Diego Mantovani, PhD, FBSE
Lab. Biomaterials and Bioengineering
Dept of Min-Met-Materials Eng.Research Center, CHU de Québec
Laval University
Innovative Cardiovascular DevicesBiocompatible Nano-Materials
www.lbb.ulaval.ca 2
Outline
Historical/prospective perspective
Nanotechnology for Medical Implants
Advanced Materials with Extreme Properties
Tissue Engineering and Regenerative Medicine
Conclusions
D. Mantovani, 3 www.lbb.ulaval.ca
Life expectancy
D. Mantovani, 4 www.lbb.ulaval.ca
Introduction
D. Mantovani, 5 www.lbb.ulaval.ca
Global challenges for humans
EnergyFoodMedicalEnvironment
D. Mantovani, 6 www.lbb.ulaval.ca
D. Mantovani, 7 www.lbb.ulaval.ca
D. Mantovani, 8 www.lbb.ulaval.ca
LBB: Sciences in MedicineWhy ?
© www.fda.gov
The Medico-Social Problem:• Atherosclerosis represents the main cause for 35 to 38 % of total death (North America & Europe)• Pharmacological treatment, angioplasty, stent implantation and vessel replacement constitute the modern surgical approaches
LBB works aim to improve the performances of medical devices, and to develop and explore the feasibility of new strategies for the replacement and the regeneration of patient diseased tissue that today are utopia but tomorrow could generate new treatments and therapies
D. Mantovani, 9 www.lbb.ulaval.ca
Stents: How?
D. Mantovani, 10 www.lbb.ulaval.ca
Clinical Complications Restenosis
Re-narrowing or blockage of an artery at the site of treatment leading up to 30% of failure after 3 months of implantation [1]. They cannot be explanted.
Toxicity and degradationCorrosion causes a degradation of the mechanical properties of the device [2] and presents a high risk for the release of potentially toxic metallic compounds [3].
[1] Wieneke, et al., Herz, 2002. 27(6): p. 518-26.[2] Bertrand, et al., J. of the American College of Cardiology, 1998. 32 (3): p. 562-571.[3] Uo, M., et al., Biomaterials, 2001. 22(7): p. 677-85.
D. Mantovani, 11 www.lbb.ulaval.ca
Clinical and Scientific Strategy
A surface modification protocol has been developed
Stainless steel is the material widely used (70 to 75%) for the fabrication of stent
Pretreatment + Plasma deposition of a Teflon-like ultra-thin film
Drug Eluting Stents help to prevent restenosis
Delamination
Cracks
But …How to graft bioactive molecules to metallic surfaces while preserving the bioactivity ?
Current Polymer Coatings
Otsuka, Y.;et al, Journal of Invasive Cardiology 2007, 19, 71.
Research Project
Deposit a coating on a stent material -« biocompatible » adherent, stable and impermeable
General objective
Deposition of fluorocarboncoating via
plasma
Amination of polymer
coating via plasma
Attachment of biomolecules
(phosphorylcholine)
Pretreatmentof stainless
steelsubstrateSS316L
Resistant to deformation Stable in pseudo-physiological medium Corrosion inhibitor
D. Mantovani, 14 www.lbb.ulaval.ca
Multistep process
12.7mmt = 0.5 mm
• Electropolishing- To clean the surface- To minimize the roughness- To reduce and uniformize the oxide layer thickness
• Acid dipping- To remove the contaminants due to electropolishing
• Plasma etching (H2 or C2F6 gas precursors)- To further reduce the oxide layer thickness
1 – Pretreatment
D. Mantovani, 15 www.lbb.ulaval.ca
A procedure was established in our labs to optimize the characteristics ofthe electopolished surface.
Haidopoulos et al. (2005). Surf. Coat. Technol. 197(2-3): 278
Achieved Results
SurfacesAFM Analyses
Topography
As Received
Electropolished
Roughness (102 nm)
D. Mantovani, 16 www.lbb.ulaval.ca
2-Plasma deposition
• Development of a pulsed in-house RF plasma reactor•C2F6 as gas precursor•Time of Plasma deposition•Sample distance to antenna •Pressure •Gas flow•Duty cyle
•Setting of a characterization routine for the deposited film•Chemical analysis (XPS, FTIR)•Surface observation (SEM, AFM, contact angle)
Haidopoulos et al (2005). Plasma Process. Polym. 2(5): 424Haidopoulos et al. (2006) J. Mater. Sci. - Mater. Med. 17, 647
SubstrateAt. %
F CrAs-received Not coated - 6
Coated 52 -Pre-treated Not coated - 11.8
Coated 52 -
Preliminary results
Optimization of the plasma parameters
• Objective: Obtain a highly fluorinated and ultra thin film• F content and chemical binding evaluated by XPS and FTIR• Thickness measured by ellipsometry
• Pulsed RF glow discharge on flat specimens – Precursors: C2F6 + 6% H2
– Duty cycle (Ton/ Toff): 5/90 ms– RF Peak power (13,56 MHz): 150 W– Total gas flow: 20 sccm– Pressure: 700 mTorr– Position: afterglow
Lewis et al. (2008) J. Phys. D: Appl. Phys. 41, 045310
D. Mantovani, 17 www.lbb.ulaval.ca
D. Mantovani, 18 www.lbb.ulaval.ca
3-Film adhesion and cohesionEstablish a procedure to characterize the adhesive and cohesiveproperties of the fluoropolymer film after plastic deformation of thesubstrate.
Small Punch Test
Lewis et al. (2007). Adhesion Aspects of Thin Films 3: 1
25% plastic deformation
D. Mantovani, 19 www.lbb.ulaval.ca
Achieved Results•No metallic compounds by XPS analysis were detected at the surface after the deformation suggesting that the film did not delaminate or crack.
•The film surface and bulk compositions after deformation were not altered according to XPS and FTIR analyses.
Sample % F % C % O F/C
No deformation 50.9 ± 0.6 47.4 ± 0.6 1.7 ± 0.5 1.07 ± 0.02
25% deformation 49.6 ± 0.8 47.8 ± 0.5 2.6 ± 0.8 1.04 ± 0.02
Lewis et al. (2007). Adhesion Aspects of Thin Films 3: 1
D. Mantovani, 20 www.lbb.ulaval.ca
SEM
No metallic compounds are detected with XPS
< 1%
25% deformation
Touzin et al. (2010) Mater. Sci. Forum 2009 638-642: 10
Bismuth electrodeposition
High chemical contrast by scanning electron microscopy
Detectable at very low concentration by XPS
Easy to deposit
400 200 00
10
20
30
40
C
Inte
nsity
(x10
3 pho
tole
ctro
ns)
E (binding energy)
25 % deformed substrateelectroplated with Bi at -850 mV
35 nm thick film 100 nm thick film
Bi
XPS spectrum
Holvoet et al. (2010). Electrochim. Acta 55(3): 1042
Corrosion rates
D. Mantovani, 22 www.lbb.ulaval.ca
Corrosion rates (µm/year)Samples Flat Deformed
As-received SS316L 4.6 ± 0.2 6.6 ± 0.1Electroplished SS316L 1.1 ± 0.3 4.1 ± 0.6Coated electropolished
SS316L0.46 ± 0.01 0.8 ± 0.3
Coated H2 etched SS316L 1.3 ± 0.4 1.9 ± 0.3
Coated X8 etched SS316L 13 ± 2 2.6 ± 0.2
Effect of the interface on the corrosion behaviour of the coating/substrate system
•Decrease of the corrosion rates for both flat and deformed coated samples•Etching effect onto the oxide layer and the corrosion rate
Conclusions
NextDLC-BASED COATINGS FOR ANTIBACTERIAL
APPLICATIONS
Shifting the paradigm
Degradable metals …
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BIOMATERIALS
Classes:Metals (corrosion resistant...)Polymers (synthetic, natural, permanent, degradable ...)CeramicsCompositesGlasses
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PHASE I:EXPLORING MAGNESIUM
ALLOYS2002-2005
(J Levesque, D Dubé, D Mantovani)
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J. Lévesque, H. Hermawan, D. Dubé, D. Mantovani, Design of a pseudo-physiological test bench specific to the development of biodegradable metallic biomaterials, Acta Biomaterialia 2008;4:284-295
Schematic view of a simulated coronary artery test-bench for testing degradation behaviour of candidate materials for metallic
biodegradable stent
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Surface morphology of specimens tested under the different conditions after 6, 12, 24, 48, 84 and 168 h: (a) static condition, (b)
dynamic condition (s = 0.88 or 4.4 Pa), (c) dynamic cond. (s = 8.8 Pa)
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SEM images of the cross-section of surface layers on the specimens tested for 168 h at a shear stress of (a) 0.88 Pa, (b) 4.4 Pa and (c) 8.8 Pa
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PHASE II:DEVELOPING
FE-BASED ALLOYS
2004-ongoing
(co-supervised respectively by Profs. D. Dubé & M. Fiset)
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a- Fe-based Alloy
DESIGN AND FABRICATION PROCESSES FOR METALLIC DEGRADABLE BIOMATERIALS
H. Hermawan, D. Dubé and D. Mantovani
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Fabrication
Starting powders
Step-1: Mixing, 1 hStep-2:
Compacting, 10 T
Step-3: Sintering, 1200C, 2h, Hydrogen
Step-2 Step-3 Step-4 Step-3+4
Step-4: Cold rolling
Starting powders
Mn35%
Fe65%
Lubricant0.5%
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www.lbb.gmn.ulaval.caLaboratory for Biomaterials & Bioengineering
Mechanical properties
-yield (MPa)Fe35Mn = 228SS316L = 235e (%)Fe35Mn = 32SS316L = 56E (GPa)Fe35Mn = 179SS316L = 193
The strength of Fe35Mn* is comparable to SS 316L** Fe35Mn is ductile enough for stent material
* Densified P/M alloy (annealed); ** Wrought alloy (hot rolled); the tests were performed based on ASTM E8
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www.lbb.gmn.ulaval.caLaboratory for Biomaterials & Bioengineering
Non-magnetic behaviour
Fe35Mn has low magnetic susceptibility (non-magnetic) It’s magnetic susceptibility is not altered by plastic deform.
The tests were performed by an Alternating Gradient Magnetometer.
0.00
0.50
1.00
1.50
0% 5% 15%
Degree of plastic deformation
Mag
netic
Sus
cept
ibilt
y (
m3 /k
g)
Fe35MnSS316L
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Cross sectional profile of polished Fe-Mn specimens: (a) before and (b, c) after 1 week and 3 months of degradation test respectively, and (d, e) etched Fe25Mn and Fe35Mn specimens
after 3 months of degradation test respectively
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Concentration of iron and manganese ions in test solution as a function of immersion time for specimens of Fe25Mn and Fe35Mn alloys measured by the AAS
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Phase II-bBOTTOM-UP APPROACH
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M. Moravej, M. Fiset and D. Mantovani
INVESTIGATION OF FABRICATING BIODEGRADABLE CORONARY IRON STENT BY ELECTROFORMING
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www.lbb.gmn.ulaval.caLaboratory for Biomaterials & Bioengineering
Electroforming method
• ASTM B 374 : production orreproduction of articles byelectrodeposition upon amandrel or mould that issubsequently separated fromthe deposit.
Electroforming [1]
[1] J. A. McGeough et al, Annals of the CIRP, 2001
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www.lbb.gmn.ulaval.caLaboratory for Biomaterials & Bioengineering
Fabrication of pure iron films by electroforming
•Manufacturing of complex shapes and surfaces•Fabrication of parts with different size, thickness and properties•Production of high purity materials•Fabrication of thin walled materials with dimensional precision
Electrodeposition of stent tubes directly on a dissolvable cathode with a bottom-up method
- +
Cathode Anode
Electrolyte
_
+Cations
Anions
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Iron electroformed foils (100 microns)
Surface morphology Cross section
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Microstructure
Electroformed Fe annealed at 550°C
Average grain size: 2 microns
Fe fabricate by casting annealed at 550°CAverage grain size: 30 microns
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Degradation rate
Material Electroformed Fe
E-Fe annealed
CTT-Fe annealed
Fe-35Mn alloy
AM60B-F Mg alloy
DR(mm/y)
0.40 0.25 0.14 0.26 2.78
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Electroformed iron minitube
D= 5 µm D= 25 µm
Electro-formed iron stent316L stainless steel stent1
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• Design and development of• New Fe-based alloys;• New processes for high purity alloys;• New processes for bottom-up fabrication of stents;
• New surface treatments for positively controlling the corrosion;
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Ongoing Works
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H. Hermawan, D. Dubé, D. Mantovani. Acta Biomaterialia 6 (2010) 1693–1697
Concept in cardiovascularapplications
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Concept in musculoskeletalapplications
Mg Implant
From Frank Witte
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1st Berlin2009
2nd Maratea2010
3rd Quebec2011
May 2010
4th Maratea2012
ww
w.b
iode
grad
able
met
als.o
rg
Intl Symposium on Biodegradable Metals
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Génie tissulaire vasculaire
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Les approches
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L’approche par échaffaudage
D. Seifu, A. Purnama, K. Mequanint, D. Mantovani. Nature Cardiology. 2013, in press.
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Méthodologie
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Materials and Methods
1. Mechanical Stimulation System
Flexcell international corporation
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Collagen ScaffoldSEM
L. Levesque, Advanced Materials Research, 2012
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SEM
Collagen + Cells DynamicCondition
L. Levesque, Advanced Materials Research, 2012
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SMCs’ Collagen Production
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L. Levesque, 9th World Biomaterials Congress, 2012
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Co-culture statique : Qui, quoi et quand ?
Cellules endothélialesCellules musculaires lisses
CRSNG-FONCER, Collaboration avec Jayachandran Kizhakkedathu, UBC
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Co-culture statique : Qui, quoi et quand ?Gélification (collagène + CML) = 30 minMaturation gel endothélialisé = 24h
Coloration au Trichrome de MassonVert : collagène ; Rouge : cytoplasme ; Noir/brun ; noyau
Tapis de CE à la surface du gel
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Co-culture dynamiqueLa surface du gel va être soumis à une contrainte de cisaillement afin d’observer l’adhésion des cellules endothéliales et leur orientation dans le sens du flux.
Système de perfusion avec une pompe Masterflex et une chambre de flux Ibidi.
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Comment peut-on définir le remodelage ?
Production de matrice extracellulaire.
Production de facteurs de croissance.
Réorientation des cellules et des fibrilles de collagène
Amélioration des propriétés mécaniques
Dégradation des protéines.
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Culture dynamique 2 semaines
+Contrainte
de cisaillement
Pression+
Meilleures
propriétés mécaniques
Culture statique 2 semaines
Remodelage par les cellules
Effet des cultures statique et dynamique sur le remodelage des gels de collagène
Remodelage par les cellules
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Dispositif expérimental
ÉCHAFAUDAGECollagène sans cellules
CONSTRUCTION ARTÉRIELLE Culture statique 1 SEMAINE
CONSTRUCTION ARTÉRIELLECollagène + cellules, t = 0
CONSTRUCTION ARTÉRIELLE Culture statique 2 SEMAINES
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Combinaison
500 μm
Culture statique– Microscopie de fluorescence
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www.lbb.ulaval.caLaboratory for Biomaterials & Bioengineering
SEM
25 X
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SEM
100 X
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Moteur rotatif à5 tours/min
Réservoir demilieu de culture
Espaceur en siliconepour assurer un axe derotation constant
Endothélialisation d’une construction artérielle à base de collagèneConception d’un bioréacteur à parois rotatives
Bouchon avec filtre 0.22 μm
Roulement à billes(Ø = 4,7mm)
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Measuring mechanical property
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Relaxation test of cell seeded tubular construct using Instron 5848 Microtester, where SLSC9D is Single Layer Static Culture of 9 Days and DLSC9D Double Layer Static Culture of 9 Days
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Immunohistochemistry
Red: PSMS with Calponin, Blue: Nuclei, Green: HUVECs with CD31 and actin and collagen Green with Alex fluor green.
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Masson Trichrome staining
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SEM
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Con
train
te (P
a)
Temps (s)
106 cellules/mLCulture statique1 semaine
Pas de cellulesCulture statique2 semaines
Tests de relaxation en circonférentiel
Culture statique – Propriétés mécaniques
106 cellules/mLCulture statique1 semaine
ε = 0,1 ε = 0,2 ε = 0,3
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Laser guided thickness measurement
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LaserMike 136 Thickness and external diameter measurement of cell seeded construct.
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Measuring mechanical properties
ε = 0,1 ε = 0,2 ε = 0,3 ε = 0,4 ε = 0,5 ε = 0,6 ε = 0,7 ε = 0,8
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Measuring mechanical properties
Relaxation test of cell seeded tubular construct using Instron 5848 Microtester, where SLSC9D is Single Layer Static Culture of 9 Days and DLSC9D Double Layer Static Culture of 9 Days
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Futur Collagène-Élastineversion 1
Collagène
Résistances aux tissus.
ELP(VPGVG)
HELP(VAPGVG)
Élasticité aux tissus.
Ce motif est responsable de la prolifération cellulaire et d'autres activités biologiques. Des résidus de lysine et de la glutamine présents dans les domaines riche d’alanine permet deux
types de spécifique, réticulation enzymatique, en utilisant la lysyl oxydase et / ou de latransglutaminase, afin pour obtenir une matrice.
Capacité d'auto-assemblage et d'auto-organisation dans polymères réticulés avec des propriétésphysiques et mécaniques remarquablement similaires à l'élastine native.
Collagène-HELP
Prof. A. Bandera
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Prof. Marisa Beppu
Structural layer: mechanical resistance, elasticity, anti-bacterial
capacity Konjac glucomannan
and chitosanmicrostructured with
silk fibroin.
Bioactive layer :growth factor stimulation, re-epithelialization, drug
release
Dressings high biological performance
Collagen or gelatin, cells and drugs
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Conclusions
Structures d’échafaudages avec ensemencement de cellules VS structures d’échafaudages a base de cellules!
Un cycle de culture est la clef pour emmener les cellules a structurer le tissus régénéré
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“The Human Being can do all things if He will”
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Remerciements
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Our students are our force• 4 associate researchers, 6 (24 depuis 2000) post-docs, 18 (47) PhD and 3 (48) MSc students, from 13 (32) countries, speaking more than (23) languages and representing (7) religions, constitute the LBB;
In this mixture of identities, cultures and nationalities we found each day the inspiration
required to push innovation in surgery and in the connected fields;
• 40 % of our students hold a merit scholarship;