Politecnico di Torino

Department of Mechanical and Aerospace Engineering

Prof. Gianluca Ciardelli

by Brett Ryder

Three-dimensional growth of

adipose-derived stem cells on

scaffolds for cardiovascular

applications

Activities of the Industrial Bioengineering Group:

- Development and characterization of biomaterials

- Fabrication and functionalization of scaffolds

- Drug delivery system

- Bionanotechnology and Nanomedicine

Industrial Bioengineering Group @ POLITO

G. Ciardelli

Mechanical/electrical cuesGrowth factors/biomolecules

Nutrients

Tissue engineering

Eschenhagen, T. and Zimmermann, W.H., Circ Res, 2005. 97(12): p. 1220-31.Rabkin, E. and F.J. Schoen, Cardiovasc Pathol, 2002. 11(6): p. 305-17.Leor, J. et al., Pharmacol Ther, 2005. 105(2): p. 151-63. G. Ciardelli

TRADITIONAL APPROACH

Tissue Engineering/Regenerative Medicine (TERM) aims to develop a bioengineeredconstructs that can provide a physical support to a damaged tissue/organ by replacingcertain functions of the damaged extracellular matrix (ECM) and prevent adverseremodeling (fibrosis, scar formation) and dysfunction.

Tissue engineering

Leor, J. et al., Pharmacol Ther, 2005. 105(2): p. 151-63. G. Ciardelli

Non-cellularized scaffold

CELLS SCAFFOLD BIOENGINEERED TISSUE/SCAFFOLDIn vivo remodeling

CELLS

Cellularized scaffold

Cell therapy (cell injection)

+

BIOENGINEERED TISSUE/SCAFFOLDIn vivo remodeling

+

SCAFFOLD BIOENGINEERED TISSUE/SCAFFOLDIn vivo remodeling

+

OTHER APPROACHES

CRITICAL ISSUES:

- The bioartificial system starts to interact with the surrounding tissue immediately after implantation

- Need to guarantee a fast perfusion of the bioengineered construct

- Need to identify adequate and highly available cell sources

Tissue engineering

G. CiardelliVunjak Novakovic, G. et al., Cold Spring Harb Perspect Med, 2014. 4(3).

Porous scaffolds serve as a mimic of thenative ECM, provide a temporarystructural and mechanical support to therepairing tissue, induce cell recruitment,homing, adhesion, proliferation and, incase, differentiation toward to desiredphenotype.

Tissue models serve as representationof tissue development, regeneration anddisease progression and can be used inearly stage research to study theefficacy, safety, and mode of action oftherapeutic agents.

Tissue engineering

G. CiardelliMashayekhan S. et al. (2013). Stem Cells in Tissue Engineering, Pluripotent Stem Cells, Dr.Deepa Bhartiya (Ed.), ISBN: 978-953-51-1192-4, InTech.

INFARCTED AREA

CELL INJECTION in THE INFARCTED

REGION

INTRACORONARY CELL INJECTION

HEART

PATCH SUTURED on THE INFARCTED

REGION

CYTOKINES

GROWTH FACTORS

SCAFFOLDS withISOTROPIC

PROPERTIES

SCAFFOLDS withANISOTROPIC PROPERTIES

HEART BONE MARROW

BLOOD

MUSCLE

EMBRYONIC STEM CELLS

ADIPOSE TISSUE

Possible CELL EXPANSION in a BIOREACTOR

Cardiac tissue engineering

G. CiardelliA Silvestri, M. Boffito et al. Macromol Biosci , 2013, 8, 984-1019.

Cardiac Tissue Engineering/Regenerative Medicine (TERM) aims to develop a bioengineered constructs that can provide aphysical support to the damaged cardiac tissue by replacing certain functions of the damaged extracellular matrix (ECM) andprevent adverse cardiac remodeling and dysfunction after myocardial infarction.

Cardiac tissue engineering: requirements

G. Ciardelli

MECHANICAL PROPERTIES

GEOMETRYSURFACE

PROPERTIES

The scaffold should reproduce ECM properties from several points of view:

- interconnected porous structure- pore size ranging from tens to onehundred μm to induce cell penetrationand migration, vascularization anddiffusion of nutrients.

A sample from rat ventricle. Staining forf-actin (green) and nuclei (blue).

-reproduce the mechanicalproperties of the native tissue Young Modulus from tens kPato a few MPa.

- be able to support both systolicand diastolic forces generated ateach cardiac cycle. ELASTOMERIC PROPERTIES

Scaffold surface is the first elementthat interfaces with cells. itinfluences cell response and tissueregeneration.

Wettability plays a key role on celladhesion and spreading. betteradhesion and spreading on surfaceswith moderate hydrophobicity.

A Silvestri, M. Boffito et al. Macromol Biosci , 2013, 8, 984-1019.M Boffito et al. Polym Int, 2014, 63, 2-11.Jin Ho Lee et al. J Colloid Interface Sci, 1998, 205, 323-330.Davis M.E. et al. Circulation Res, 2005, 97, 8-15.

A suitable scaffold should:

Selection of scaffold-forming materials

G. Ciardelli

NATURAL POLYMERS SYNTHETIC POLYMERS

- ECM components- biocompatibility- weak inflammatory response in vivo- promotion of cell adhesion and proliferation- poor mechanical properties- rapid degradation kinetics- poor reproducibility

- good workability- biocompatibility- degradability- tunable mechanical properties

PLA, PGA, PCL and their copolymers highstiffness and poor strength to cyclicdeformation.

Polyurethanes (PURs)

NH C

O

O O C

O

NH NH C

O

O

A large family of polymers, characterizedby the presence of repeating urethanegroups along the polymeric chain.

Urethane group

Poly(glycerol sebacate) (PGS)

Poly(1,8-octanediol-co-citric acid) (POC)

Polyurethanes (PURs) as biomaterials

O C N R N C O2 + HO R' OH

OCN R NH C

O

O R' O C

O

NH R NCO

Prepolymer

HO R'' OH

R'' O C

O

NH

HN C

O

O R''

Polyurethane

Diisocyanate Macrodiol

Chain Extender (diol)

H2N R NH2

N R'' NH C

O

NH

HN C

O

NH R'' N

Chain Extender (diamine)

Polyurethaneurea

Two Steps

″

G. Ciardelli

POLYURETHANE SYNTHESIS:

Polyurethanes (PURs) as biomaterials

• Linear block copolymers

• Soft segment Elastomeric mechanical properties

• Hard segments Gives stability and favorsremodeling

Fast and easy synthesis

Great variety of possible surface and bulk modifications

Possibility of modulating the final properties

of the material, by properly selecting the

reagents:

Tunable composition

Biodegradability

Biocompatibility

Modulation of final properties

Phase separation (soft and hard phases)

Soft

Hard

- Macrodiol

- Diisocyanate

- Chain extender

G. Ciardelli

Selection of scaffold-forming PUR

G. Ciardelli

PURs with different composition were synthesized and characterized to study the influence of the chain extender intheir physicochemical properties and biological response.

POLYOL

Poly(ε-caprolactone) diol Mn = 2000 Da

HO CH2 C O

O

CH2 CH2 O C CH2

O

O H

xy

5 5

C2000

DIISOCYANATE

1,4-butandiisocyanate (BDI)

OCN CH2 NCO4

B

S. Sartori, et al. React Funct Polym 73(2013)1366-1376.

NH

CH CH2CH2 OHOH

C

O

O

CH3

CH3

CH3

NH2CH

C

CH2

OEt

O

CH2CH2CH2NH2

H2N Ala Ala NH CH2 NH24

1,4- Cyclohexane dimethanol (CDM)

L-lysine ethyl esterdihydrochloride

N-Boc SerinolCHAIN

EXTENDERS

Ala-Ala

K

C

NSHO CH2

CH2 OH

A

PU POLYOL DIISOCYANATE CHAIN EXTENDER MOLAR RATIO

K-BC2000 PCL2000 BDI L-lysine ethyl ester dihydrochloride 1:2:1

NS-BC2000 PCL2000 BDI N-Boc Serinol 1:2:1

A-BC2000 PCL2000 BDI Ala-Ala 1:2:1

C-BC2000 PCL2000 BDI CDM 1:2:1

Selection of scaffold-forming PUR

G. CiardelliS. Sartori, et al. React Funct Polym 73(2013)1366-1376.

0

50

100

150

200

K-BC2000 NS-BC2000 A-BC2000 C-BC2000

Yo

un

g M

od

ulu

s (M

Pa)

**

*

0

100

200

300

400

500

600

700

800

K-BC2000 NS-BC2000 A-BC2000 C-BC2000

Stra

in a

t B

reak

(%

)

*

**

*

0

2

4

6

8

10

12

14

K-BC2000 NS-BC2000 A-BC2000 C-BC2000

Stre

ss a

t B

reak

(M

Pa)

**

*

K-BC2000 and NS-BC2000: good candidates as scaffoldmaterials for contractile tissues mimicking or repair.

A-BC2000: unsuitable for muscle tissue, but adapt asscaffold material for other soft tissues, such as tendonsand ligaments.

Mechanical properties

Durability tests (1Hz, 15%, 5 days) on K-BC2000

No macroscopic damages or fracture.

Selection of scaffold-forming PUR

G. CiardelliS. Sartori, et al. React Funct Polym 73(2013)1366-1376.

K-BC2000 NS-BC2000 C-BC2000 A-BC2000

C% = 22.3 C% = 43.8 C% = 36.6 C% = 46.9

Crystallinity correlates with morphologies: PURs with the highest crystallinity showed highorder spherulitic morphologies; PURs with lower crystallinity showed a clear phase separationbut no spherulites and lamellar structures were detected.

A better biological response was observed for PURs having surface with less orderedstructure, as those observed in CBC2000 and KBC2000 by Atomic Force Microscopy (AFM).

Actin cytoskeleton of myoblasts C2C12 cultured for 7 days on the synthesised PURs.

Selection of scaffold-forming PUR

G. CiardelliS. Sartori, et al. React Funct Polym 73(2013)1366-1376.

0

0,5

1

1,5

2

2,5

3

Ctrl K-BC2000 NS-BC2000 A-BC2000 C-BC2000

Ab

s (4

90

nm

)

24h 3d 7d

*

*

**

**

*

**

*

Skeletal myoblast growth on polystyrene cellculture plates and the synthesized PURs. Opticalabsorbance at 490 nm is directly proportional tothe amount of viable cells.

Vinculin, actin and PCNA protein expression after 3 days of cell culture.

Vinculin and PCNA protein expressionapproximately comparable to the positive controlK-BC2000 and C-BC2000

With the exception of NS-BC2000,increase in viability from day 3 to day 7after cell seeding, indicating the abilityof cells to proliferate on thedeveloped materials.

Cell tests with myoblasts C2C12

In collaboration with the Health Sciences Department of Università del Piemonte Orientale (Novara, Italy).

Selection of scaffold-forming PUR

G. CiardelliS. Sartori, et al. React Funct Polym 73(2013)1366-1376.

The effect of different chain extenders on mechanical and biological behaviour has beendemonstrated.

Biodegradable PURs have been successfully synthesized.

The most promising polymer for the design of porous matrices mimickingmuscle tissue properties was K-BC2000, since:

it better matched the elastomeric behaviour of muscle tissues.

cell tests performed with myoblasts on this substrate showed highviability, adequate cell adhesion, spreading and proliferation.

Scaffold design

G. Ciardelli

INFARCTED AREA

CELL INJECTION in THE INFARCTED

REGION

INTRACORONARY CELL INJECTION

HEART

PATCH SUTURED on THE INFARCTED

REGION

CYTOKINES

GROWTH FACTORS

SCAFFOLDS withISOTROPIC PROPERTIES

SCAFFOLDS withANISOTROPIC PROPERTIES

HEART BONE MARROW

BLOOD

MUSCLE

EMBRYONIC STEM CELLS

ADIPOSE TISSUE

Possible CELL EXPANSION in a BIOREACTOR

- easily accessible in large quantities with minimal invasive harvesting procedure.

- cell isolation yields a high amount of stem cells.

Bioreactors are key factor for a successful application of tissue engineering principles in cardiacregenerative medicine, where state-of-the-art mechanostimulation protocols have definitely proven tocontrol cell proliferation, differentiation, and electrical coupling in engineered 3D cardiac tissue.

Scaffold design

G. CiardelliIn collaboration with Swiss Stem Cell Foundation (SSCF, Lugano, Switzerland)

Scaffold fabrication and

sterilization

ADCS seeding

STATIC cell culture for 1 week

DYNAMIC cellculture for 10 days

BIOENGINEERED SUBTRATE

ADSC

Adipose tissue

GMP-optimized procedure to extract

ADSCs from subcutaneous adipose tissue

harvested during elective liposuction

procedures.

in SERUM-FREEE cell culture medium.

in dynamic conditions in the

presence of SERUM-FREE cell culture medium enriched with a CARDIOGENIC

cocktail.

NON-INDUCED CELLS CARDIO–INDUCED CELLS

ADSCs cultured in 2D

Scaffolds produced by thermally induced phase separation

starting from a PUR solution quenched at -

20°C and surface functionalized with recombinant type I

collagen.

Scaffold fabrication and functionalization

G. Ciardelli

PUR solubilization indimethyl sulfoxide (DMSO).

The solution is poured in amold and quickly cooleddown at -20°C.

Solvent extraction in EtOH/H2O(70:30) followed by freezedrying.

FREEZE-DRYINGand

Thermally Induced Phase Separation (TIPS)

1° STEP

ARGON ACRYLIC ACID EDC/NHS PROTEINA

ARGONEDC/NHS

PROTEIN

PU PU-PAA PU-PAA-PROTEIN

2° STEP

EDC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimideNHS N-hydroxysuccinimide

Conclusions

G. Ciardelli

The selected polyurethane showed appropriate mechanical properties for the production of porous scaffoldsmeeting the strict requirements for cardiac tissue engineering application.

Porous structures mimicking cardiac tissue mechanical properties have been successfully producedthrough Thermally Induced Phase Separation (TIPS).

The designed scaffolds were successfully surface functionalized with recombinant type I collagen (ε. Coli),mimicking the composition of the native ECM.

Dynamic cell culture on a 3D support seems to be a promising tool to favour andaccelerate ADSC commitment towards the cardiac phenotype, accompanied bycell alignment in the direction of the applied mechanical stimulus, that can provideto the resulting patch an anisotropic muscle-like morphology.

Polyurethane-based biomaterials have demonstrated potential for the design of 3D patient-specific cellularizedpatches for cardiac tissue engineering/regenerative medicine applications.

Sources of Funding :-European Commission (6-7FP; ERANET)-Italian Gov. (PRIN 2011 “MIND - Engineering physiologically and pathologically relevant organ Models for theINvestigation of age related Diseases”, FIRB)-Piedmont Regional Gov. (Piattaforme Innovative (F.E:S.R. 2007-2013) “Active-Advanced CardiovascularTherapies”)-Private Companies-Foundations (Swiss Stem Cell Foundation -SSCF-)-Joint Projects of Internationalization of Research financed by Compagnia di San Paolo (Smart Injectable Drug-Delivery systems for Parkinson’s and Alzheimer’s Disease Treatment (PAD-INJ))

gianluca.ciardelli@polito.it