vascular prothesis material modification to enhance endothelial cell adhesion t. markkula, f. pu,...
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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