Organ and bio 3D printing

le centre collectif de l’industrie technologique belgeThe Collective Centre of the Belgian Technology Industry
Additive Manufacturing Department
3D Bio-printing

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The future is not that far away...
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02/05/2013© Sirris | www.sirris.be | info@sirris.be |

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Bio-printing
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Agenda
• What is « bio-printing » ?
• What is the market and who would be interested?
• State of the art – current results and achievements
• What is required and how to print 3D bio-materials ?
• Conclusions
• Bibliography

What is « bio-printing » ?
02/05/2013© Sirris | www.sirris.be | info@sirris.be | 4
What is “bio-printing”?
“ The ability to print various biological materials and cells along with various
tissue scaffold materials”
Tissue Engineering:
Its goal is to produce functional cell, tissue and organ to repair, replace or
enhance biological function that has been lost by disease and injury. It is also
one of the most promising approaches to solve the problems of shortage of
suitable organs for transplantation.
Engineered and « 3D printed » bio-scaffolds:
Temporal architectural 3D structures for cell adhesion and cell growth
Usually: biodegradable (poly-glycolic acid and poly-lactic acid)
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What is « bio-printing » ?
Bio-scaffolds & cells:
• Cells are seeded onto the scaffolds and cultured in vitro in advance before
implantation.
• After cell adhesion, the cell-adhered scaffolds are implanted into the
recipients.
• After implementation, biodegradable materials are degraded and finally only
the implanted cells remain and form functional tissues in vivo.
Remaining obstacles to overcome & reach the goal:
• Find cells that will not be rejected by the host’s immune system
• How to organize specialized cells into 3D tissues ?
• How to bring nutriments (oxygen, minerals, blood, cell signals, …) to the
printed cells?
• How to transport them and keep them sterilized to the O.R. ?
• Results are depending on the 3D printing technology !
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Bio-printing
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Agenda
• Conclusions
• Bibliography

What is the market and who would be
interested?
Examples:
• Hair
• Contact lenses
• Artificial eye, cornea, cristalline
• Surgery in general (artifical organs, tissue repair, …) in forms of implants:
• External implants, internal implants, bone subistitutes, …
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Examples:
• Gynecology, obstetrics, urology
• Aesthetic surgery
• Tissue graft assistance : liver, kidney, pancreas, nerve, spinal cord,...
• Pharmaceutical : "Drug Delivery Devices"
• Cardiovascular field : vessels, pacemaker, artificial heart, valves, stents, coils,
balloon, glue, mirobeads, blood, catheters,...
• Orthopaedic surgery : plate, rode, screw, pin, cement, protheses, ligaments,
tendons
• Cell chips (electronics)
• Cosmetic applications (direct cosmetic print on skin)
interested?

interested ?
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• Alternative to donor waiting list
• Limited available « material » resource (skin, organs, cells, …)

interested ?
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Some facts:
• It would take 1,690,912,929,600 hours to print a liver for every member of
the human race using today’s processes.
• Every year, the number of people on the waiting list for an organ transplant
increases, yet the amount of donors and available organs remains at a low.
• USA: more than 114,300 (2012) people on the waiting list as candidates
• More than 73,000 active waiting list candidates
• In 2005, 1848 patients died waiting for a donated liver to become available
(USA)
• 17000 adults and children have been medically approved for liver transplants
and are waiting for donated livers to become available (January 2012)
• Drug industry problem:
• Each year, the industry spends more than $50 billion (USA, 2012) on R&D
and approximately 20 new drugs are approved by the FDA
• A new drug, on average costs $1.2 billion and takes 12 years to develop

interested ?
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• From a monetary standpoint, tissue engineering and regenerative medicine
research are now mainstream disciplines that receive nearly $200 million a
year in funding from the United States alone (2001).
Why is 3D important?:
• 2D cell culture: unnatural behavior in monolayers on plastic
• 3D cell culture: mimics natural state for tissues in the body
• A reliable, easy to use and inexpensive method for 3D cell growth will
make 2D methods obsolete !
Use of 3D cell culture: explosive growth:
• Total cell culture market: $520M (2004)
• Market growing at 13%/year to $780M (2007)
• 3D product citations (PubMed) and market share growing exponentially
• Growth limited by product availability
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interested ?

Organ printing: the future of medecine !
Conclusions:
3D bioprinting technology has the potential to significantly impact the speed,
predictiability and consequently the cost of successful drug discovery !
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Bio-printing
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Agenda
• Conclusions
• Bibliography

State of the art – current results and
achievements
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achievements
Evolution of Tissue Engineering and « Bioprinting »
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Charles Hull invented SLA
Dr. Gabor Forgacs (ONVO founder) and
colleagues made the observation that cells
stick together during embryonic
development and move together in clumps
with liquid-like properties. Manufacturing
program
The first human patients
underwent urinary bladder
augmentation using a synthetic
scaffold seeded with the
patient’s own cells (engineering,
not printed)
Thomas Boland’s lab at
Clemson modified an inkjet
printer to accomodate and
dispense cells in scaffolds
DR. Forgacs developed
new technology to
engineer 3D tissue with
only cells, no scaffolds
Organovo creates the
NovoGEN MMX Bioplotter
using Forgacs technology
Organovo prints the first
human blood vessel
without the use of scaffolds
Organovo develops multiple
drug discovery platforms,
3D bioprinted disease
models made from human
cells
1984 1996 2000 2003 2004 2006 2009 2010 2011
World’s first “3D
printed” artificial
bladder implanted
First lung tissue
patch
2012
First cardiac
sheet or
patch
First nerve
guides

achievements
Evolution of Tissue Engineering and « Bioprinting »
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Today 2011-2012:
Small-scale tissues for
drug discovery and
toxicity testing
Tomorrow 2013-2015:
Simple tissues for implant
(e.g. cardiac patches or
segments of tubes, like
blood vessels
Future 2015-2030:
Lobs or pieces of
organs
Very future 2030>:
Full organs

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02/05/2013
Case study (USA): Artificial bladder (2006)
• World’s first articial bladders built by an 3D printing
technology in lab !
• Development of artifical bladder first tested on dogs (1999)
• Transplantation was successful (but no post-reports)
• 2006: Transplantation on 7 human patients
• No risk of transplantation rejection (patient’s own cells)
• Procedure: « Orthotopic Neobladder Procedure »
• CT-Scan of the bladder (for the geometry)
• Tissue sample is taken from the patient’s bladder
• Biodegradable scaffolds are built using ink-jet printer
• Cells are grown into biodegradable scaffold (hydrogels)
• Transplantation of the artifical bladder
• Scaffold is safely degraded within the patient’s body
• Ink-jet printer: cartridges used stem cells & cross-linker
[Source: Wake Forest University School of Medicine]
© Sirris | www.sirris.be | info@sirris.be |
achievements

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02/05/2013
Case study (USA): Artificial liver (2009)
• Bio-engineering of human liver (5.7g)
• 3D printing of collagen skeleton
• Application of human liver cells on skeleton matrix
• Artificial liver is then placed in a bioreactor (nutrients & oxygen)
• After one week: widespread cell growth inside the bioengineered organ
with progressive formation of liver tissue as well as liver-associated
functions.
achievements

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02/05/2013
Case study (Europe): Artifical trachea (2011)
• World’s first articial trachea built by an additive
manufacturing technology !
• Bioartifical matrix (CT-Scan to CAD file)
• Stem cells sprayed on matrix
• Swedish hospital & european team (University Hospital
of Karolinska – Prof. Paolo Macchiariini)
• Operation time: 12h
• Trauma: cancer (size of a golf ball)
• Material of the matrix: Synthetic nanocomposite polymer
[Source: Karolinska Institute in Stockholm ]
achievements

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02/05/2013
Case study: In situ skin bio-printing for burn wounds (2011)
• Portable skin printing system
• Uses living cells to create tissue-engineered skin grafts to cover burn wounds
• Future application: battlefield burn wounds
• Fibroblasts and keratinocytes are printed directly onto skin
• Suspensions with cells are mixed with fibrinogen, type 1 collagen and
thrombin at the moment of application
• Application already tested on mice
achievements

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Bio-printing
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Agenda
• Conclusions
• Bibliography

What is necessary to print an organ ?
• The need to consider:
• Histology and anatomy in general
• What cells should be used?
• Conservation of the functionality
• What scaffold should be printed?
• What technology should be used?
• Organs are:
1) 3D structures
2) They have characteristic micro-structures required to fulfill the particular
function of an organ
3) They are composed of multiple type of cells and extra-cellular matrices
4) They have a complex vascular network to sustain the cells in the organs
What is required and how to print?
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1. Preprocessing:
• CAD, blueprints, preconditioning
2. Processing:
• Actual 3D printing, solidification
3. Postprocessing:
• Perfusion (bioreactor), postconditioning, accelerated tissue maturation
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Three potential human stem cell sources are emerging:
• Human embryonic stem cells (politically controversial)
• Resident stem cells (isolated from organs)
• Circulated bone marrow derived adult stem cells
So far: 30 =/= Cells have already been tested (2D array):
• Chondrocytes, C17.2, PC6, AML12, C166, HL1, HeLa, neural precursor cells,
mesenchymal stem cells, mouse embryonic stem cells, …)
• No significant difference in cell viability (all >95%) compared to manually
plated cells, suggesting that our cell printing technique can be generally
applied to most of cell types
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Requirements (2D-array bio-printing:
• use of a pneumatically-driven electromechanical valves for the use of liquid
materials (viscosity up to 200 Pa*s)
• Independant temperature control for the dispenser itself
• A wide range of hydrogel materials that needs to be crosslinked
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Requirements for direct 3D-Printing of organs:
• Include growth factors in the scaffolds
• Have biodegradable scaffolds rather than non-ones (bioactive as well?)
• Having nano-modifications on the scaffolds for better cell ingrowth?
• Needs to be included in the scaffold: extracellular matrix, growth factors,
vascular network and different cell types)
• Living cells will be patterned into hydrogel tissue scaffolds
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+ growth factors
(
…
)Cell type A Cell type Z
+ agents
layer 01
layer 10^9

Current and possible technologies:
1. Scaffold based approaches
1. Porogen leaching
2. Phase-separated scaffolds
3. Gas foaming
2. Textile technologies
1. Electrospinning
2. Knitting and braiding
3. Direct « 3D-printing » technologies
1. Stereolithography
2. Selective laser sintering
3. Three-dimensional printing (3DP) (inkjet printing)
4. Systems based on extrusion/direct writing
5. Indirect 3D printing
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1. Scaffold based approaches: Basic principles
Combination of:
• Viable cells
• Biomolecules
• Structural scaffold
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Scaffolds serve a purpose:
• Support cell migration
• Growth and differentiation
• Guide tissue development
• Organization into a mature and healthy state
Requirements of scaffolds:
• Biomechanical (e.g.: wound contraction forces; vascularization; in vitro and most
of all in vivo growth dynamics)
• Chemical (cell and tissue remodeling: interaction between cell type and scaffold
type material (e.g.: skin v.s. bone cell type))
• Physical (mechanical strength & stiffness)
• Biological (cell attachment, mass transfer, degradation and resporption kinetics)
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Current state of progress:
• still infancy (experimental);
• no certain knowledge about the scaffold geometries…
• What about patient-custom tissue engineering ? And patient activity ?
• What about multi-material printing (scaffolds) ?
Drawbacks:
• Cell distribution and cell composition inside of the scaffold cannot be
controlled
• Morphogenesis of essential tissue architecture especially microstructures is
completely dependent upon cells alone: Very difficult to fabricate
physiologically functional tissues which have special micro-3D structures
using the scaffold based approach.
• A new approach is necessary! – Or more basic research on scaffolds…
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Characteristics that need to be understood, defined and verified:
• Material compositions
• Porous architecture
• Structural mechanics
• Surface properties
• Degradation properties and their resulting products in the body
• Composition with other agents? (multi-material printing)
• Changes of all these factors in time (kinetics)
• Manufacturing technology and technology readiness
Conclusions:
• There is no scaffold serving universal applications !
• Scaffolds should be defined in function of the future growing cells?
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Morphology and architecture;
• Mechanical properties of porous scaffolds depend on:
• Relative density (depending on stiffness and yield strength in
compression)
• Properties of the material (pore edges & walls need to be considered)
• Stiffness : E
• Yield strength in compression: s
E=C1(100-P)^n ; s=C2(100-P)^n
Porosity definition:
• Compromise between porosity and mechanical properties
• Pore interconnectivity is a critical factor !
• Necessary for: cell migration and proliferation (initial stages)
• Scaffolds should have 100% interconnecting pore volume !
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Porosity definition:
• Recent studies demonstrate that pore size is less important for bone
formation
• Optimal pore sizes of 200-600µm are required to support bone growth (local
support for vasculature)
• This is true for 3D scaffolds with nondesigned, random collection of pores,
varying in size and interconnectivity
• With 3D scaffolds built by 3D printers with 100% of interconnecting pores and
homogeneous pore architecture, no significant difference in bone formation
with varying pore size between 300-1200µm
Question:
• Is there an optimal biomaterial and architecture for regeneration of specific
tissues ?
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1. Scaffold based approaches: Porogen leaching
Principle:
• Dispersing a template (particles) within a polymeric
or monomeric solution, gelling or fixing the structure,
and removal of the template
Result:
• Porous scaffold
Technical aspects:
• Cheap technique
• Possible to produce structures with locally porous
internal architectures
• Porosities up to 300µm in diam.
• Only local interconnection and non-controlled possible
• No control on the shape/geometry of the pores and the mechanical properties
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1. Scaffold based approaches : Phase-separated scaffolds
Principle:
• Relies on the controlled phase separation of polymer
solutions into high and low concentration regions
upon cooling. (high concentration = solidification);
(low concentration = formation of pores)
Result:
• Porous scaffold with variable morphological nature
Technical aspects:
• Liquid-liquid phase separation
• Solid-liquid phase separation
• Polymerization-induced phase separation
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1. Scaffold based approaches : Gas foaming
Principle:
• Solvent-free formation of porous materials through generation of gas bubles
within a polymer.
• The polymers are pressurized with a gas (CO2) until saturation.
• The release of the pressure results in nucleation
Result:
• Porous scaffold with more or less controlled pore sizes but uncontrolled
interconnectivity
Technical aspects:
• Pore sizes up to 100µm in diam.
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Limitations of these scaffold based approaches:
• inability to control cell distribution in the 3D structures
• inability to control positioning of multiple cell types
• inability to control composition of the scaffold at specific locations
• inability to control local concentration of growth factors
• inability to control induction of blood capillaries
• inability to control enhancement of the target organ cells at specific locations
• inability to control biodegradation of the scaffold material (not always true)
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1. Porogen leaching
3. Gas foaming
1. Electrospinning
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2. Textile technologies: Electrospinning
Principle:
• A continuous fiber is produced by extruding a polymer
melt or solution through a spinneret and then
mechanically drawn onto a winder or a series of winders
and collected on a spool.
Result:
• Polymeric sheets with high permeability
Technical aspects:
• The diameter can be controlled by the extrusion diameter
• The extrusion will affect the crystallinity of the polymer, this will influence the
mechanical strength and degradation behavior
• Possible to control the orientation of the fiber segments (neural tissue
engineering)
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2. Textile technologies: Knitting and braiding
Principle:
• Individual fibers or multifilament yarns are woven, knitted or braided into
patterns with variable pore sizes.
Result:
• Structures with poor mechanical strength that need to be filled with a
secondary scaffold such as collagen gel or electrospun fibers.
Technical aspects:
• The knitted structures only serve as a mechanical support for a secondary,
interstitial scaffold that might be damaged otherwise.
• Still experimental
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1. Porogen leaching
3. Gas foaming
1. Electrospinning
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3. Direct « 3D-printing » technologies :
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3. Direct « 3D-printing » technologies :
Principle:
• Various “3D-Printing” methods.
Result:
• Scaffolds with highly reproducible architecture and compositional variation
across the entire matrix due to CAD controlled fabrication.
Technical aspects:
• Results highly depending on the technology and materials that can be
processed !
• Most of the technologies have to be adapted (Bioprinter, SLA, SLS, …)
• Possible to fabricate biphasic or triphasic matrix systems (SLA): high potential
of micro-SLA (future)
• SLS: mostly used for calcium-phosphate scaffolds (or others: PEEK; PEAK;
PEKK, …)
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3. Direct « 3D-printing » technologies : Powder based technologies
Main drawbacks of powder based technologies :
• Open pores must be able to allow the internal unbound powders to be
removed (SLS, ZCorp, LBM, EBM, …) if the part is designed to be porous.
• Surface roughness and the aggregation of the powdered materials affect the
efficiency of removal of trapped materials.
• Resolution of printers is limited by the specification of the nozzle size and
position control (print head movement).
• Particle size of the powder used defines the layer thickness (100-400µm)
• Most materials are not available or suited for tissue engineering (need to be
established in-house).
Conclusions:
• Inkjet-printing is promising but must be adapted for tissue engineering
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3. Direct « 3D-printing » technologies : Extrusion technologies
Principle:
• Extrusion of filaments or plotting of dots in 3D without
incorporation of cells.
• Variety of polymers possible
• Hot melts as well as pastes/slurries possible
Main drawbacks of powder based techniques :
• Only idea here is to build a physical scaffold
• Only a certain range of thermoplastics for tissue engineering usable
• Cells or other biological agents cannot be encapsulated into the scaffold
matrix during fabrication process
• Design of pores is limited (diameter of extruded filament, physical connection
between the layers, turning points, …)
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3. Direct « 3D-printing » technologies : Ink-jet technologies
Ink-jet technologies : Considered as the « true organ printing »
Two possible methods: ink-jet printing or laser-printing technology
Principle:
• Gelation technique of ink-jet printing:
• Need to use two different types of gel solution, gel precursor and gel reactant.
• Aqueous sodium alginate solution forms a hydrogel in contact with Ca2+ ions.
(0.8-1% Sodium alginate on 2% CaCl2 solution)
• Alginate hydrogel is one of the biocompatible hydrogels
• They provide both structural strength for 3D structures and an aqueous
environment for cells
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Ink-jet:
• Modified commercial version of buble jet or piezoelectric printer to dispense
cells on the hydrogel material
Laser-technology:
• Based on focusing a high-energy laser pulse onto a post above the cell-laden
gel and subsequent dispensing of the cells underneath the evaporated spot.
• -
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Why hydrogels:
• Good candidate for non-skeletal tissue engineering
• Facilitate the transport of oxygen through diffusion and integrate readily into
the surrounding extracellular matrix
• Controllable dissociation/biodegradation of hydrogels in physiological
environments
• Useful for ex for chondrocytes and hepatocytes
Drawbacks:
• ink drying (inkjet droplets are so small that they dry immediately)
• ink bleeding in wet conditions (if cells are printed onto wet substrates to
prevent drying, the printed cells spread out and lose print resolution)
• how to fabricate 3D structures with an inkjet printer?

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Advantages for tissue engineering fabrication:
• High resolution fabrication (pico-liter sized ink droplets)
• Fabrication of composite products with different cells, materials and growth
factors
• Fabrication of large-sized products (rapidly)
• Easy to apply CAD bio-fabrication
• Printable onto gels, aqueous solution, cells or directly onto the targets
wounds during surgical operation
• Usability of reactive gel material and reactive two materials.
• Biomaterials: cells, proteins, DNA, bio-polymers, drugs, …
• Direct cell printing, handling and positioning

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Future directions:
• Main challenge: obtaining a homogenous distribution of cells throughout the
entire 3D scaffold volume.
• Two possibilities of incorporating cells into scaffolds (organ printing):
1) Seeding of cells onto the surface of the scaffold (after fabrication)
2) Incorporation of cells onto the scaffold fabrication process
• -

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Bio-printing
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Agenda
• Conclusions
• Bibliography

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Organ printing – Conclusions and perspectives

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« Medical Additive Manufacturing & Rapid Prototyping »
« Sirris »

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Sirris Additive Manufacturing: Contact
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Carsten ENGEL
Biomedical Engineer
Department of Additive Manufacturing
Mail: carsten.engel@sirris.be
Mobile: +32 498 91 94 50
Skype: Carsten-Engel
SIRRIS
Rue Auguste Piccard, 20
B-6041 GOSSELIES BELGIUM
http://www.sirris.be

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Agenda
• Conclusions
• Bibliography

Bibliography
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Engineering”, Biomaterials 33 (2012) 6020-6041
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processes”, CIRP Annals - Manufacturing Technology 61 (2012) 635–655
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bone tissue engineering: A review”, Acta Biomaterialia 8 (2012) 1401–1421
• R. Gaetani et al., “Cardiac tissue engineering using tissue printing technology and human
cardiac progenitor cells”, Biomaterials 33 (2012) 1782-1790
CENG

Bibliography
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CENG

Bibliography
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European Cells and Materials, Vol. 5, 2003, (pages 29-40)
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Bibliography
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454 (2004) 383–387
• R. E. Saunders et al., “Delivery of human fibroblast cells by piezoelectric drop-on-demand
inkjet printing”, Biomaterials 29 (2008) 193–203
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engineering”, Acta Biomaterialia 6 (2010) 2494–2500
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(2009) 2164–2174
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in Biotechnology Vol.22 No.12 December 2004
• B. da Graca et al., “Vascular Bioprinting”, The American Journal of Cardiology (2011)
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tomography of vascular constructs within thick hydrogel scaffolds”, Biomaterials 33 (2012)
5325-5332
• C.M. Mota et al., “Bioextrusion”, RPD 2008 – Rapid Product Development
CENG

Bibliography
• T. Xu et al., “Hybrid printing of mechanically and biologically improved constructs for cartilage
tissue engineering applications”, Biofabrication 5 (2013) 015001 (10pp)
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submerged in a hydrophobic high-density fluid”, Biofabrication 5 (2013) 015003 (11pp)
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Bioprinting and Biofabrication in Bordeaux (3B’09)”, Biofabrication 2 (2010) 010201 (7pp)
CENG

Organ and bio 3D printing

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