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

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.

The overall goal of this procedure is to provide a wet chemistry-based surface treatment for the floral carbon polymer polytetrafluoroethylene, or PTFE. This method can help answer key questions in the biomaterials field as how to improve the bio-compatibility of medical polymers. The main advantage of this technique is that only minimum of equipment is required, and that the method is more generally applicable compared to other methods.

The preparation of the sodium naphthalenide solution needs strictly anhyrdrous conditions. Cut 0.25 grams of sodium metal into small pieces to enhance dissolution. Add the sodium metal to a solution of 1.4 grams of naphtheline in 20 milliliters of tetrahydrofuran in a screw-capped 100 milliliter glass bottle, equipped with a PTFE-coated magnetic stirring bar.

Modestly heat the solution to further enhance dissolution. The final solution has a dark, slightly greenish color, and may be stored under strictly dry conditions. Punch out PTFE disks of 12 millimeter diameter from 0.5 millimeter thick foil material.

Mark one side, and clean is iso-propanol. Incubate PTFE samples individually in the activating solution for one to two minutes, using forceps. The color change from white to dark brown indicates successful treatment.

Subsequently rinse twice with THF, and then with iso-propanol. Oxidize the treated samples in 30%hydrogen peroxide, containing 20%trichloroacedic acid for three hours. Wash with water, and dry.

The surface now has a slight brownish appearance. Treat the oxidized disks with 50%HMDI in dry THF for two hours. Then, rinse with THF and leave to dry.

Finally, hydrolize the iso-cyanate bearing samples in water for two to three hours, and dry. Place each aminated disk with the marked side up into the individual wells of a 24 well plate. Add 1.5 milliliters of the epoxide solution, ensuring full coverage of the samples.

After incubating for two hours, wash two times with water, and once with carbonate buffer. To the bottoms of individual wells of a fresh 24 well plate, add 50 microliters of 0.5 milligram per milliliter peptide, and 50 millimolar carbonate buffer, pH nine, containing 0.1%sodium azide. Carefully place the epoxy-functionalized disks upside down onto the drops of the peptide solution.

Make sure that the space between the bottom of a well, and the PTFE disk is completely wetted due to capillary action. Incubate the plate for at least three hours, or overnight in a wet chamber with a humidified atmosphere, achieved by placing wet tissue paper on the bottom of the chamber. Following incubation, wash three times with water, and sterilize in 50%iso-propanol water for at least 30 minutes.

Prior to cell seeding, as described in the text protocol, rinse the samples in sterile phosphate buffered saline. The results of the crucial chemical reaction steps were monitored by infrared spectroscopy. The initial activation with sodium naphthalamide generates double bonds, and to a minor extent, hydroxyl functionalities.

The signal indicating carbon double bonds disappears upon oxidation, yielding a surface bearing almost exclusively hydroxyl groups. The color changes due to activation and oxidation are in agreement with the expected chemistry. Conjugated double bonding systems are expected to be brownish, and loss of the double bonding systems results in brightening.

In addition, the possible outcome of activation and oxidation on the surface morphology was investigated by means of scanning electron microscopy. Virtually no detrimental effect of the treatment was observed. Immobilization of the endothelial cell adhesive peptide, arginine, glutamine, aspartic acid valine, onto the polymer surface, supports endothelial cell growth.

Whereas virtually no cell adhesion and proliferation occurs on untreated material, the modification strongly supports colonization over a two week period. Exemplified for a clinical application, the modification was identically performed on original material from a commercially available graft made of expanded PTFE with similar results over a period of one week. Once mastered, this technique takes about 12 hours.

It should be kept in mind that this procedure is specific for PTFE. After watching this video, you should have a good understanding how to surface activate PTFE, and how to immobilize peptides in subsequent steps. Following this procedure, other molecules, such as polysaccharides or cord factors can be immobilized in a similar fashion.

Keep in mind that working with highly toxic and corrosive chemicals is dangerous, and that standard precautions must be taken.