#3DPrintConfChi

1. 3D Printing Technologies for
Tissue Regeneration and
Biomedical Science
Jonathan T. Butcher, Ph.D.
Department of Biomedical Engineering
Cornell University
July 10, 2013

2. Tissue Failure is a Tremendous
Clinical Burden
• Approximately 5 million
surgeries/yr in US to replace
damaged tissues
– 3M orthopaedic/reconstructive
(bone, cartilage, soft tissue)
– 1M cardiovascular (blood
vessel, valve)
– 300K internal organ
– 200K neural
• Tissue transplant supply is
insufficient
• Synthetic implants fail from
wear, fatigue, biocompatibility
“Rex”

3. Tissue
Engineering:
Living
Replacement
Tissues
Capable
of
Growth
and
Remodeling
Cell Isolation
Expansion
Scaffold Seeding
In Vitro
Conditioning Langer and Vacanti, Science 1993

4. Challenges of Tissue Engineering
• Cells, Scaffolds, Conditioning
• Rapid, scalable methods for
fabrication of living tissues
• Minimize time, resources, cost,
expertise needed for tissue
production
• Cellular uniformity, QA/QC
• Fabrication of customized/
personalized tissues vs. “Off the
shelf” replacements
• Effective business models
– FDA, Insurance reimbursement

5. Tissues Exhibit Complex Natural
Engineering: The Aortic Valve
S
L
O
R
L
L
S
R = root, L = leaflet, S = sinus, O = Ostia
Bicuspid
Aortic Valve
Valve
Calcification
How can we engineer this macro- and
micro-scale complexity within living tissue
replacements?

6. 3D Biofabrication Methods
Injection Molding Tissue Injection Molding
(Chang+, JBMR 2001)
3D Printing/FDM Tissue Printing
(Cohen+ Tissue Eng 2006)
Sintering/HIP Cell-Mediated Sintering
(Mercier+ Ann Biomed Eng 2003)
Spray Coating Tissue Painting
(Roberts+ Biotech Bioeng 2005)
Soft Lithography Living Lithography
(Choi+ Nature Med 2007)

7. Tissue Injection Molding
Tissue biopsy
or stem cells
Cells suspended in
alginate solution
+ CaSO4
Intervertrebral Disc
(Bowles et al, PNAS
2011)
Ear
(Reiffel et al, PLoS
One2013)
Trachea
(Kojima et al, J
Thoracic Cardio
Surg 2002)
Meniscus
(Ballyns et al,
Biomaterials,
2010)
Mold from
positive model
Chang et al, J Biomed Mat Res 2001

8. Image-Guided Mold Design
Mold DesignData ConversionµCT Image
Molded
Alginate
Printed
ABS Plastic
Cultured Meniscus
Implant
Ballyns et al, Tissue Eng Part A 2008

9. 3D Tissue Printing Technology
Micro CT/MRI Threshold Reconstruction
Bioprinter
Crosslinkable
monomer
Photoinitiator
Cell
Crosslinkable
macromer
UV LED
Bioink
Deposited and Crosslinked Bioink
Cohen et al, Tissue Engineering 2006; Hockaday et al, Biofabrication 2012

10. 3D Printing “Inks” for Controllable Biological
Response of Encapsulated Cells
Me-HA
MO0.05HA
MO0.1HA
Cell
Cell adhesion site
HA (MOHA) HA (MOHA)+Me-Gel
Mw ↓
Me-HA (MOHA) Me-Gel PEGDA
Stiffness ↑
Provide mechanical
strength
Provide cell
adhesion cites
Mimic ECM
PEGDA+Me-Gel

11. UV LED
Array
Root
Leaflets Nozzles
Direct 3D Printing of
Photocrosslinked Hydrogel Tissues
Tri-Leaflet Heart Valves Gradient Tissues

12. Optimal Deposition Rate and Path
Space Scale with Nozzle Diameter
0.000
0.002
0.004
0.006
400 600 800 1000 1200
DepositionRate
Nozzle Diameter (µm)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
400 600 800 1000 1200Pathspace(mm)
Nozzle Diameter (µm)
Kang et al, Biofabrication 2013

13. Comparison of 3D Biofabrication
Technologies
Injection Molding 3D Tissue Printing
High spatial resolution
Rapid fabrication
Fewer “ink” material
requirements
Mold printed anywhere
Resolution tied to nozzle
diameter
Significant “ink” material
requirements
“In-house” printing only (?)
No ability to fabricate internal
inclusions/voids
Only homogeneous material
formulations
Must extract safely/sterily
from mold
Can fabricate virtually any
geometry
Can fabricate multiple
materials and blends of
materials
No need to extract tissue

14. Image Based Quantification of
Shape Fidelity
Hockaday et al Biofabrication 2012
Surface Deviation
Maps
80% ± 10% match
Scaled Printed Valves Slice-by-Slice Overlay
74%
Match
89%
Match
Inner Diameter 22mm 17mm 12mm

15. 70
80
90
100
0 5
%Accuracy
Circular Diagonal
Base
Design
Print
Base Middle Top
Design
Print
High Fidelity Micro-scale 3D
Tissue Printing - Gradients
Diagonal Gradient
Spherical Gradient
Middle Top

16. Dynamic Gradients of Cells in 3D
Printed Hydrogel Tissues
Cells Fluorescently Labeled Red or Green
Printed in a 3D vertical gradient
50x
0
0.5
1
1.5
0 20
Intensity(au)
Position (mm)
High Throughput 3D Culture Screening

17. Density Thresholds
for Material Regions
Layer Specific
Heterogeneous
Material Domains
Initial Layer Mid-print Final
Heterogeneous
printed valve
shown in stages
CT image slice
Base Sinus Aorta
Combined Macro- and Micro-Scale
3D Tissue Printing: Heart valves

18. Tissue Engineered Meniscus
Ballyns et al, Tissue Eng Part A 2008
Cells remodel alginate and produce collagen in culture

19. Anatomically Appropriate
Mechanical Stimulation
CompressiveStrain
Loading Platen Loading Tray Bioreactor
Load
Cell
High
Linear Poroelastic
FE Model
Low
Ballyns et al, J Biomechanics 2010

20. Mechanical Conditioning Accelerates
Biomechanical Remodeling
Puetzer et al, Tissue Eng Pt A 2013

21. Tissue Engineered Intervertebral
Disc via Hybrid Printing
Bowles et. al., Tissue Eng Pt A 2010

22. In Vivo Evaluation in Rat Tail
6 Weeks 6 Months
N = 24
N = 12 MRI Signal
Disc Height
Histology
Mechanics
N = 48
Discectomy N = 6
Native Disc Re-implant N = 6

23. TE-IVD Maintains Mechanical
Integrity After 6 Months In Vivo
Bowles et. al., PNAS 2011

24. TE-IVD Tissue Generation and
in vivo Integration

25. Ear Reconstruction via
Photogrammy Based 3D Printing
• Combined laser-scan and panoramic photograph
– Non-invasive, no ionizing radiation
– Scan time < 30 seconds, 250 micron resolution
3D Reconstruction Molded Tissue

26. 3 Months In Vivo Results in
Cartilage-like Structure
26
Reiffel et al, PLoS ONE 2013
1 month 3 months

27. In Situ 3D Tissue Printing for
Bone/Cartilage Defects
Osteochondral DefectMounting and CT Scan
In Line
Scan and
Print
Cohen et al, Biofabrication 2010

28. Matrix Stiffness Directs Stem Cell
Differentiation
Cells differentiate on substrates
mimicking native stiffness
Reilly et al J Biomech. 2010, Kloxin et al Biomaterials 2010, Engler et al Cell 2006
Cells reside in matrix environments
with specific stiffness ranges

29. Mechanical Tunability PEGDA/Me-
HA/Me-Gel Hydrogels
PEGD700/Me-
HA/Me-Gel
PEGD3350/Me-
HA/Me-Gel
PEGD8000/Me-
HA/Me-Gel
Irgacure 0.1%
Irgacure 0.05%
Irgacure 0.025%
0
20
40
60
80
100
120
Young'sModulus(kPa)

30. A.Lc (human) A.Sc (human)
P.Sc (porcine)
P.Lr (porcine)
PEGDA3350/
Me-Gel/Alg
PEGDA8000/
Me-Gel/Alg
P.Sc (pediatric)
P.Lc
(pediatric)
A. Aortic
P. Pulmonary
L: Leaflet
S: Sinus
c: circumferential
r: radial
Material Formulations that Mimic
Physiological Valve Tissue Mechanics
0 5 10 15 20 25 30 35 40 45 50
0
20
40
60
80
100
Stress(MPa)
Strain (%)
k

31. 0
25
50
75
100
0.5 0.75 1
Viability[%Live]
VA086 Concentration [w/v%]
0
25
50
75
100
0.05 0.075 0.1
Irgacure 2959 Photoinitator
Concentration [w/v%]
Sensitivity to Encapsulation Conditions
Dependent on Cell Type and Photoinitiator
DAY 7
A
P<0.05
A
B
A
B
B A
A
A
A AB AA A AA
B
B
HAdMSC HAVIC HAsSMC

32. Stiffness and Adhesion Control
Myofibroblast Phenotype of VIC
0
2
4
6
Relative
Expression
αSMA
0
2
4
6
Relative
Expression
Vimentin
0
5
10
15
20
25
30
Relative
Expression
Periostin
0
5
10
15
20
25Relative
Expression Hyaluronidase I
MO0.1HA
MO0.05HA
Me-HA
MO0.1HA/Me-Gel
MO0.05HA/Me-Gel
Me-HA/Me-Gel

33. Stiffness Directs Stem Cell Differentiation Towards
Heart Valve Phenotypes

34. Fabricated
chamber
C
3D Printed Fluid Bioreactor Enables Direct
Stimulation of TEHV in Minimal Volumes
Bioprosthetic “Stiff” Valve Physio-Valve

35. 3D Printed Vascularized Tissue Grafts
for Reconstructive Surgery
Wound
MRI
CAD
Print
Design
Print
Implant

36. Colloidal Gels
Hydrogels
‘Fugitive’ Inks
Barry, Shepherd et. al (2009)
Therriault, Shepherd et. al (2005)
Printing ~1 µm hydrogel filaments
under UV light.
Next Generation Designer “Inks”
Hanson-Shepherd et. al (2010)
pHEMA
Primary rat neuron cells

37. µ-Fluidic Particle Synthesis for Novel
3D Printing Nozzles
Shepherd et. al, Adv. Mat. (2008)
Shepherd et. al, Langmuir (2006)
*unpublished
Single Emulsion: Sheath
Flow
Double Emulsion: Co-flow Microcapillary
Single Phase: Stop Flow
Lithography

38. Where We Are Now
Skin: Michael+ PLoS One 2013
Ear: Reiffel+ PLoS One 2013
Heart Valve: Hockaday+
Biofabrication 2012
IVD: Bowles+ PNAS 2012
Meniscus: Ballyns+ Tissue Eng
2010
Bone: Ciocca+ Comp
Med Imag 2009

39. • Total body scan (data storage)
• Marrow stem cell biopsy
Cell storage
Cell-seeded
polymer “ink”
Tissue
printer
Living
implant
Data Gathering Injury/Disease/Defect Treatment
Where We Hope to Be

40. How Do We Get There?
• New 3D Printing Technology
– Multiple printing modes
– Controllable curing systems
– Direct clinical printing options
– Cost and revenue models
• Improved “inks” for printing
– Significant but KNOWN material requirements
– Shear thinning for more rapid deposition
• Improved Image based geometry/material
retrieval and deposition algorithms

41. Acknowledgments
Cornell
Prof. Hod Lipson
Prof. Larry Bonassar
Prof. Rob Shepherd
Duan Bin, PhD
Robby Bowles, PhD
Jeff Ballyns, PhD
Bobby Mozia
Heeyong Kang
Laura Hockaday
CWMC
Roger Härtl, MD
Harry Gephard, MD
Jason Spector, MD
Alyssa Reiffel
HSS
Suzanne Maher, PhD
Tim Wright, PhD
Russ Warren, MD
Hollis Potter, MD