Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Summary

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Abstract

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.

This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Introduction

Various polymers used in medicine that have been approved for some time do not exhibit enhanced biocompatibility, i.e., lack of cell-adhesiveness, induction of fibrotic encapsulation and thrombogenicity, to mention a few. Interactions between the biomaterial and the biological system takes place mainly at the surface of the implant. As a consequence, research has focused on surface modification in order to create appropriate properties for a desired application while leaving the bulk properties of the material unaffected. Polytetrafluoroethylene (PTFE) as a physiologically inert polymer is used in many medical fields such as hernia surgical mesh ¹,....

Protocol

1. Preparation of Sodium Naphthalenide Activating Solution and Surface Activation

Note: Carry out reactions in a well-ventilated fume hood. Follow general rules for handling highly flammable solvents and corrosive metals like metallic sodium. Naphthalene has a very unpleasant smell (mothball), even in very small amounts! If not indicated otherwise reactions are performed at room temperature. Sodium azide is highly toxic! THF (99.9%, see List of Materials) was stored over approximately 20% (by volume) molecular sieve. Distill THF with a noticeable water content over sodium. The formation of sodium naphthalenide does not occur if trace amounts ....

Results

The results of the crucial chemical reaction steps were monitored by IR spectroscopy (Figure 1). The initial activation with sodium naphthalenide generates double bonds — and to a minor extent — OH-functionalities. The signal indicating C=C bonds disappear upon oxidation, yielding a surface bearing almost exclusively hydroxyl-groups. Analysis of further standard conjugation steps are not shown here. The color changes due to activation and oxidation are in agreement with t.......

Discussion

The detailed description of surface modification protocol of PTFE consists of successive steps starting with the elimination of fluorine from the polymer backbone as depicted in Figure 6. As a result, a layer is formed that contains an abundant amount of conjugated carbon-carbon double bonds in accordance with the dark brownish color that developed upon naphthalenide treatment. Standard oxidation with acidic hydrogen peroxide yields a hydroxylated surface accompanied by brightening to a pale brown, this .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PTFE foil 0.5 mm	Cadillac Plastic	n/a
REDV peptide	Genecust	n/a	custom synthesis >95% purity
iso-propanol	Sigma Aldrich	34965
tetrahydrofurane (THF)	Sigma Aldrich	401757
dimethylsulfoxide	Sigma Aldrich	D8418
molecular sieve 3 Å	Sigma Aldrich	208574
sodium metal	Sigma Aldrich	483745
phosphate buffered saline (PBS)	Sigma Aldrich	D8537
naphthalene	Sigma Aldrich	147141
hydrogen peroxide 30%	Sigma Aldrich	95321
trichloroacetic acid	Sigma Aldrich	T6399
diethylene glycol diglycidyl ether	Sigma Aldrich	17741
hexamethylene diisocyanate (HMDI)	Sigma Aldrich	52650
Calcein-AM	Sigma Aldrich	56496
sodium bicarbonate	Sigma Aldrich	S6014
sodium azide	Sigma Aldrich	71290
24 well plates	Greiner-Bio-One	662 160
ATR-FTIR spectrophotometer Nicolet Magna-IR 850	Nicolet	n/a
fluorescence microscope Olympus X-70	Olympus	n/a
humbilical vein endothelial cells (HUVECs)	Lonza	n/a
ePTFE vascular graft	Gore	n/a