#3dprintconfsc

1. Cornell University
Butcher Lab
Bioprinter Team
Laura Hockaday, PhD
Cardiovascular Developmental Bioengineering Laboratory
Inside 3D Printing Santa Clara
October 22, 2014: 3:30pm – 4:15pm
3D Printing Heart Valves

2. Preview
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3D Printing for Tissue Engineering
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Heterogeneous Fabrication
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Recent success
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Optimization of an extrusion printing technology for heart valve bioprinting
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Hydrogel, cells, and image processing into printable formats
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3D printing for a custom bioreactor
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Developing a prototype for dynamic culture of 3D printed valve tissue
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Use commercial rapid prototyping
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Next steps for the field to advance

3. 3D Printing for Tissue Engineering
Tissue Engineering
3D Biomaterial Scaffold
Template for Cells
Position Living Cells
Self Organization and Matrix Creation
Applications for Fabricated Living Tissue
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Study mechanism
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In vitro models for drug testing
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Clinical scale replacement/repair
3D Printing
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Complex molds
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Direct cell and scaffold printing
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Bioreactor parts to mimic complex loading
Unique advantages of 3D printing: Multi-material deposition and elaborate spatial control

4. TE Relevant 3D Printing Technologies…. All of Them
Laser Based Systems
Nozzle-Based Systems
Inkjet Printer Based Systems
Photo- polymerization
Extrusion Dispensation
Droplet or liquid stream deposition of a binder into layer of powder or particles or sheets
Billiet et al. Biomaterials 2012
Unique advantages of 3D printing: Multi-material deposition and elaborate spatial control

5. Recent 3D Printing Advances in Heterogeneous Tissue Fabrication
Enabling of higher order structure and multiple cell types into engineered tissue which in native tissue key for efficient function
Cartilage + Bone
Bioelectronics
Vasculature
Miller et al Nat Mater. 2012
Mannoor et al Nano Lett 2013
Bone
Meniscus
Intervertebral
Liver
Neural
Myocardium
Kidney
Cornea
Skin
Cartilage + Vessel
Fedorovich et al Tiss Engr A 2011
Fedorovich Tiss Engr A 2012

6. Tremendous Global Burden of Pediatric Valve Disease
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United States Valve Disease
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Predominately affects adults1
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5 million per year
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Congenital valve defects
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1/100 live births
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40,000 per year
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Worldwide Valve Disease
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Valve disease predominantly affects children and young adults2
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Rheumatic valve disease
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15 million cases per year
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282, 000 deaths per year
(1) Hinton and Yutzey et al 2010. (2) Zilla, Brink et al. 2008 (3) Carapetis, Steer et al. 2005 (4) Howell and Butcher 2012
Critically diseased or malformed heart valves require prosthetic replacement

7. Grim Inadequacy of Valve Replacement for Pediatrics - Need for TEHV
Jameson et al. Ann Thor Surg 1998
Younger Patient = Faster Bio-prosthetic Deterioration
Bio-prosthetic valves
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No anticoagulants
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Deterioration
Mechanical valves
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Durability
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Anticoagulant treatment
Within 10 years children with valve prosthetics
•40% need repeat surgeries
•20% mortality
Need for living and growing valve replacement

8. Complex Shape and Mechanics Impacts Valve Function
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Ostia deliver blood to heart
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Vortex formation in sinuses
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Stiff root wall
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Flexible strong leaflets
Merryman et al. J. Biomech. 2004; Courtney et al. Biomat. 2006; Dagum et al., Circ. 1999; Sacks et al. J. Biomechanics 2009; Butcher et al, JHVD 2004; Stephens et al. Acta Biomater. 2010.
Root
Sinus
Leaflet
Ostia

9. Persistent Problem for Polymeric TEHV is Fabricating Complex Shape and Mechanics
While limited to a single material TEHV studies found
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Invitro dynamic culture prior to implantation improves performance
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Autologous stem cells can differentiate into endothelial-like and interstitial-like cells
Sutured Electrospun1
Molded Fibrin3
Layered Electrospun2
Leaflets stiff
Contraction
Leaflet tissue thickening
Reduced pliability
Reached limits of classical fabrication  Need for rapid prototyping
(1)Sutherland et al Circ 2005; (2) Schmidt et al J Am Coll Cardiol. 2010; (3)Flanagan et al Tiss Eng A 2009

10. Native Structure Suggests Design Criteria for TEHV
•Non obstructive
•Closure prompt and complete
•Non-thrombogenic and non-immunogenic
•Accommodate growth of recipient
•Last life time of recipient
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Mimic the natural anatomic 3D geometry
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Replicating regionally heterogeneous tissue compliance of the root and cusp
To have durable and ongoing remodeling

11. Optimization of an Extrusion 3D Printer for a Specific Biological Application
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Adapt a better fabrication technique for TEHV
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Shape and multiple region mechanics
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Better control and distribution cells within TEHV
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Multi-cell function
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Develop custom bioreactor for dynamic conditioning of TEHV
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Format for a reiterative approach to study remodeling
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Strengthen and mature 3D bioprinted valves

12. 3D Extrusion Printing of PEGDA in Hydrogel Scaffolds
Micro CT/MRI
Threshold
Reconstruction
Extrude and Photocrosslink
Crosslinkable PEGDA
Photoinitiator
UV LED
Hydrogel Precursor
Deposited and Crosslinked Bioink
(1)Kloxin et al Biomaterials 2010;(2)Hutson et al Tiss Engr A 2011; 3)Benton et al Tiss Engr A 2009;
Bioprinter
Hydrogel Scaffold
-Tunable mechanics1
- Cell-mediated degradation2,3

13. Compliant Leaflets and Stiff Root Can be 3D Printed into Valve Shape
Root
Leaflet
PEG-DA hydrogels
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Nonlinear elastic mechanics in tensile testing
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Modulus can be tuned with polymer mass and molecular weight ratio
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700MW stiff
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8000MW compliant
1% w/v Irgacure photoinitiator
-Photocroslinking of both formulations 30-60 sec per layer

14. Aortic Valve Shape and Pediatric Scale Can Be Replicated Using 3D Printing and Assessed for Volumetric Fidelity Using μCT
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Print time
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45min, 30min, 14 min
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Majority of surface point deviation falls within ±10% tolerance
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By printing alternative shapes
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sensitivity for smaller scaffolds
Hockaday et al Biofabrication 2012
Scaled Hydrogel Valves
22mm
17mm
12mm
Volumetric Fidelity Analysis
1cm

15. 3D Printed Hydrogel Valves Seeded with Valve Interstitial Cells are Cytocompatible
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VIC main populating cell type of valve leaflets
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Post fabrication seeded
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Cytocompatible
91% Viabilty
100% Viability
Live/Dead
2mm
Hockaday et al Biofabrication 2012

16. Major Findings of 3D Valve Printing Study
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3D printing and photocrosslinking hydrogels fabricated into anatomical and cytocompatible scaffolds
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μCT evaluation of adult and pediatric sized valve constructs was performed to assess fidelity
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Valve geometries printed were interlocking STL type files
•Heterogeneity existed between the root parts and leaflet parts
(1) Hockaday, Kang et al. 2012; (2) Duan, Hockaday et al. 2013; (3) Duan, Kapetanovic, Hockaday et al. 2014

17. Remaining Challenge to Control Cell Distribution Throughout TEHV
21 day sections show very few cell in interior
Root
Leaflet
Live/Dead
Post-Fabrication Seeding
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Poor cell infiltration
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No control of cell location
Direct 3D bio printing of encapsulated cells
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Multi cell spatial control
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Cells must tolerate fabrication conditions
100μm

18. 3D Bioprinting with Photo-crosslinkable Hydrogels for Controlled Shape and Cell Distribution
Crosslinkable monomer PEGDA
Photoinitiator
Valve cell
Crosslinkable macromer
MEGEL
UV LED
Bioink
Deposited and Crosslinked Bioink
PEGDA
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Tunable scaffold mechanics1
MEGEL
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Cell-mediated degradation2,3 and attachment sites
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Cell encapsulation with fabrication4
(1)Kloxin et al Biomaterials 2010; (2) Hutson et al Tiss Engr A 2011; (3)Benton et al Tiss Engr A 2009; (4)Chan et al Lab Chip 2010 (5);Chandler, Berglund et al. 2011; (6) Rouillard, Berglund et al. 2011
Irgacure 2959
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Cytocompatible photoinitiator
VA086
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Less toxic than Irgacure5,6

19. Encapsulated Viability for Photo- crosslinked Hydrogels
Cell Source for TEHV
Adipose Derived Mesenchymal Stem Cell
0.05 w/v% Irgacure
LIVE/DEAD
0.5 w/v% VA086

20. Higher Order Complex Structure Determines Biomechanics which Impacts Valve Function
Need for rapid prototyping control of deposition within valve shape
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Layered internal structure
Merryman et al. J. Biomech. 2004; Courtney et al. Biomat. 2006; Dagum et al., Circ. 1999; Sacks et al. J. Biomechanics 2009; Butcher et al, JHVD 2004; Stephens et al. Acta Biomater. 2010.
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Regional stiffness differences
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Dynamic response to load
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Regional cellular response
Smooth Muscle Cells
Endothelial Cells
Interstitial Cells
Image courtesy of Jen Richards

21. Control Structure within Solid Shapes Dither Images Into Vector Format
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User specified vector format
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Paired coordinates
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Compatible with Model 1 and 2 Fab@Home
Photograph of
Fabric
Binary Image
Extruded Material Along Vector Paths
A

22. Control Structure within Hydrogel Shapes Apply Gradient Convert to Vectors
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Function defined layers
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2D and 3D gradients
Completely arbitrary and user defined internal pattern of material within a shape can be printed using this 2nd algorithm

23. Internal Structure in Root and Leaflet Controlled by Converting Tissue Heterogeneity into Vectors
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Combination algorithm identifies regions of heterogeneity in images
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Isolates anatomic shape from background
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Heterogeneous material gradient applied throughout valve shape based on the intensity values present in each image slice
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Printed in dye labeled hydrogel
Hockaday, Duan, Kang, Butcher. 3D-Printed Hydrogel Technologies for Tissue-Engineered Heart Valve. 3D Printing and Additive Manufacturing. 2014 Sept 19.

24. High Pattern Fidelity for Cell Distribution within Printed Hydrogel Layers
Cell Tracker green HADMSC Cell Tracker Red HADMSC Megel/PEGDA
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Scale for swelling by expanding pathwidth
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Compare to different edge mixing models
1mm
78%
Kang, Hockaday, Butcher J. Quantitative optimization of solid freeform deposition of aqueous hydrogels. Biofabrication. 2013 Sep 5. Hockaday, Duan, Kang, Butcher. 3D-Printed Hydrogel Technologies for Tissue-Engineered Heart Valve. 3D Printing and Additive Manufacturing. 2014 Sept 19.

25. Major Findings: Direct 3D Printing of Cells and Image Processing to Control Internal Structure within Valve Features
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Vector printing
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Enables image and function generated material control within printed structures
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Combination algorithm
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Separates intrinsic tissue heterogeneity present in medical imaging scans of anatomical tissue into printable format files
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Printing using the Fab@HomeTM platform enabled multi-material fully cellularized heterogeneous tissue fabrication
Hockaday LA*, Kang KH*, et al. Arbitrary and anatomically based control of internal heterogeneity of 3D printed tissues. In preparation.

26. Development of a Dynamic Conditioning System to Culture 3D Printed Valves
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Determine the effects of heterogeneity and remodeling on function
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Mimic hemodynamic loading
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Aortic
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Across root
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Outflow
Hydrogel valve mechanics not sufficient directly after fabrication, bioreactor needed to remodel and reinforce tissue
Hockaday, Duan, Kang, Butcher. 3D-Printed Hydrogel Technologies for Tissue-Engineered Heart Valve. 3D Printing and Additive Manufacturing. 2014 Sept 19.

27. Fabricating Conditioning Chamber with Heart Valve Cell Compatible Materials
Ponoko®
Quickparts
Quickparts
Hapco
Prototype flow and pressure validation
Material screening for sterilization and cell compatible
Condition 3D printed valves
Fabricate in biocompatible materials
SLA printed part to form Cast SteralloyTM
Conditioned media and direct contact metabolism assay (MTT)
3D printed parts
Stem cells + hydrogel 3D printed

28. Conclusions
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Tools to incorporate heterogeneity directly into engineered tissues
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A means to evaluate remodeling through dynamic conditioning and ultrasound, μCT evaluation of the tissue
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3D printing has enabled this technology but there are still critical needs to be addressed
Hockaday et al Bioreactor for parallel dynamic conditioning of adult and pediatric sized 3D printed hydrogel heart valves. In preparation.

29. Critical Needed Studies and Technologies
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Study the effects of heterogeneity and the interactions and remodeling behaviors within living hydrogel
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Need for predictive swelling, growth, and remodeling models
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Guide 3D printing of complex geometries
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Evaluating the biological consequences of complex heterogeneity in a hemodynamic environment G
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eo
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MRI Study to Evaluate engineered valve heterogeneity compared to native geometries
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More materials that are biocompatible for 3D printing

30. Acknowledgments
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Funding
The Hartwell Foundation
National Science Foundation
Morgan Family Foundation
American Heart Association 0833840N
National Instituites of Heath HL110328
Cornell Center for Materials Research DMR-1120296
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Help, space, equipment
Fischbach Lab
Shuler Lab
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Collaborators
Hod Lipson
Chih-Chang Chu
Larry Bonnasar
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Cardiovascular Developmental Bioengineering Laboratory
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31. Acknowledgments
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Jeffrey Ballyns
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Marc Riccio
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Sydney Moise, Bruce Kornreich , and Flauvia Giacomazzil
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Warren Zipfel
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Rebecca Williams
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Sam Portnoff /Widetronix, Inc
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Jennifer Puetzer
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Jeffrey Lipton
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Yi Wang, Wenming Luh, Emily Qualls
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Xiaofan Luan, Bruce Land
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Bioprinter Team
Special Thanks to
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Jonathan Butcher, Heeyong Kang, Kevin Yeh, Bin Duan , Phillip Cheung
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Harshal Sawant, Mohammed Cherakoi, Scott Newman, Alain Kaldany, Kang Li, Dan Cheung, Shoshana Das, Patrick Armstrong,
Kevin Lamott