Ldb Convergenze Parallele_Mantovani_03

Diego Mantovani, PhD, FBSE
Lab. Biomaterials and Bioengineering
Dept of Min-Met-Materials Eng.
Research Center, CHU de Québec
Laval University
Innovative Cardiovascular Devices
Biocompatible 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

Introduction

Global challenges for humans
Energy
Food
Medical
Environment

LBB: Sciences in Medicine
Why ?
© 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

Stents: How?

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 degradation
Corrosion 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.

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
fluorocarbon
coating via
plasma
Amination of
polymer
coating via
plasma
Attachment of
biomolecules
(phosphorylcholine)
Pretreatment
of stainless
steel
substrate
SS316L
 Resistant to deformation
 Stable in pseudo-physiological medium
 Corrosion inhibitor

Multistep process
 12.7mm
t = 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

A procedure was established in our labs to optimize the characteristics of
the electopolished surface.
Haidopoulos et al. (2005). Surf. Coat. Technol. 197(2-3): 278
Achieved Results
Surfaces
AFM Analyses
Topography
As Received
Electropolished
Roughness (102 nm)

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): 424
Haidopoulos et al. (2006) J. Mater. Sci. - Mater. Med. 17, 647
Substrate
At. %
F Cr
As-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

3-Film adhesion and cohesion
Establish a procedure to characterize the adhesive and cohesive
properties of the fluoropolymer film after plastic deformation of the
substrate.
Small Punch Test
Lewis et al. (2007). Adhesion Aspects of Thin Films 3: 1
25% plastic
deformation

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

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 0
0
10
20
30
40
C
Intensity(x10
3
photolectrons)
E (binding energy)
25 % deformed substrate
electroplated 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
Corrosion rates (µm/year)
Samples Flat Deformed
As-received SS316L 4.6 ± 0.2 6.6 ± 0.1
Electroplished SS316L 1.1 ± 0.3 4.1 ± 0.6
Coated electropolished
SS316L
0.46 ± 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
Next
DLC-BASED COATINGS FOR ANTIBACTERIAL
APPLICATIONS

Shifting the paradigm
Degradable metals …

www.ulaval.ca
BIOMATERIALS
Classes:
Metals (corrosion resistant...)
Polymers (synthetic, natural, permanent, degradable ...)
Ceramics
Composites
Glasses
25

www.ulaval.ca
PHASE I:
EXPLORING
MAGNESIUM
ALLOYS
2002-2005
(J Levesque, D Dubé, D Mantovani)
26

www.ulaval.ca 27
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

www.ulaval.ca 28
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)

www.ulaval.ca 29
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

www.ulaval.ca
PHASE II:
DEVELOPING
FE-BASED
ALLOYS
2004-ongoing
(co-supervised respectively by Profs. D. Dubé & M. Fiset)
30

a- Fe-based Alloy
DESIGN AND FABRICATION
PROCESSES FOR METALLIC
DEGRADABLE BIOMATERIALS
H. Hermawan, D. Dubé and D. Mantovani

www.ulaval.ca
Fabrication
Starting powders
Step-1: Mixing, 1 h
Step-2:
Compacting, 10 T
Step-3: Sintering, 1200C, 2h, Hydrogen
Step-2 Step-3 Step-4 Step-3+4
Step-4: Cold rolling
Starting powders
Mn
35%
Fe
65%
Lubricant
0.5%

www.ulaval.ca
www.lbb.gmn.ulaval.caLaboratory for Biomaterials & Bioengineering
Mechanical properties
-yield (MPa)
Fe35Mn = 228
SS316L = 235
e (%)
Fe35Mn = 32
SS316L = 56
E (GPa)
Fe35Mn = 179
SS316L = 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

www.ulaval.ca
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
MagneticSusceptibilty(m
3
/kg)
Fe35Mn
SS316L

www.ulaval.ca 35
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

www.ulaval.ca 36
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

www.ulaval.ca
Phase II-b
BOTTOM-UP APPROACH
37
M. Moravej, M. Fiset and D. Mantovani
INVESTIGATION OF FABRICATING
BIODEGRADABLE CORONARY IRON STENT
BY ELECTROFORMING

www.ulaval.ca
Electroforming method
• ASTM B 374 : production or
reproduction of articles by
electrodeposition upon a
mandrel or mould that is
subsequently separated from
the deposit.
Electroforming [1]
[1] J. A. McGeough et al, Annals of the CIRP, 2001

www.ulaval.ca
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

www.ulaval.ca
Iron electroformed foils
(100 microns)
Surface morphology Cross section

www.ulaval.ca
Microstructure
Electroformed Fe annealed at 550°C
Average grain size: 2 microns
Fe fabricate by casting annealed at
550°C
Average grain size: 30 microns

www.ulaval.ca
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

www.ulaval.ca
Electroformed iron minitube
D= 5 µm D= 25 µm
Electro-formed iron stent
316L stainless
steel stent1

www.ulaval.ca
• 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;
D. Mantovani, 44 www.lbb.gmn.ulaval.ca
Ongoing Works

www.lbb.ulaval.ca 45
H. Hermawan, D. Dubé, D. Mantovani. Acta Biomaterialia 6 (2010) 1693–1697
Concept in cardiovascular
applications

46
Concept in musculoskeletal
applications
Mg Implant
From Frank Witte

www.lbb.gmn.ulaval.ca
1st Berlin
2009
2nd Maratea
2010
3rd Quebec
2011
May 2010
4th Maratea
2012
www.biodegradablemetals.org
Intl Symposium on
Biodegradable Metals

Génie tissulaire vasculaire

Les approches

L’approche par échaffaudage
D. Seifu, A. Purnama, K. Mequanint, D. Mantovani. Nature Cardiology. 2013, in press.

Méthodologie

Materials and Methods
1. Mechanical Stimulation System
Flexcell international
corporation

Collagen Scaffold
SEM
L. Levesque, Advanced Materials Research, 2012

SEM
Collagen + Cells Dynamic
Condition
L. Levesque, Advanced Materials Research, 2012

SMCs’ Collagen Production
56
L. Levesque, 9th World Biomaterials Congress, 2012

Co-culture statique : Qui, quoi et
quand ?
Cellules endothéliales
Cellules musculaires lisses
CRSNG-FONCER, Collaboration avec Jayachandran Kizhakkedathu, UBC

Co-culture statique : Qui, quoi et quand ?
Gélification (collagène + CML) = 30 min
Maturation gel endothélialisé = 24h
Coloration au Trichrome de Masson
Vert : collagène ; Rouge : cytoplasme ; Noir/brun ; noyau
Tapis de CE
à la surface
du gel

Co-culture dynamique
La 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.

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.

 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

Dispositif expérimental
ÉCHAFAUDAGE
Collagène sans cellules
CONSTRUCTION ARTÉRIELLE
Culture statique 1 SEMAINE
Collagène + cellules, t = 0
Culture statique 2 SEMAINES

Combinaison
500 μm
Culture statique– Microscopie de fluorescence

www.ulaval.ca
www.lbb.ulaval.caLaboratory for Biomaterials & Bioengineering
SEM
25 X

www.ulaval.ca
www.lbb.ulaval.caLaboratory for Biomaterials & Bioengineering
SEM
100 X

Moteur rotatif à
5 tours/min
Réservoir de
milieu de culture
Espaceur en silicone
pour assurer un axe de
rotation constant
Endothélialisation d’une construction
artérielle à base de collagène
Conception d’un bioréacteur à parois rotatives
Bouchon avec filtre
0.22 μm
Roulement à billes
(Ø = 4,7mm)

Measuring mechanical property
67
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

Immunohistochemistry
Red: PSMS with Calponin, Blue: Nuclei, Green: HUVECs with CD31 and actin and collagen Green with
Alex fluor green.
68

Masson Trichrome staining
69

Contrainte(Pa)
Temps (s)
106 cellules/mL
Culture statique
1 semaine
Pas de cellules
Culture statique
2 semaines
 Tests de relaxation en circonférentiel
Culture statique – Propriétés mécaniques
106 cellules/mL
Culture statique
1 semaine
ε = 0,1 ε = 0,2 ε = 0,3

Laser guided thickness measurement
72
LaserMike 136 Thickness and
external diameter measurement
of cell seeded construct.

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

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

Futur Collagène-Élastine
version 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 la
transglutaminase, afin pour obtenir une matrice.
 Capacité d'auto-assemblage et d'auto-organisation dans polymères réticulés avec des propriétés
physiques et mécaniques remarquablement similaires à l'élastine native.
Collagène-HELP
Prof. A. Bandera

Prof. Marisa Beppu
Structural layer:
mechanical resistance,
elasticity, anti-bacterial
capacity
 Konjac glucomannan
and chitosan
microstructured with
silk fibroin.
Bioactive layer :
growth factor stimulation,
re-epithelialization, drug
release
Dressings high biological performance
 Collagen or gelatin,
cells and drugs

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

“The Human Being can do all things if He will”

Remerciements

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;

Ldb Convergenze Parallele_Mantovani_03

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