3D Printing in Tissue Engineering
Christopher Barnatt, ExplainingTheFuture.com
Bioprinting
3D-printing with cells and/or bioactive
components
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Bioink
+Cell/Bioactive
componentsMaterials
Ji and Guvendiren. Front
Bioeng Biotechnol. 2017
Inkjet Printing
Heater Piezoelectric
Actuator
Bioink
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Inkjet Printing
Considerations
• Crosslinking/gelation
– Speed to maintain structural integrity
– Methodology compatibility with printing process
• Nozzle geometry and printing speed
– Effects shear and thermal stress on materials and cells
– Frequency of blockage
• Viscosity
– Necessarily low for inkjet
• Thermal printing droplets
– May be mixed, unordered, and unequal in size
• Piezeloectric printing droplets
– Droplets generally more regular and equal size
– Can cause damage to cell membrane and cell lysis Lie, et al. J Transl
Med. 2016.
Inkjet Printing
Advantages:
• Typically low-cost
• Capable of printing cells with
good viability, though challenges
still exist
• Thermal-based cartridges found to be reasonably amenable to cell
viability
• Multimaterial fabrication available
Disadvantages:
• Pore development in cell
membranes
• Piezoelectric cartridges hamper
cell viability
• Bioinks must exhibit low viscosity, limiting material choices
• Shear stress can negatively affect
cells
• Must be quick-gelling/crosslinking
drop-by-drop
Inkjet Printing
Applications:
• Cell patterning
• Organoids and blood
vessels
• In situ printing
Materials:
• Hydrogels
• Proteins
• DNA
• Cells (in suspension)
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Extrusion-Based Printing
Pneumatic Piston Screw
Bioink
Extrusion-Based Printing
Fused Deposition Modeling:
• Solid feed material is melted through
deposition and assembled after
extrusion and cooling
• Good mechanical strength and no
solvent required
• High temperatures required for
melting may prohibit inclusion of
cellular and growth factor
components
Nozzle
Extrusion-Based Printing
Solution-Based
Deposition:
• Scaffold deposition
takes place through
extruded solution
• Allows incorporation of
growth factors and cells
(not thermal based)
Bioink
Crosslinker
reservoir
IT Osbolat, M Hospodiuk. Biomaterials. 2016.
Nozzle
Bioink
Crosslinker
Pre-
crosslinked
BioinkCrosslinker
Coaxial-nozzle
Bioink
Aerosolized
Crosslinker
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Extrusion-Based Printing
Considerations
• Crosslinking/gelation
– Speed to maintain structural integrity
– Methodology compatibility with printing process
• Shear thinning effects
– Cell viability
– Gellation
• Extrusion and printing speed
– Effects integrity of extruded fibers
• Viscosity
– Effected by gelation and temperature
• First-layer anchoring
Extrusion-Based Printing
Malda, et al. Advanced
Materials. 2013.
Considerations
Extrusion-Based Printing
Yuk and Zhao.
Advanced
Materials. 2017
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Extrusion-Based Printing
Advantages:
• Good for rapid prototyping
• Materials can be set with
temperature, photocrosslinkable,
chemically crosslinkable, or simply
viscous enough to form structures upon deposition
• Can fabricate basic scaffolds for
tissue engineering applications
• Some options for printing with cells
• Good for direct production of
components for structural strength or
prototypes with high strength
• Multimaterial fabrication available
Disadvantages:
• Materials must have low enough
viscosity to be deposited
• Resolution and feature size can be
limited depending on printing
technique
• Difficult to include cells, growth
factors, and other more “fragile”
components in high-temperature
techniques
• Solidification rate varies
Extrusion-Based Printing
Applications:
• Tissue engineering
scaffolds with and without
cells
• Durable components for
bioreactors
• Surgical planning models
and tools
• Prototype devices
• Customized
prosthetics/accessories
Materials:
• Synthetic and natural
materials
• Thermoplastics
• Silicon
• Hydrogels
• Cell-laden materials
• Composite materials
LIFT Bioprinting
Laser
Substrate
Ribbon
Objective
Biological Layer /
Deposition Material
Bubble
Laser
Absorption
Layer
Laser-Induced Forward Transfer
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LIFT Bioprinting
Guillemot, et al.
Future Medicine.
2010.
LIFT Bioprinting
Considerations
• Thickness of biological materials on films– Can alter effectiveness of bubble formation and necessary energy
– Can control materials deposited on printing substrate
– Mechanical protection of cells
• Rheological properties– Effects bubble formation and collapse
– Too viscous; no transfer
• Energy of laser pulse– Initiates bubble formation
– Irradiation of cells possible
• Wettability of substrate– May affect splashing and spreading of bioink
Guillemot, et al.
Future Medicine.
2010.
LIFT Bioprinting
Advantages:
• High resolution printing
(single cell)
• Can use high-viscosity
bioink (no nozzle)
• Good for microscale cell
patterning
Disadvantages:
• Limited printing in z-axis
• Heat generated from laser
may damage cells or
affect cell biology
• Lengthy fabrication time
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LIFT Bioprinting
Applications:
• Cellular constructs
• In situ printing
Materials:
• Cells
• Hydrogels
• Biopolymers
• Peptides
• DNA
Vat Photopolymerization
Laser
Tray with material
resin
Base plate
Construct
Stereolithography
Vat Photopolymerization
Projector
Tray with material
resin
Base plate
Construct
Digital light projection
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Vat Photopolymerization
Considerations
• Photocrosslinking
– Photoinitiators effects on biological components
– UV/Vis light effects
• Extraneous crosslinking
– May necessitate use of photoinhibitors
• Exposure
– Can modulate mechanical strength and structural integrity
• Resins
– Uncrosslinked moieties must be compatible with any included biological components
• First-layer anchoring
Vat Photopolymerization
Polymeric Properties Desired for Vat
Photopolymerization
Methods Used to Achieve Properties
Low Viscosity
0.25–10 Pa s
•Polymer Architecture
•Oligomers
•Stars
•Hyperbranched/Dendrimers
•Liquid comonomers
•Non-reactive diluents (plasticizers/solvents)
Fast Cure Times
2–100 s
•Many photopolymerizable functionality
•More reactive end groups
•Higher intensity of light
Crosslinkable Materials
Functionality >2
•Multifunctional monomers/polymers
Photopolymerizable Functionality •Acrylate/methacrylates
•Epoxides
•Electron deficient alkene for 2 + 2
cycloaddition
Mondschein, et al.
Biomaterials.2017.
Vat Photopolymerization
Photoinitiator Wavelength Peak Properties
Irgacure 2959 257 – 276 nm One of the most
common
Least toxic of Irgacures
Irgacure 184 246, 280, 333 nm More cytotoxic than
Irgacure 2959
Irgacure 651 DMPA 250, 340 nm More cytotoxic than
Irgacure 2959
Camphorquinone 285, 400-500 nm Can absorb blue light
LAP 375 nm Relatively high water
solubility
Initiate in visible light
region
Eosin Y disodium salt 514 nm Usable with green light
Less toxic than Irgacure
2959
Mondschein, et al. Biomaterials.2017.
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Vat Photopolymerization
Advantages:
• High resolution printing, good for
complex features
• Mechanical properties controlled
through crosslinking and post-
processing
Disadvantages:
• Material resins must be photo-
crosslinkable, requiring photo-
initiators and -inhibitors
• Materials may require solvents
and raw resins are not always biocompatible or particularly
environmentally/people friendly
• May be difficult to impossible to
include cells
• Generally limited to single
materials
Vat Photopolymerization
Applications:
• Surgical planning
• Surgical tools
• Prosthetics and implants
(bone, cardiac)
• Tissue engineering
scaffolds
• Cell constructs for tissue
engineering
Materials:
• Photo-crosslinkable
natural and synthetic
materials and polymers
• Hydrogels (limited)
• Elastomers
• Ceramic composites
(infused resins)
Future of 3D Printing in Tissue
Engineering
Post-Processing:
• Tissues and cells may not
be fully mature after
printing
• Lack of cell-cell
connections
• Questionable mechanical
integrity
• Need time for full cell
maturation
3D printing
should be
considered 4D
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Future of 3D Printing in Tissue
Engineering
Post-Processing:
Zone
1Zone
2
Zone
3
Zone
4
Adapted from BioCell
Printing, CIRP Ann Manuf
Technol 2011
Choosing a printing process
Types of Technique Resolution Form of material deposition
Piezoelectric/Thermal Inkjet
Printing
Electro Hydrodynamic Jetting
Acoustic Droplet Ejection
BioLP/AFA-LIFT/MAPLE-DW
100 μm
10–20 μm
37–150 μm
10–100 μm
•Droplets jetted onto substrate
•Continuous droplets
deposited to form line
Mechanical/Pneumatic
Extrusion
15–400 μm • Extrude continuous hydrogel
line
• Continuous droplets
deposited to form line
Stereolithography (SLA) ∼1 mm • Shapes (line/dot) form
through selective curing of
photopolymer
Digital Light Processing (DLP) 20–200 μm
Lee and Yeong. Advanced
Healthcare Materials. 2016.
Choosing a printing process
Strategies MethodBioprinting
TechniquesApplication
Direct Printing Optimizing viscosity via
semi-crosslinked
precursor, use of
thickening agent
Extrusion Cartilage
Skin
In-Process Crosslinking Pre-mixture of precursor
with crosslinker (Co-
extruder)
Extrusion Lumen construct for
nutrient delivery
Extrusion Femur, Arteries, Heart,
Brain
Deposition of precursor
into crosslinker
Inkjet
Deposition of precursor
and crosslinker
sequentially
Microvalve
Post-Process
Crosslinking
Expose printed construct
to crosslinker after
printing
Extrusion Aortic Valve
Cartilage
Lumen construct for
nutrient delivery
Indirect Printing Printing of bio-ink with
support mold or within a
support bath
Extrusion Vasculature
Femur, Arteries, Heart,
Brain
Inkjet Vasculature
Microvalve
Hybrid Printing Cross-technology
deposition of bio-ink and
scaffolding material
Extrusion Bone, Cartilage, Muscle
Inkjet Cartilage
Lee and
Yeong.
Advanced
Healthcare
Materials.
2016.
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Printer Availability
Commercially
AvailableOpen Source
+Out of box functionality
+Software generally straightforward
+Standardized documentation
between researchers
-Limited materials
-Hardware costs
-Software limitations
-Hardware limitations
+Hardware and software customizability
+Reduced cost
+Custom materials
+/-No
subscriptions/registrations/warranties
-Troubleshooting
-Hardware and software limitations
based on user
Conclusions
• Important to consider goals of research
• Match goals with individual material and printing technique
advantages and limitations
• Consider post-processing techniques
– Sterilizability
– Biocompatibility
– Culturing and maturation
Resources
Bartolo P, Domingos M, Gloria A, Ciurana J. Biocell printing: integrated automatic assembly system for tissue engineering constructs. CIRP Annals.
2011;60(1):271-274. (http://bit.ly/2EZPCT7)
Chimene D, Lennox KK, Kaunas RR, Gaharwar AK. Advanced bioinks for 3D printing: a materials science perspective. Annals of Biomedical
Engineering. 2016;44(6):2090-2102. (http://bit.ly/2H8Q2XS)
Cui X, Boland T, D’Lima DD, Lotz MK. Thermal inkjet printing in tissueengineering and regenerative medicine. Recent Pat Drug Deliv Fromul.
2012;6(2):149-155. (http://bit.ly/2EBLkmU)
Guillemot F, Souquet A, Catros S, Guillotin B. Nanomedicine. 2010;5(3). (http://bit.ly/2H6JngZ)
Ji S, Guvendiren M. Recent advances in bioink design for 3D bioprinting of tissues and organs. Front Bioeng Biotechnol. 2017;5:23.
(http://bit.ly/2EjDICs)
Lee and Yeong. Design and printing strategies in 3D bioprinting of hydrogels: a review. Advanced Healthcare Materials. 2016;5(22):2856-2865.
(http://bit.ly/2nUEGic)
Malda J, Visser J, Melchels FP, Jungst T, Hennink WE, Dhert WJA, Groll J, Hutmacher DW. 25th anniversary article: engineering hydrogels for
biofabrication. 2013;25(36):5011-5028. (http://bit.ly/2CgdIG0)
Mondschein RJ, Kanitkar A, Williams CB, Verbridge SS, Long TE. Polymer structure-property requirements for stereolithographic 3D printing of soft
tissue engineering scaffolds. Biomaterials. 2017;140:170-188. (http://bit.ly/2ElmGI2)
Ozbolat IT, Hospodiuk M. Current advances and future perspectives in extrusion-based bioprinting. Biomaterials. 2015:76:321-343.
(http://bit.ly/2nUruKk)
You F, Eames BF, Chen X. Application of extrusion-based hydrogel bioprinting for cartilage tissue engineering. Int J Mol Sci. 2017;18(7):1597.
(http://bit.ly/2BTYQl6)
Yuk H, Zhao X. A new 3D printing strategy by harnessing deformation, instability, and fracture of viscous inks. 2017. (http://bit.ly/2BUPJjU)
