AMSUS 2018
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Advancement of Bio-printing/fabrication technology: Military Medicine and Technology into the Future
4D Bio3: Four Dimensional Bioprinting, Biofabrication, &
Biomanufacturing Joel Gaston, PhD
Disclosure statement
∎Disclaimer: The views expressed in this presentation are those of the authors and do not necessarily reflect the official policy of the Department of Defense nor the U.S. Government
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4D Bio3 Vision, Mission, & Goals
VisionTo be a leading DoD resource for innovation, integration and application of biofabrication technologies.
MissionTo discover, develop and deliver biofabrication technologies and novel solutions for advancement of military medicine, biomedical/medical education, and multi-disciplinary collaboration within the DoD, but also with other federal agencies, academia, and industry.
Goals To provide intramural (DoD) expertise in biological printing/fabrication research and product
development. To engage and foster research collaboration with other DoD, Federal and/or non-Federal
scientists to facilitate advancement of biofabrication technologies for military medicine. To develop next generation of DoD expertise in biofabrication by providing educational
opportunities.
4D Bio3 Federal Strategic Council
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4D Bio3: Unique Characteristics
∎ Technology “Centric”: Crosses typical disease- / field-based funding silos Enhanced funding opportunities (e.g. all JPCs)
Truly a “Core” or “Foundational” Program supporting all USU departments
∎ Internally focused mission (DoD…Federal) Enhance USU research and educational capabilities
Augment DoD education (USMA training, MSC recruitment/retention)
Operational focus (e.g. austere environments)
Federal lab (e.g. benchmarking, independent verification and validation)
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4D Bio3 Leadership & Program Management
Leadership
Vincent B Ho, MD MBAProgram DirectorUSUHS/WRNNMC
Russell “Kirk” Pirlo, PhDNRL Principal Investigator
NRL
Stuart K. Williams II, PhDTechnical Officer-RM,
The Geneva Foundation/University of Louisville
Bradley Ringeisen, PhDFederal Strategic Council Chair
DARPA
Joel Gaston, PhDSenior Research ScientistThe Geneva Foundation
Program Management and Consultation
Kelli Blaize-WiseProgram Manager
The Geneva Foundation
Linzie WagnerGrants & Contracts Manager
The Geneva Foundation
Gerald Grant, DDS MSProgram Consultant
The Geneva Foundation/University of Louisville
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4D Bio3 Team
Principal Investigators
Alexandra Miller, PhDPI
USUHS
John Kalinich,PhDPI
USUHS
Kyle Packer, MDPI
USUHS
Lee Johnson, PhDCo-PI
Meadowave
Joe McCabe, PhDPI
USUHS
Angela Melton-Celsa, PhD
PIUSUHS
Joseph Mattapallil, PhDPI
USUHS
Tom Darling, MDPI
USUHS
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Research Scientists and Lab Technicians
Jordan Betz, PhDResearch Scientist
The Geneva Foundation
Frank Alexander, PhDResearch Scientist
The Geneva Foundation
Trey Picou, PhDResearch Scientist
The Geneva Foundation
Kevin Dicker, PhDResearch Scientist
NRL
Don Adube, PhDResearch Scientist
NRL
Kim Smith, MSLab Technician
NRL
Shonnette Grant, MSLab Technician
The Geneva Foundation
4D Bio3 Facilities
∎ USU - 4D Bio3 Facility – A 4,000 ft2 State of The Art Biofabrication/Bioprinting Facility located in Rockville, MD that includes:
1000 ft2 Biofabrication Suite
Tissue Culture Room
Bioreactors and Sensors Facility
Dark Room w/ microscopy
Medical/Surgical Simulation inclusive of Electrospinning
∎ NRL – A 3,000 ft2 facility that has 13 years of expertise in Bioprinter and Bioreactor Development, Biomaterials and Bioprinting Patented Technology
∎ AFRRI – 600 ft2 Cell Processing lab to allow for direct access to WRNMMC.
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Ribbon Cutting – March 28th, 2018(Pictured L-R) CAPT Mark Kobelja, Dr. Yvonne Maddox,
Ms. Elise Huszar, Dr. Terry Rauch, Dr. Vincent Ho, & Dr. George Ludwig
4D Bio3 Capabilities/Technology
∎ 3D Bioprinting of Cells, Hydrogels, and Thermoplastics
Laser forward transfer, extrusion, and microvalve techniques
Multimodal printing
Match material properties to cell and tissue type
∎ Induced Pluripotent Stem Cell Culture and Differentiation
Differentiate into any adult cell type
All cell types in tissue model derive from single source
Human cell lines with normal chromosomal karyotype
Ability to add immune component without cross-reactivity
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Adapted from: doi:10.1038/s41419-017-0028-1
Biofabrication and in vitro testing
In vitro testing
∎ Increased physiologic relevance
∎ Patient specific cells
Site specific
High fidelity
Similar phenotype
∎ Tissue specific customization
Based on tissue requirements
∎ High throughput
Biofabrication
∎ Scalability
∎ Standardization
∎ Industrialization
∎ Building blocks
Cells
Biomaterials
bioreactors
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Custom bioreactors for in vitro modeling
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∎ Customizable for specific use Microscope
Blood Brain barrier
Lung
Microbiome
∎ 3D printing Compact geometries
Internal channels
Rapid prototyping
∎ Biocompatible
∎ Integrated components Integrated sensors
Fluid flow
Electrospinning
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Electrospun vs traditional scaffolds
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Traditional PET transwell
Electrospun gelatin
Extrusion based printing
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Printing to one side of framed biopaper Showing bioprinted hydrogel on both sides of electrospun biopaper
4D Bio3 Capabilities/Technology
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∎ Tight barrier separating central nervous system from circulatory system
∎ Comprised of unique cells
Microvascular endothelium
Astrocytes
Pericytes
∎ Inaccessible for patient testing
∎ Involved in diverse disease states
Blunt trauma
Infection
4D Bio3 In Vitro Blood Brain Barrier Model
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iPS derived brain microvascular endothelial cells CD31 stained red
Present at intercellular junctions
Nucleus stained blue
iPS derived astrocytes GFAP stained green
Intermediate filament present in astrocytes
Nucleus stained blue
BBB: Study Design
∎ Rationale: Gene expression analysis for biopaper compared to PET 26 genes investigated
much more than traditionally investigated for the BBB
Genes chosen based on barrier impact/integrity or cell function
∎ Experimental setup: Between subjects design 2 groups
Astrocytes/endothelial cells on PET transwell
Astrocytes/endothelial cells on biopaper transwell
5 time points (Day 3, 7, 14, 21, and 28)
6 replicates of each sample per variable and time point
∎ Statistical analysis Two tailed t-test between transwell material (biopaper vs PET) at each time point
Results considered significant at p < .05
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qPCR results
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∎ 16 of 26 genes had significant difference in at least 1 time point
∎ PET had more genes with higher expression at early time points
∎ Biopaper had more genes with higher expression at later time points
∎ Transmembrane proteins (OCLDN, CLDN5, other CLDNs) at tight junctions had equivalent expression
∎ Some accessory proteins more highly expressed on biopaper; none higher on PET
∎ Adherens junction transmembrane proteins generally higher expressed on biopaper
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0.01
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Day 3 Day 7 Day 14Day 21Day 28
OCLN
BP
PET
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0.20
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Day 3 Day 7 Day 14Day 21Day 28
TJP2
BP
PET
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Day 3 Day 7 Day 14 Day 21 Day 28
VE-cadherin
BP
PET
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4D Bio3 BBB Culture Model: Experiments in Progress
∎ Military relevant metals and the BBB Investigate the effect of military relevant metals
Sub-toxic concentrations
Effect on cellular gene expression
Effect on barrier ability and metal translocation
∎ Radiation exposure Dose response curves and BBB permeability
BBB cellular histone changes due to radiation
Radiation bystander effect and cellular crosstalk
Radiation effect on BBB permeability and oncology-drug diffusion
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4D Bio3 Bioprinting and Direct Write Electrospinning
∎ Direct write electrospinning is a method of printing nanoscale fibers to form tissue structures and scaffolds for bioprinting.
∎ Complex system integration development using custom mechanical, optical, electrical and fluid real-time control.
∎ We have formed micro-structures with hundreds of lines per second print rates using polyethylene oxide and collagen.
∎ Our printheads can be readily modified for use in existing printers or as part of a custom printer.
1 micron collagen fibers
Meadowave Direct Write Electrospinning printhead
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4D Bio3 Sensor Technology: Optical Based Wireless Sensor Networks
∎ Optical based wireless sensor network for nearfield communication
∎ Custom Lattice FPGA based logic gate designs for compact low power.
∎ Experience with circuit design using ORCAD.
∎ Microchip packaging for biomedical use with direct to chip flexible cabling.
∎ Working towards millimeter scale networked sensors for implantation in bioprinted tissue as localized biomarker detectors and motion sensors.
∎ Uses in real time monitoring, feedback and threat detection for medical and surgical simulation.
bioprinted vascularized tissue
array of mm scale biomarker readout units
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4D Bio3 Current Programs
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Work in Progress:Establish USU lab for DoD bioprinting/fabrication research/collaboration
Modify existing “organ-on-chip” models (e.g. BBB) for DoD research
Develop new models (e.g. gut microbiome, skin, eye)
Develop educational opportunities (MS4)
4D Bio3 Accomplishments & Future Directions
∎ Where We Have Been:Goals
Facility -To provide intramural (DoD) expertise in biological printing/fabrication research and product development.
R & D- To engage and foster research collaboration with other DoD, Federal and/or non-Federal scientists to facilitate advancement of biofabricationtechnologies for military medicine.
Governance Structure – A Federal Strategic Council that combines the expertise and federal stakeholders in the field of Biofabrication and Regenerative Medicine
Where We are Going: Education & Training -To develop next
generation of DoD expertise in biofabrication by providing educational opportunities through R&D advancements.
New 2400 ft2 Facility co-located with 4D Bio3 for Advanced R&D, Distance Learning, and Advance Development partnerships
Public & Private Partnerships – Advanced Technical Councils
Future Combined Technology Research and Development (R&D) – AI, microsensors, combined with biofabrication/RM needs.
Validation and Commercialization Pathway through R&D Public-Private Partnerships.
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4D Bio3 Accomplishments & Future Directions
∎ Blood brain barrier model Infection and countermeasure testing
Precision medicine outcome: directed treatment and patient susceptibility
∎ Microbiome model Combined eukaryotic/prokaryotic model
Precision medicine outcome: directed bacterial and drug treatment
∎ Skin model Skin grafts and barrier tissue permeability testing
Precision medicine outcome: directed wound treatment and disease susceptibility
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Future Directions
Education & Training -Academies Collaborations
Future Directions: FabLAB Austere Environments: FAME
bioprinting collaboration with NASA/ISS
WH Briefing on 18 Jun 2018:
https://youtube.com/watch?v=pqFuI1zQD4s&feature=youtu.be
Austere Environment – Djibouti – May 2019
FabLAB AE-sports injury: scientific and educational collaboration with West Point in bioprinting for sports injury
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4D Bio3 – FAB AE- SI TeamLTC Jason Barnhill, PhD, Dr. Ken Wickiser, PhD,
LTC Jonathan Dickens, MD andLTC Matthew Posner, MD
* Neither the Department of Defense, USU, nor any of its components endorse The companies, or any product, service, or event connected to the organization.
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4D Bio3 - RM
4D Bio3 - RM
∎ Industry, Academic, Government, and Military collaborators
155 partners in 6 regions
∎ Initiatial 3 RM Areas:
1.) Blood
2.) Musculoskeletal
3.) Vascularization
Acknowledgments
We would like to thank:
Russell “Kirk” Pirlo, PhD – NRL Site PI for 4D Bio3
Jordan Betz, PhD – Research Scientist for 4D Bio3
Dr. Vincent B. Ho, MD, MBA – Chair, Professor, USU Radiology and Director of 4D Bio3
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Questions
For More Information or Tour of the Facility:https://www.usuhs.edu/4dbio3/about-us
Please contact:
Dr. Vincent Ho, MD, MBA, [email protected]
Dr. Joel Gaston, PHD, [email protected]
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