inside3dprinting_jonathanbutcher
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3D Printing Technologies for Tissue Regeneration and
Biomedical Science
Jonathan T. Butcher, Ph.D. Department of Biomedical Engineering
Cornell University July 10, 2013
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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”
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Tissue Engineering: Living Replacement Tissues Capable of Growth and Remodeling
Cell Isolation
Expansion
Scaffold Seeding
In Vitro Conditioning
Langer and Vacanti, Science 1993
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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
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Tissues Exhibit Complex Natural Engineering: The Aortic Valve
S
L
O
RL
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?
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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)
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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
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Image-Guided Mold Design
Mold Design Data Conversion µCT Image
Molded Alginate
Printed ABS Plastic
Cultured Meniscus Implant
Ballyns et al, Tissue Eng Part A 2008
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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
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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
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UV LED Array
Root Leaflets Nozzles
Direct 3D Printing of Photocrosslinked Hydrogel Tissues
Tri-Leaflet Heart Valves Gradient Tissues
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Optimal Deposition Rate and Path Space Scale with Nozzle Diameter
0.000
0.002
0.004
0.006
400 600 800 1000 1200
Dep
ositi
on R
ate
Nozzle Diameter (µm)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
400 600 800 1000 1200 Pa
thsp
ace
(mm
) Nozzle Diameter (µm)
Kang et al, Biofabrication 2013
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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
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Image Based Quantification of Shape Fidelity
Hockaday et al Biofabrication 2012
Surface Deviation Maps 80% ± 10% match 50% 18%
Scaled Printed Valves Slice-by-Slice Overlay
74% Match
89% Match
Inner Diameter 22mm 17mm 12mm
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70
80
90
100
0 5
% A
ccur
acy
Circular Diagonal
Base
Design
Base Middle Top
Design
High Fidelity Micro-scale 3D Tissue Printing - Gradients
Diagonal Gradient
Spherical Gradient
Middle Top
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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
Inte
nsity
(au)
Position (mm)
High Throughput 3D Culture Screening
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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
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Tissue Engineered Meniscus
Ballyns et al, Tissue Eng Part A 2008
Cells remodel alginate and produce collagen in culture
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Anatomically Appropriate Mechanical Stimulation
Com
pressive Strain
Loading Platen Loading Tray Bioreactor
Load Cell
High
Linear Poroelastic FE Model
Low Ballyns et al, J Biomechanics 2010
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Mechanical Conditioning Accelerates Biomechanical Remodeling
Puetzer et al, Tissue Eng Pt A 2013
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Tissue Engineered Intervertebral Disc via Hybrid Printing
Bowles et. al., Tissue Eng Pt A 2010
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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
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TE-IVD Maintains Mechanical Integrity After 6 Months In Vivo
Bowles et. al., PNAS 2011
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TE-IVD Tissue Generation and in vivo Integration
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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
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3 Months In Vivo Results in Cartilage-like Structure
26
Reiffel et al, PLoS ONE 2013
1 month 3 months
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In Situ 3D Tissue Printing for Bone/Cartilage Defects
Osteochondral Defect Mounting and CT Scan
In Line Scan and
Cohen et al, Biofabrication 2010
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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
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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
Youn
g's
Mod
ulus
(kP
a)
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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 45 500
20
40
60
80
100
Stre
ss(M
Pa)
Strain (%)
k
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0
25
50
75
100
0.5 0.75 1
Viab
ility
[% L
ive]
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 AA
A
A AB AA A A A B
B
HAdMSC HAVIC HAsSMC
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Stiffness and Adhesion Control Myofibroblast Phenotype of VIC
0
2
4
6
Rel
ativ
e Ex
pres
sion
αSMA
0
2
4
6
Rel
ativ
e Ex
pres
sion
Vimentin
0 5
10 15 20 25 30
Rel
ativ
e Ex
pres
sion
Periostin
0 5
10 15 20 25
Rel
ativ
e Ex
pres
sion
Hyaluronidase I
MO0.1HA MO0.05HA Me-HA
MO0.1HA/Me-Gel MO0.05HA/Me-Gel Me-HA/Me-Gel
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Stiffness Directs Stem Cell Differentiation Towards Heart Valve Phenotypes
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Fabricated chamber
C
3D Printed Fluid Bioreactor Enables Direct Stimulation of TEHV in Minimal Volumes
Bioprosthetic “Stiff” Valve Physio-Valve
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H
3D Printed Vascularized Tissue Grafts for Reconstructive Surgery
Wound
MRI
CAD
Design
Implant
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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
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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
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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
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• 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
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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
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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