3D Printing Technologies for Tissue Regeneration and

Biomedical Science

Jonathan T. Butcher, Ph.D. Department of Biomedical Engineering

Cornell University July 10, 2013

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”

Tissue  Engineering:  Living  Replacement  Tissues  Capable  of  Growth  and  Remodeling  

Cell Isolation

Expansion

Scaffold Seeding

In Vitro Conditioning

Langer and Vacanti, Science 1993

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

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?

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)

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

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

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

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

UV LED Array

Root Leaflets Nozzles

Direct 3D Printing of Photocrosslinked Hydrogel Tissues

Tri-Leaflet Heart Valves Gradient Tissues

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

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

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

70

80

90

100

0 5

% A

ccur

acy

Circular Diagonal

Base

Design

Print

Base Middle Top

Design

Print

High Fidelity Micro-scale 3D Tissue Printing - Gradients

Diagonal Gradient

Spherical Gradient

Middle Top

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

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

Tissue Engineered Meniscus

Ballyns et al, Tissue Eng Part A 2008

Cells remodel alginate and produce collagen in culture

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

Mechanical Conditioning Accelerates Biomechanical Remodeling

Puetzer et al, Tissue Eng Pt A 2013

Tissue Engineered Intervertebral Disc via Hybrid Printing

Bowles et. al., Tissue Eng Pt A 2010

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

TE-IVD Maintains Mechanical Integrity After 6 Months In Vivo

Bowles et. al., PNAS 2011

TE-IVD Tissue Generation and in vivo Integration

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

3 Months In Vivo Results in Cartilage-like Structure

26

Reiffel et al, PLoS ONE 2013

1 month 3 months

In Situ 3D Tissue Printing for Bone/Cartilage Defects

Osteochondral Defect Mounting and CT Scan

In Line Scan and

Print

Cohen et al, Biofabrication 2010

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

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)

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

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

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

Stiffness Directs Stem Cell Differentiation Towards Heart Valve Phenotypes

Fabricated chamber

C

3D Printed Fluid Bioreactor Enables Direct Stimulation of TEHV in Minimal Volumes

Bioprosthetic “Stiff” Valve Physio-Valve

H

3D Printed Vascularized Tissue Grafts for Reconstructive Surgery

Wound

MRI

CAD

Print

Design

Print

Implant

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

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

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

•  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

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

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