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

Replacement of blood vessels with artificial implants PTFE and PET most commonly used >6 mm Ø prosthesis OK Smaller grafts thrombosis Vascular grafts

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

Problems  In vivo endothelial cells become detached inflammation thrombosis  Role of leucocytes in detachment process?

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

Immuno- globulin superfamily Integrins Selectins Adhesion molecules

Endothelial cells to other cells Endothelial cells to Endo- thelial cells Endothelial cells to material surface Interactions 1 2 3

Materials PET poly(ethylene terephthalate) -[CF 2 -CF 2 ] n - PTFE poly(tetrafluoroethylene) -[CH 2- CH 2 -O-C- -C-O] n - OO

RF-plasma systems 1.Inductive coil glass tube 3 W 2.Capacitor plate glass barrel 80 W Gases Ammonia - NH 3 Nitrogen - N 2 Oxygen – O 2 Argon - Ar Nitrous oxide - N 2 O Air Treatment times min

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

DCA - PET receding contact angles

DCA - PTFE receding contact angles

XPS - PET atomic composition

XPS - PTFE atomic composition

Range of wettabilities and chemistries Surface characterization results Wettability does not necessarily follow the introduction rate of O and N on the surface

Interactions 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 analysis Cell numbers and morphology

In vitro cell culturing Endothelial 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 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.

Expression of adhesion molecules of endothelial cells on PET and PTFE

NH 3 -plasma treated PET Untreated PET CD54 - ICAM, P1, D1 Immunohistochemical staining

NH 3 -plasma treated PTFEUntreated PTFE CD54 - ICAM, P1, D1 Immunohistochemical staining

Cell adhesion and proliferation

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 conclusions

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