Transcript No Slide Title

Vascular prothesis material
modification to enhance endothelial
cell adhesion
T. Markkula, F. Pu, R.L. Williams, J.A. Hunt
Department of Clinical Engineering
University 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
Vascular grafts
Replacement of blood vessels with
artificial implants
PTFE and PET most commonly used
>6 mm Ø prosthesis OK
Smaller grafts
thrombosis
Improvements
 Find a new material
 Modify existing materials
 Engineer new tissue
• Endothelial cell lining of inner
surface of prosthesis
What we did:
 Plasma treatment of polymer
surface
 Endothelial cells seeded on the
new surface
Endothelial cells
 Seeded on the surface in vitro
before operation
 Importance of adhesion to graft
material
Endothelial cell adhesion
Endothelial cell adhesion
Endothelial cell adhesion
Problems
 In vivo endothelial cells become
detached
inflammation
thrombosis
 Role of leucocytes in detachment
process?
Endothelial cell adhesion
Endothelial cell adhesion
Macrophages
Endothelial cells
Material surface
Improvements
 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 adhesion
Macrophages
Endothelial cells
Material surface
• Immunoglobulin
superfamily
• Integrins
• Selectins
Adhesion molecules
• Endothelial
cells to other
cells
3
• Endothelial
cells to Endothelial cells
• Endothelial
cells to
material
surface
2
1
Interactions
Materials
• PET poly(ethylene terephthalate)
-[CH2-CH2-O-CO
-C-O]nO
• PTFE poly(tetrafluoroethylene)
-[CF2-CF2]n-
RF-plasma systems
1. Inductive coil glass
tube 3 W
2. Capacitor plate glass
barrel 80 W
Treatment times
• 1 - 30 min
Gases
• Ammonia - NH3
• Nitrogen - N2
• Oxygen – O2
• Argon - Ar
• Nitrous oxide - N2O
• Air
Surface analysis
• Surface chemistry - XPS, SIMS
• Wettability - DCA
• Surface morphology - AFM
90
DCA - PET receding contact angles
Advancing contact angle
80
Receding contact angle
Contact angles [°]
70
60
50
40
30
20
10
0
Untreated
Oxygen
Ammonia
Air
Nitrous
Oxide
Nitrogen
Argon
DCA - PTFE receding contact angles
140
Contact angles and hysteresis [°]
Advancing contact angle
Receding contact angle
120
100
80
60
40
20
0
Untreated
Oxygen
Air
Nitrous
Oxide
Nitrogen
Argon
Ammonia
XPS - PET atomic composition
0.45
0.40
O/C
N/C
0.007
0.080
0.35
O/C and N/C
0.005
0.004
0.021
0.30
0.25
0.20
0.15
0.387
0.410
0.416
0.408
0.360
0.336
0.255
0.10
0.05
0.00
Untreated
Nitrous
oxide
Oxygen
Air
Nitrogen
Argon
Ammonia
2.303
F/C
2
0.117
O/C
1.825
1.5
N/C
0.16
0.14
0.12
0.114
1.434
0.096
1.429
0.10
0.086
1.258
0.077
F/C
0.152
0.148
1
0.08
0.893
0.06
0.044
0.544
0.5
0
0.022
0.015
0
0
Untreated Oxygen
0.02
0.015
0
Air
Argon
0.04
0.00
Nitrous
Oxide
Nitrogen Ammonia
O/C & N/C
2.5
XPS - PTFE atomic composition
Surface characterization results
• Range of wettabilities and chemistries
• Wettability does not necessarily follow the
introduction rate of O and N on the surface
Interactions
3
2
1
Material surface properties
Interaction with endothelial cells
Endothelial cells (EC) interacting
EC interacting with blood cells
Thrombosis or no
Cell culture analysis
• Cellular interaction by expression of adhesion
molecules (Flow cytometry, FACS)
(immunohistochemistry)
• Cell numbers and morphology
Endothelial cell adhesion
In vitro cell culturing
Plasma
treated
PTFE
Untreated
PTFE
Plasma
treated
PET
Untreated
PET
PS cover
slip
(control)
Endothelial cell adhesion
 Cell culturing of endothelial cells
alone
 Co-culture of endothelial cells
with macrophages
 Endothelial cells express adhesion
molecules depending on external
stimuli
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.
Expression of adhesion molecules of
endothelial cells on PET and PTFE
120
120
P1D7
% Positives
100
Control
TNF-α
T-PET
N-PET
T-PTFE
N-PTFE
G
80
60
P
P
P
P
40
P
G
P
P
P
P
100
D
G
80
P
P
60
P
P
P
P
40
P
Control
TNF-α
T-PET
N-PET
T-PTFE
N-PTFE
G
G
% Positives
P1D1
D
G
P
G
20
20
0
0
CD 54
CD 106
Adhesion Molecules
CD 62E
CD 54
CD 106
Adhesion Molecules
CD 62E
G
Immunohistochemical staining
NH3-plasma treated PET
CD54 - ICAM, P1, D1
Untreated PET
Immunohistochemical staining
NH3-plasma treated PTFE
CD54 - ICAM, P1, D1
Untreated PTFE
Cell adhesion and proliferation
10
No. Cells x10000 / sq.cm
Control
8
Passage 1
G2
TNF-a
T-PET
N-PET
6
T-PTFE
N-PTFE
4
P
G1
2
G1
G1
G1
0
0
1
Time (days)
7
Cell growth conclusions
• 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
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...
• Plasma treatment alters the attachment
of macrophages to endothelial cells...
• Macrophage numbers, attachment site
and endothelial cell adhesion molecule
expressions are altered