Vascular prothesis material Vascular prothesis material modification to enhance modification to enhance endothelial cell adhesionendothelial cell adhesion

Vascular prothesis material Vascular prothesis material modification to enhance modification to enhance endothelial cell adhesionendothelial cell adhesion

T. Markkula,T. Markkula, F. Pu, R.L. Williams, J.A. Hunt F. Pu, R.L. Williams, J.A. HuntT. Markkula,T. Markkula, F. Pu, R.L. Williams, J.A. Hunt F. Pu, R.L. Williams, J.A. Hunt

Department of Clinical EngineeringDepartment of Clinical EngineeringUniversity of LiverpoolUniversity of Liverpool

L929 fibroblasts on PET

PET surface cleaned ultrasonically for 30 min with 70 % Ethanol and for 30 min with water prior to cell culturing

Replacement of blood vessels Replacement of blood vessels with artificial implantswith artificial implants

PTFE and PET most commonly PTFE and PET most commonly usedused

>6 mm >6 mm ØØ prosthesis OK prosthesis OKSmaller grafts thrombosisSmaller grafts thrombosis

Vascular graftsVascular grafts

ImprovementsImprovements

Find a new material

Modify existing materials

Engineer new tissue

• Endothelial cell lining of inner surface of prosthesis

What we did:What we did:

Plasma treatment of polymer surface

Endothelial cells seeded on the new surface

Endothelial cellsEndothelial cells

Seeded on the surface in vitro before operation

Importance of adhesion to graft material

Endothelial cell adhesionEndothelial cell adhesion

Endothelial cell adhesionEndothelial cell adhesion

Endothelial cell adhesionEndothelial cell adhesion

ProblemsProblems

In vivo endothelial cells become detached inflammation thrombosis

Role of leucocytes in detachment process?

Endothelial cell adhesionEndothelial cell adhesion

Endothelial cell adhesionEndothelial cell adhesion

Macrophages

Endothelial cells

Material surface

ImprovementsImprovements

Modify the surface to become more ‘endothelium friendly’

Improved adhesion is not enough. Cells need to stay on surface even in vivo.

Try to change interaction of endothelial cells with inflammatory cells through surface modification

Endothelial cell adhesionEndothelial cell adhesion

Macrophages

Endothelial cells

Material surface

•Immuno-globulin superfamily

•Integrins

•Selectins

Adhesion molecules

•Endothelial cells to other cells

•Endothelial cells to Endo-thelial cells

•Endothelial cells to material surface

InteractionsInteractions

1

2

3

MaterialsMaterials

•PET poly(ethylene terephthalate)

-[CF2-CF2]n-

•PTFE poly(tetrafluoroethylene)

-[CH2-CH2-O-C- -C-O]n- O O

RF-plasma systemsRF-plasma systems1. Inductive coil

glass tube 3 W

2. Capacitor plate glass barrel 80 W

GasesGases• Ammonia - NH3

• Nitrogen - N2

• Oxygen – O2

• Argon - Ar

• Nitrous oxide - N2O

• Air

Treatment timesTreatment times

• 1 - 30 min

Surface analysisSurface analysis

• Surface chemistry - XPS, SIMS• Wettability - DCA• Surface morphology - AFM

DCA - PET receding contact anglesDCA - PET receding contact angles

0

10

20

30

40

50

60

70

80

90

Untreated Oxygen Ammonia Air NitrousOxide

Nitrogen Argon

Con

tact

an

gle

s [°

]

Advancing contact angle Receding contact angle

DCA - PTFE receding contact anglesDCA - PTFE receding contact angles

0

20

40

60

80

100

120

140

Untreated Oxygen Air NitrousOxide

Nitrogen Argon Ammonia

Con

tact

an

gle

s an

d h

yste

resi

s [°

] Advancing contact angle Receding contact angle

XPS - PET atomic compositionXPS - PET atomic composition

0.255

0.3870.410 0.408

0.336

0.416

0.360

0.004

0.080 0.021

0.007

0.005

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Untreated Nitrousoxide

Oxygen Air Nitrogen Argon Ammonia

O/C

an

d N

/C

O/C N/C

XPS - PTFE atomic compositionXPS - PTFE atomic composition2.303

1.434 1.429

1.258

0.893

0.544

1.825

0.022

0.077

0.114

0.096

0.1480.152

0 0

0.015

0

0.015

0.044

0.086

0.117

0

0.5

1

1.5

2

2.5

Untreated Oxygen Air Argon NitrousOxide

Nitrogen Ammonia

F/C

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

O/C

& N

/C

F/C O/C N/C

• Range of wettabilities and chemistries

Surface characterization resultsSurface characterization results

• Wettability does not necessarily follow the introduction rate of O and N on the surface

InteractionsInteractions

1

2

3

Material surface properties

Interaction with endothelial cells

Endothelial cells (EC) interacting

EC interacting with blood cells

Thrombosis or no

• Cellular interaction by expression of adhesion molecules (Flow cytometry, FACS) (immunohistochemistry)

Cell culture analysisCell culture analysis

• Cell numbers and morphology

In vitro cell culturing

Endothelial cell adhesionEndothelial cell adhesion

Plasma treated PTFE

Untreated PTFE

Plasma treated

PET

Untreated PET

PS cover slip

(control)

Cell culturing of endothelial cells alone

Co-culture of endothelial cells with macrophages

Endothelial cells express adhesion molecules depending on external stimuli

Endothelial cell adhesionEndothelial cell adhesion

Flow cytometry (FACS)

Mouse antihuman monoclonal antibodies conjugated with FITC, RPE and CyC were used to target CD31, CD54, CD51/61, CD106, CD62E, CD62P and CD62L.The isotope IgG1-k was used for negative control

Immunohistochemistry

ABC immunostaining protocol was used to visualise the quantified expression.

Flow cytometry (FACS)

Mouse antihuman monoclonal antibodies conjugated with FITC, RPE and CyC were used to target CD31, CD54, CD51/61, CD106, CD62E, CD62P and CD62L.The isotope IgG1-k was used for negative control

Immunohistochemistry

ABC immunostaining protocol was used to visualise the quantified expression.

0

20

40

60

80

100

120

CD 54 CD 106 CD 62EAdhesion Molecules

% P

osi

tives

Control

TNF-α

T-PETN-PET

T-PTFE

N-PTFE

P1D1

G G

G

P

P

P P

P

D

P P

P P P

0

20

40

60

80

100

120

CD 54 CD 106 CD 62EAdhesion Molecules

% P

osi

tives

Control

TNF-α

T-PETN-PET

T-PTFE

N-PTFE

P1D7

G

G

G

P P

P P P

D

P

G

P

G

Expression of adhesion molecules Expression of adhesion molecules of endothelial cells on PET and of endothelial cells on PET and PTFEPTFE

NH3-plasma treated PET Untreated PET

CD54 - ICAM, P1, D1

Immunohistochemical staining

NH3-plasma treated PTFE Untreated PTFE

CD54 - ICAM, P1, D1

Immunohistochemical staining

Cell adhesion and Cell adhesion and proliferationproliferation

0

2

4

6

8

10

0 1 7Time (days)

No.

Cel

ls x

1000

0 / s

q.cm

Control

TNF-a

T-PET

N-PET

T-PTFE

N-PTFE

Passage 1

G1 G1

G1

G1

G2

P

•Plasma treatment of PET and PTFE with ammonia appeares to be a powerful method to enhance cell attachment

•The modification of PET and PTFE slightly alter the profile of adhesion molecules expressed but not significantly

•Plasma treatment of PET and PTFE with ammonia appeares to be a powerful method to enhance cell attachment

•The modification of PET and PTFE slightly alter the profile of adhesion molecules expressed but not significantly

Cell growth conclusionsCell growth conclusions

What will be done...What will be done...

• Surface chemistry of samples will be determined using CHEMICAL DERIVATIZATION with XPS...

• The whole range of treatments will be tested with endothelial cell / macrophage co-cultures

What wasn’t presented here...What wasn’t presented here...

• Plasma treatment alters the attachment of macrophages to endothelial cells...

• Macrophage numbers, attachment site and endothelial cell adhesion molecule expressions are altered