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 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


Introduction


Global challenges for humans

EnergyFoodMedicalEnvironment



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


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 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.


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


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


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)


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



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


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 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


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 …

www.ulaval.ca

BIOMATERIALS

Classes:Metals (corrosion resistant...)Polymers (synthetic, natural, permanent, degradable ...)CeramicsCompositesGlasses

25

www.ulaval.ca

PHASE I:EXPLORING MAGNESIUM

ALLOYS2002-2005

(J Levesque, D Dubé, D Mantovani)

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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)

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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 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%

www.ulaval.ca

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

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

Mag

netic

Sus

cept

ibilt

y (

m3 /k

g)

Fe35MnSS316L

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-bBOTTOM-UP APPROACH

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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 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

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

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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

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 stent316L 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 cardiovascularapplications


46

Concept in musculoskeletalapplications

Mg Implant

From Frank Witte

www.lbb.gmn.ulaval.ca

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

www.lbb.gmn.ulaval.ca


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 ScaffoldSEM

L. Levesque, Advanced Materials Research, 2012


SEM

Collagen + Cells DynamicCondition

L. Levesque, Advanced Materials Research, 2012


SMCs’ Collagen Production

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L. Levesque, 9th World Biomaterials Congress, 2012


Co-culture statique : Qui, quoi et quand ?

Cellules endothélialesCellules musculaires lisses

CRSNG-FONCER, Collaboration avec Jayachandran Kizhakkedathu, UBC


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


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.


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

ÉCHAFAUDAGECollagène sans cellules

CONSTRUCTION ARTÉRIELLE Culture statique 1 SEMAINE

CONSTRUCTION ARTÉRIELLECollagène + cellules, t = 0

CONSTRUCTION ARTÉRIELLE 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 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)


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


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


Laser guided thickness measurement

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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-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


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


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”


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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

Small Business & Entrepreneurship

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