1. Introduction to Bioprinting
Biofabrication Workshop
Biomaterials Lab and Center for Engineering Complex Tissues
Anthony J. Melchiorri, Ph.D.
Associate Director, Biomaterials Lab
Rice University

2. 3D Printing in Tissue Engineering
The most commonly recognized goal of 3d printing in medicine is the manufacture of a whole organ. It’s hoped that this can address the organ donor shortage and immune compatibility issues present with the current clinical paradigm in dealing with damaged or failing tissues and organs. But while that image is certainly an attractive and suitably fits in a good many science fiction films, at least for most organs, we’re far from achieving clinical relevancy. Certainly some organs have eben easier to recapitulate than others and can be readily achieved now, but organ manufacturing is not the only useful application or consideration for 3d printing in tissue engineering.
Christopher Barnatt, ExplainingTheFuture.com

3. Bioprinting 3D-printing with cells and/or bioactive components
-use bioprinting not just for tissue/organ replacement, but also drug discovery, microfluidics work, and other in vitro experiments.

4. Bioink
Cell/Bioactive components +
Materials

5.
Ji and Guvendiren. Front Bioeng Biotechnol. 2017

6. Inkjet Printing Bioink Heater Piezoelectric Actuator
all air bubbles generated by heating in the printhead collapse to provide pressure pulses to eject ink drops with various volumes from 10 to 150 pL out of the nozzle
Thermal inkjet printers electrically heat the printhead to produce air-pressure pulses that force droplets from the nozzle, whereas acoustic printers use pulses formed by piezoelectric or ultrasound pressure.
the actuator of polycrystalline piezoelectric ceramic in each nozzle provides the transient pressure to eject the ink drops onto the substrate [24]. These inkjet printing technologies have been widely used in electronics and micro-engineering industries for printing electronic materials and complex integrated circuits

7. 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

8. 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
Commercial inkjets can b e modified
Benign side effect of pore development in cell membrane could actually enable gene transfection and drug delivery
3D fab must use drop-by-drop quick polymerization or stabilization
Must be fast gelling
Can support high-resolution printing of intricate structures
 The well-documented damage to the cell membrane and cell lysis after sonification at 15–25 kHz is within the range of frequencies employed by piezoelectric inkjet printers [28]. As for thermal inkjet printers, although the heating element in each nozzle raises the local temperature to 300°C and lasts for a few microseconds during printing [23], ejected mammalian cells are heated for only 2 µs with a temperature rise of 4–10°C above ambient and an average cell viability of 90% [29]. Therefore, thermal inkjet printing technology is more biocompatible to the living system comparing to piezoelectric printing. Furthermore, the thermal inkjet printers are usually more convenient than piezoelectric inkjet printers in terms of modification, access, and maintenance. Therefore, many research groups including us are utilizing thermal inkjet printers for tissue engineering and regenerative medicine applications.

9. Inkjet Printing Materials: Hydrogels Proteins DNA
Cells (in suspension)
Applications:
Cell patterning
Organoids and blood vessels
In situ printing
In situ printing accomplished with skin and cartilage
Blood vessel/organoid = heart valves and vessels, though maturation needed in hydrogels
Muscle patch printing
Wound healing/skin

10. Extrusion-Based Printing
Pneumatic
Piston
Screw
Bioink
. For example, inkjet bioprinting requires low viscosities to avoid clogging and low thermal conductivity to prevent heat damage to the cells.
In contrast, extrusion bioprinting can accommodate much higher viscosities but shear thinning materials are often necessary to prevent mechanical damage to the cells
Often limited to resolution of fiber diameters 100 microns or above

11. 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
More commonly associated with filament extrusion printers like makerbots but not amenable to cells. Possible to include some bioactive components but must withstand high Ts.

12. Extrusion-Based Printing
Solution-Based Deposition:
Scaffold deposition takes place through extruded solution
Allows incorporation of growth factors and cells (not thermal based)
Nozzle
Bioink
Crosslinker reservoir
Crosslinker
Coaxial-nozzle
For non Fused Depositiom odeling, there must be some kind of crosslinking methodology to connect printer fibers. This might include printing of alginate, for instance, where crosslinking occurs in the presence of a calcium ion (from calcium chloride solution).
Other methods include photocrosslinking which have their own challenges regarding chemical and irradiation effects on cells.
Bioink
Bioink
Pre-crosslinked
Bioink
Crosslinker
Aerosolized
Crosslinker
IT Osbolat, M Hospodiuk. Biomaterials

13. 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
Extursion/printing speed – affect diameter, structural integrity at inersections and corners within scaffold, layer height, resolution

14. Extrusion-Based Printing
Considerations
Schematic representation of shear thinning and yield stress in plotting gelatin methacrylamide (gelMA)/gellan gum. In the syringe the gellan chains (in white) form a temporary network and induce gel-like viscosity (i). Upon dispensing through a needle, the temporary network is broken up by shear and all polymer chains align, reducing the viscosity by orders of magnitude (ii). Directly after removal of shear stress, the temporary network is restored and the plotted filament solidifies instantly (iii).
Malda, et al. Advanced Materials

15. Extrusion-Based Printing
DIW 3D printing of viscoelastic inks. a) Schematic illustration of typical setup and parameters for DIW 3D printing. The viscoelastic ink is extruded out of the nozzle tip, with inner diameter D, at a speed of C while the nozzle tip moves at a speed of V and a height of H. b) Diverse modes in new 3D printing strategy by varying selections of printing parameters. Fibers with resolution unlimited by the nozzle diameter can be achieved in a highly controllable manner.
Predictable and reproducible printing of various fibers and patterns by a single nozzle. a) The phase diagram with marked symbols (solid circle or star) for corresponding printing parameter selections. b) Single nozzle printing of fibers with various diameters and complex patterns with parallel straight nozzle motions by adopting the new modes of DIW 3D printing. Each printing condition corresponds to solid circle symbols along the dotted line in (a). c) Single nozzle printing of various nonlinear patterns and fiber diameters in a single printed fiber by continuous transition between different modes. Each printing condition corresponds to solid star symbols in (a).
Yuk and Zhao. Advanced Materials. 2017

16. 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

17. Extrusion-Based Printing
Materials:
Synthetic and natural materials
Thermoplastics
Silicon
Hydrogels
Cell-laden materials
Composite materials
Applications:
Tissue engineering scaffolds with and without cells
Durable components for bioreactors
Surgical planning models and tools
Prototype devices
Customized prosthetics/accessories
Ceramic – ceramic paste
Metals –

18. LIFT Bioprinting Laser-Induced Forward Transfer Laser Absorption Layer
Substrate
Ribbon
Objective
Biological Layer / Deposition Material
Bubble
Laser Absorption Layer
Laser-Induced Forward Transfer
laser-induced cavitation bubble is central to the mechanism behind MAPLE-DW and AFA-LIFT. Cavitation bubble collapse is a well-documented source of energy which has been shown to cause damage to turbine blades or cells within a bioreactor.4
Laser beam pulsed at desired time lengths, and donor material ribbon is printable material. This is supported on a transport layer, such as gold or titanium, which absorbs the laser energy and transfer it to the ribbon. When laser pulses on ribbon, focused energy generate an incredibly small, high pressure bubble that propels droplet onto collecting substrate.
No nozzle in this type of printing which prevents clogging issues. Very high resolution due to diameter of laser and resulting energy transfer to bubble.

19. LIFT Bioprinting
A vapor bubble is generated (see II) by vaporization of the absorbing layer and/or the first molecular layers of the liquid film. At given bioink viscosity and film thickness, jetting (see III.b) is observed for intermediary values of laser fluences (Γ1 < Γ < Γ2). For a lower fluence (Γ > Γ2), the bubble collapses far from the free surface without generating a jet (see III.a). For a higher fluence (Γ < Γ1), the bubble bursts to the surface, generating sub-micrometer droplets (see III.c). Increasing film thickness or bioink viscosity leads to increased threshold Γ values.
the fluence is defined as the time-integrated flux of some radiation or particle stream. Fluence = optical energy per unit area.
1. Mechanism for laser-induced droplet ejection. A vapour bubble is generated by vaporization of the absorbing layer and/or the first molecular layers of the liquid film. At a given viscosity, depending on bubble dynamics, jetting (b) is observed for intermediary values of fluences. For lower fluences (a), the bubble collapses far from the free surface without generating a jet. For high fluences (c), the bubble bursts to the surface, generating sub-micrometer droplets. Increasing viscosity leads to increased threshold values between the subthreshold, jetting and plume regimes [31].
Guillemot, et al. Future Medicine

20. 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
High viscosity may also mean much higher energy transfer which can effect cells.
Guillemot, et al. Future Medicine

21. 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

22. LIFT Bioprinting Materials: Cells Hydrogels Biopolymers Peptides DNA
Applications:
Cellular constructs
In situ printing
Used to print HA bioink in calvarial defects of mice
DNA – Dan Luo at Cornell 
a linear expression plasmid is incorporated into a DNA hydrogel by using branched DNA monomers as croslinkers

23. Vat Photopolymerization
Stereolithography
Base plate
Construct
Tray with material resin
Selective exposure by laser
Conventional SLA machiens utilize the enrgy from an UV light source to drive the conversion of UV-irradiation-sensitive liquid oligomers into cross-linked solid/gel-like polymeric networks.
Two-photo STL is possible which involves two relatively lwo-intensity photons in order to excite a photo-sensitive resin to a high-energy radical state. Depends quadratically on the incident light intensity as opposed to the linear relationship for single-photon STL allowing for extremely rapid fabrication in three dimensions with submicron resolution. Uses near-IR lasers, but must use different photocrosslinkers due to chemicals demonstrating reduced photosensitiy to light in near-IR wavelength range. THE NIR femtosecond laser has resolution of ~100 nm compared to UV laser’s 1 micron
Laser

24. Vat Photopolymerization
Digital light projection
Base plate
Construct
Tray with material resin
Also singlephotoon STL
Process of photoinitiator excitation is driven by absorption of a single photon
Projector

25. 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

26. 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 cycloaddition
Mondschein, et al. Biomaterials.2017.

27. 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
Camphorquinone
285, 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.

28. 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
Supports often necessary and require part post-processing

29. Vat Photopolymerization
Materials:
Photo-crosslinkable natural and synthetic materials and polymers
Hydrogels (limited)
Elastomers
Ceramic composites (infused resins)
Applications:
Surgical planning
Surgical tools
Prosthetics and implants (bone, cardiac)
Tissue engineering scaffolds
Cell constructs for tissue engineering
Materials usually acrylate and methacrylate oligomers, epoxide, ad viny ether-based resins tha tcure rapidly upon irradiation. ‘
Many traditional SLA resins are brittle because they are composed primarily of low molecular weight monomers that react to form rigid cross-linked networks.
Many elastomers required for biomedical applications require diluents to reduce viscosity.
PEGDMA hydrogels used with this
GelMA
Multimaterial may require washing and replacing resin bath.

30. 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

31. Future of 3D Printing in Tissue Engineering
Post-Processing:
Zone 1
Zone 2
Zone 3
Zone 4
At it’s most complicated—even simplest may require washing of solvents or raw materials.
Adapted from BioCell Printing, CIRP Ann Manuf Technol 2011

32. 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
Laser Guided Direct Writing
100 nm – 10 μm
• Single cell manipulation
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

33. Choosing a printing process
Strategies
Method
Bioprinting Techniques
Application
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)
Lumen construct for nutrient delivery
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
Aortic Valve
Indirect Printing
Printing of bio-ink with support mold or within a support bath
Vasculature
Hybrid Printing
Cross-technology deposition of bio-ink and scaffolding material
Bone, Cartilage, Muscle
Lee and Yeong. Advanced Healthcare Materials

34. Commercially Available
Printer Availability
Commercially Available
Open 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

35. 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
Oftentimes 3d printing is an excerise in compromise

36. Resources
Bartolo P, Domingos M, Gloria A, Ciurana J. Biocell printing: integrated automatic assembly system for tissue engineering constructs. CIRP Annals. 2011;60(1): ( Chimene D, Lennox KK, Kaunas RR, Gaharwar AK. Advanced bioinks for 3D printing: a materials science perspective. Annals of Biomedical Engineering. 2016;44(6): ( 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): ( Guillemot F, Souquet A, Catros S, Guillotin B. Nanomedicine. 2010;5(3). ( Ji S, Guvendiren M. Recent advances in bioink design for 3D bioprinting of tissues and organs. Front Bioeng Biotechnol. 2017;5:23. ( Lee and Yeong. Design and printing strategies in 3D bioprinting of hydrogels: a review. Advanced Healthcare Materials. 2016;5(22): ( 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): ( 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: ( Ozbolat IT, Hospodiuk M. Current advances and future perspectives in extrusion-based bioprinting. Biomaterials. 2015:76: ( You F, Eames BF, Chen X. Application of extrusion-based hydrogel bioprinting for cartilage tissue engineering. Int J Mol Sci. 2017;18(7):1597. ( Yuk H, Zhao X. A new 3D printing strategy by harnessing deformation, instability, and fracture of viscous inks (