Inside3DPrintingSantaClara_LauraHockaday

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

Preview • 3D Printing for Tissue Engineering • Heterogeneous Fabrication

– Recent success • Optimization of an extrusion printing technology

for heart valve bioprinting – Hydrogel, cells, and image processing into printable

formats • 3D printing for a custom bioreactor

– Developing a prototype for dynamic culture of 3D printed valve tissue

– Use commercial rapid prototyping • Next steps for the field to advance

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 • Study mechanism • In vitro models for drug testing • Clinical scale replacement/repair 3D Printing • Complex molds • Direct cell and scaffold printing • Bioreactor parts to mimic complex loading

Unique advantages of 3D printing: Multi-material deposition and elaborate spatial control

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

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

Tremendous Global Burden of Pediatric Valve Disease

• United States Valve Disease – Predominately affects adults1

• 5 million per year – Congenital valve defects

• 1/100 live births • 40,000 per year

• Worldwide Valve Disease

– Valve disease predominantly affects children and young adults2

– Rheumatic valve disease • 15 million cases per year • 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

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

• No anticoagulants

• Deterioration

Mechanical valves • Durability • Anticoagulant

treatment

Within 10 years children with valve prosthetics • 40% need repeat

surgeries • 20% mortality

Need for living and growing valve replacement

Complex Shape and Mechanics Impacts Valve Function

• Ostia deliver blood to heart • Vortex formation in sinuses • Stiff root wall • 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

Persistent Problem for Polymeric TEHV is Fabricating Complex Shape and Mechanics

While limited to a single material TEHV studies found • Invitro dynamic culture prior to implantation improves performance • 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

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

• Mimic the natural anatomic 3D geometry • Replicating regionally heterogeneous tissue

compliance of the root and cusp

To have durable and ongoing remodeling

Optimization of an Extrusion 3D Printer for a Specific Biological Application

• Adapt a better fabrication technique for TEHV – Shape and multiple region mechanics

• Better control and distribution cells within TEHV – Multi-cell function

• Develop custom bioreactor for dynamic conditioning of TEHV – Format for a reiterative approach to study

remodeling – Strengthen and mature 3D bioprinted valves

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

Compliant Leaflets and Stiff Root Can be 3D Printed into Valve Shape

Root Leaflet PEG-DA hydrogels • Nonlinear elastic mechanics in

tensile testing • Modulus can be tuned with

polymer mass and molecular weight ratio • 700MW stiff • 8000MW compliant

1% w/v Irgacure photoinitiator -Photocroslinking of both formulations 30-60 sec per layer

Aortic Valve Shape and Pediatric Scale Can Be Replicated Using 3D Printing and Assessed for

Volumetric Fidelity Using μCT • Print time

– 45min, 30min, 14 min

• Majority of surface point deviation falls within ±10% tolerance

• By printing alternative shapes – sensitivity for smaller

scaffolds

Hockaday et al Biofabrication 2012

Scaled Hydrogel Valves 22mm 17mm 12mm

Volumetric Fidelity Analysis

1cm

3D Printed Hydrogel Valves Seeded with Valve Interstitial Cells are Cytocompatible

• VIC main populating cell type of valve leaflets

• Post fabrication seeded

• Cytocompatible

91% Viabilty 100% Viability

Live/Dead

2mm

Hockaday et al Biofabrication 2012

Major Findings of 3D Valve Printing Study

• 3D printing and photocrosslinking hydrogels fabricated into anatomical and cytocompatible scaffolds

• μCT evaluation of adult and pediatric sized valve constructs was performed to assess fidelity

• 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

Remaining Challenge to Control Cell Distribution Throughout TEHV

21 day sections show very few cell in interior

Root Leaflet Live/Dead Post-Fabrication Seeding • Poor cell infiltration • No control of cell location

Direct 3D bio printing of encapsulated cells

• Multi cell spatial control • Cells must tolerate fabrication conditions

100μm

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 • Tunable scaffold mechanics1

MEGEL • Cell-mediated degradation2,3

and attachment sites

• 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 • Cytocompatible

photoinitiator VA086 • Less toxic than

Irgacure5,6

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

Higher Order Complex Structure Determines Biomechanics which Impacts Valve Function

Need for rapid prototyping control of deposition within valve shape

• 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.

• Regional stiffness differences

• Dynamic response to load

• Regional cellular response

Smooth Muscle Cells Endothelial Cells Interstitial Cells

Image courtesy of Jen Richards

Control Structure within Solid Shapes Dither Images Into Vector Format

• User specified vector format

• Paired coordinates

• Compatible with Model 1 and 2 Fab@Home

Photograph of Fabric

Binary Image Extruded Material Along Vector Paths

A

Control Structure within Hydrogel Shapes Apply Gradient Convert to Vectors

• Function defined layers

• 2D and 3D gradients

Completely arbitrary and user defined internal pattern of material within a shape can be printed using this 2nd algorithm

Internal Structure in Root and Leaflet Controlled by

Converting Tissue Heterogeneity into Vectors

• Combination algorithm identifies regions of heterogeneity in images

• Isolates anatomic shape from background • Heterogeneous material gradient applied throughout valve

shape based on the intensity values present in each image slice

• 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.

High Pattern Fidelity for Cell Distribution within Printed Hydrogel Layers

Cell Tracker green HADMSC Cell Tracker Red HADMSC Megel/PEGDA

• Scale for swelling by expanding pathwidth

• 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.

Major Findings: Direct 3D Printing of Cells and Image Processing to Control Internal Structure within Valve

Features

• Vector printing – Enables image and function generated material control

within printed structures • Combination algorithm

– Separates intrinsic tissue heterogeneity present in medical imaging scans of anatomical tissue into printable format files

• 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.

Development of a Dynamic Conditioning System to Culture 3D Printed Valves

• Determine the effects of heterogeneity and remodeling on function

• Mimic hemodynamic loading – Aortic – Across root – 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.

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

Conclusions

• Tools to incorporate heterogeneity directly into engineered tissues

• A means to evaluate remodeling through dynamic conditioning and ultrasound, μCT evaluation of the tissue

• 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.

Critical Needed Studies and Technologies

• Study the effects of heterogeneity and the interactions and remodeling behaviors within living hydrogel

• Need for predictive swelling, growth, and remodeling models – Guide 3D printing of complex geometries

• Evaluating the biological consequences of complex

heterogeneity in a hemodynamic environment G • eo • MRI Study to Evaluate engineered valve heterogeneity

compared to native geometries

• More materials that are biocompatible for 3D printing

Acknowledgments • 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

• Help, space, equipment Fischbach Lab Shuler Lab • Collaborators

Hod Lipson Chih-Chang Chu Larry Bonnasar

• Cardiovascular Developmental Bioengineering Laboratory

•

Acknowledgments

– Jeffrey Ballyns – Marc Riccio – Sydney Moise, Bruce Kornreich , and Flauvia

Giacomazzil – Warren Zipfel – Rebecca Williams – Sam Portnoff /Widetronix, Inc – Jennifer Puetzer – Jeffrey Lipton – Yi Wang, Wenming Luh, Emily Qualls – Xiaofan Luan, Bruce Land – Bioprinter Team

Special Thanks to – Jonathan Butcher, Heeyong Kang, Kevin

Yeh, Bin Duan , Phillip Cheung – Harshal Sawant, Mohammed Cherakoi,

Scott Newman, Alain Kaldany, Kang Li, Dan Cheung, Shoshana Das, Patrick Armstrong, Kevin Lamott