Name: wet chemistry and peptide immobilization on polytetrafluoroethylene for improved cell-adhesion
Uploaded: 2016-08-15T14:00:00
Description: Watch this Scientific Journal Video about Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion at JoVE.com

The authors would like to acknowledge the help of Walter Scholdei (Max-Planck-Institute for Polymer Research, Mainz, Germany.

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 vo...

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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 ...

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, medical ports 2 and, most importantly, vascular grafts 3.
Especially in blood contacting situations the hydrophobic nature of PTFE causes unspecific adsorption of plasma components and as a consequence platelet adhesion, often resulting in thrombotic events and occlusion of the graft 4. Furthermore, PTFE, like most polymers, does not support cellular adhesion and coverage which would be a desirable feature to induce the formation of a beneficial layer of endothelial cells (ECs) on the inner (luminal) surface of the vascular graft 5. A biomimetic endothelium is expected to fulfil many of the functions of its natural equivalent, notably its antithrombogenic properties 6. A general biomimetic modification strategy is based upon the concept of exclusively endowing the material with cell-adhesiveness while leaving the materials bulk properties unaffected. In addition, platelet adhesion may be reduced by incorporating anti-adhesive (anti-fouling) attributes 7. Various peptides &#8212; mostly derived from proteins of the extracellular matrix &#8212; have been described that strongly enhance cell-adhesion by binding to cellular receptors, belonging to the class of integrins 8. The best-known example in this regard is the peptide Arg-Gly-Asp (RGD) that interacts with most cell types. Other amino acid sequences are recognized by integrins exclusively expressed on specific cells. For example, Arg-Glu-Asp-Val (REDV) and Tyr-Ile-Gly-Ser-Arg (YIGSR) have been found to bind to ECs in a specific manner 9. Covalent immobilization of such peptides has been carried out on a plethora of inherently non-adhesive materials including metals and polymers 10,11.
Porous PTFE, more precisely expanded PTFE (ePTFE) &#8212; along with polyethylene terephthalate (PET) - is the most important material for the production of vascular grafts 12. Established physical techniques for appropriate treatments, such as plasma modification 13 or by photochemical methods 14, are hampered by the fact that porous and/or tubular structures are not readily treatable inside the pores or the lumen respectively. Wet chemistry on PTFE is a difficult task because of the highly inert nature of the fluorin-containing polymer that resists most chemical attacks 15.
In this paper we describe a comparatively facile method for a covalent modification strategy. Adapted from a procedure to render PTFE bondable, functional groups were created on the materials surface that serve as anchor points for further conjugation of biologically active molecules.

<table border="0" cellpadding="0" cellspacing="0" width="700">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">PTFE foil 0.5 mm</td>
			<td style="width:119px;">Cadillac Plastic&#160;</td>
			<td style="width:145px;">n/a</td>
			<td style="width:115px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">REDV peptide</td>
			<td>Genecust</td>
			<td>n/a</td>
			<td>custom synthesis &#62;95 % purity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iso-propanol</td>
			<td>Sigma Aldrich</td>
			<td>34965</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tetrahydrofurane (THF)</td>
			<td>Sigma Aldrich</td>
			<td>401757</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dimethylsulfoxide</td>
			<td>Sigma Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">molecular sieve 3&#197;</td>
			<td>Sigma Aldrich</td>
			<td>208574</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium metal</td>
			<td>Sigma Aldrich</td>
			<td>483745</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">phosphate buffered saline (PBS)</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">naphthalene</td>
			<td>Sigma Aldrich</td>
			<td>147141</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hydrogen peroxide 30 %</td>
			<td>Sigma Aldrich</td>
			<td>95321</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trichloroacetic acid</td>
			<td>Sigma Aldrich</td>
			<td>T6399&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">diethylene glycol diglycidyl ether</td>
			<td>Sigma Aldrich</td>
			<td>17741</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hexamethylene diisocyanate (HMDI)</td>
			<td>Sigma Aldrich</td>
			<td>52650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcein-AM</td>
			<td>Sigma Aldrich</td>
			<td>56496</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S6014&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium azide</td>
			<td>Sigma Aldrich</td>
			<td>71290</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24 well plates</td>
			<td>Greiner-Bio-One</td>
			<td>662 160</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ATR-FTIR spectrophotometer Nicolet Magna-IR 850</td>
			<td>&#160;Nicolet</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fluorescence microscope Olympus X-70</td>
			<td>Olympus</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">humbilical vein endothelial cells (HUVECs)</td>
			<td>Lonza</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ePTFE vascular graft</td>
			<td>Gore</td>
			<td>n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

wet chemistry and peptide immobilization on polytetrafluoroethylene for improved cell-adhesion

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.

The results of the crucial chemical reaction steps were monitored by IR spectroscopy (Figure 1). The initial activation with sodium naphthalenide generates double bonds &#8212; and to a minor extent &#8212; 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 agre...

Watch this Scientific Journal Video about Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion at JoVE.com

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.

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

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.

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