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 volume) molecular sieve. Distill THF with a noticeable water content over sodium. The formation of sodium naphthalenide does not occur if trace amounts of water are present.
<ol>
	<li>To a solution of 1.4 g (10.9 mmol) of naphthalene in 20 ml tetrahydrofuran (THF, dried over 3 &#197; molecular sieve), add 0.25 g (10.9 mmol) sodium metal in a screw-capped 100 ml glass bottle equipped with a PTFE-coated magnetic stirring bar. 
	Note: Dissolution can be greatly enhanced by cutting the sodium into small pieces and modest heating (35 - 40 &#176;C). The final solution has a dark, slightly greenish color and may be stored under strictly dry conditions.</li>
	<li>Punch out PTFE discs of 12 mm diameter from 0.5 mm thick foil material. Mark one side (e.g., using a letter stamp punch) and clean with iso-propanol.</li>
	<li>Incubate PTFE-samples individually in the activating solution (step 1.1) for 1 - 2 min using forceps. Note: The color change from white to dark brown indicates successful treatment.</li>
	<li>Subsequently rinse twice with THF and then with iso-propanol. 
	Note: The naphthalenide solution is exhausted when its color changes to a watery light brown. Unused naphthalenide solution (possibly containing metallic sodium) has to be decomposed by slowly adding iso-propanol prior to disposal.</li>
	<li>Oxidize treated samples in hydrogen peroxide (30%) containing 20% (w/v) trichloroacetic acid for 3 hr. Wash with water and dry. The surface now has a slight brownish appearance. Note: Activation and oxidation result in a drastically increased wettability of the surface. This finding was examined in detail previously 14.</li>
	<li>Treat oxidized discs with 50% (v/v) hexamethylene diisocyanate (HMDI) in dry THF for 2 hr, rinse with THF and leave to dry.</li>
	<li>Hydrolyze isocyanate bearing samples in water for 2 - 3 hr and dry. Note: The final amino-functionalized PTFE surface is far more stable than the isocyanate-bearing samples and is compatible with many standard crosslinkers for further coupling.</li>
</ol>
2. Peptide Immobilization
<ol>
	<li>Prepare a solution of 20% (v/v) diethyleneglycol diglycidyl ether (diepoxide) in 50 mM carbonate buffer, pH 9.</li>
	<li>Place the aminated discs with the marked side up individually into a 24 well plate and add 1.5 ml of the epoxide solution. Ensure full coverage of the samples. Incubate for 2 hr and wash two times with water and once with carbonate buffer.</li>
	<li>Add 50 &#181;l of a 0.5 mg/ml peptide (e.g., REDV) in 50 mM carbonate buffer, pH 9 containing 0.01% NaN3, to the bottom of individual wells of a fresh 24 well plate and carefully place the epoxy-functionalized discs upside-down (i.e., marked side down) onto the drop of the peptide solution. Make sure that the space between the bottom of the well and the PTFE disc is completely wetted due to capillary action.</li>
	<li>Incubate in a wet chamber (e.g., any sealable plastic box with a tight closing lid) with humidified atmosphere (by placing wet tissue paper on the bottom) for at least 3 hr or overnight. 
	Note: Albeit the REDV motif may be regarded as well-characterized, a scrambled or reverse amino acid sequence may be included as an additional negative control.</li>
	<li>Wash three times with water and sterilize in 50% iso-propanol/water for at least 30 min. Prior to cell seeding, rinse the samples in sterile phosphate buffered saline (PBS).</li>
</ol>
3. Cell Seeding
<ol>
	<li>Grow HUVECs using standard cell culture procedures 17,18.</li>
	<li>Place the peptide-conjugated samples with the modified side up in a 24 well plates. Use untreated PTFE discs as a control.</li>
	<li>Seed 5 x 104 HUVECs in 2 ml medium per well and incubate for 4 hr at 37 &#176;C and 5% CO2 in an incubator.</li>
	<li>Remove the discs, rinse carefully to remove unattached cells and transfer to a fresh 24 well plate. Add 2 ml of fresh medium and incubate for the desired period (e.g., 24 hr, 1 w, etc.). Change medium every two days.</li>
	<li>Prepare a stock solution of 1 mg/ml Calcein-AM in dimethyl sulfoxide (DMSO).</li>
	<li>After growing the cells remove the medium, wash with PBS and add PBS containing 1 &#181;l/ml Calcein-AM stain (e.g., 1 &#181;l of a stock solution per ml PBS). Gently agitate and incubate at 37 &#176;C for 45 min in the dark.</li>
	<li>Wash with PBS and immediately take micrographs on a fluorescence microscope equipped with standard FITC filters (Ex 488 nm, Em 515 nm). Use 100-fold magnification. Perform triplicates from unmodified as well as treated samples.</li>
	<li>Using ImageJ software determine the area colonized by the cells. Alternatively count the cells manually or use ImageJ. Both methods will give similar information about the attachment and growth of cells on the respective surfaces.</li>
</ol>

<ol>
	<li>Doctor, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+various+prosthetic+materials+and+newer+meshes+for+hernia+repairs.">Evaluation of various prosthetic materials and newer meshes for hernia repairs.</a> J. Minim. Access Surg. 2, 110-116 (2006).</li><li>Zaghal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+totally+implantable+venous+access+devices.">Update on totally implantable venous access devices.</a> Surg. Oncol. 21, 207-215 (2012).</li><li>Niu, G., Sapoznik, E., Soker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+blood+vessels.">Bioengineered blood vessels.</a> Exp. Opin. Biol. Th. 14, 403-410 (2014).</li><li>Wang, M. -. J., Tsai, W. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomaterials in Blood-Contacting Devices: Complications and Solutions. , (2010).</li><li>de Mel, A., Jell, G., Stevens, M. M., Seifalian, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofunctionalization+of+biomaterials+for+accelerated+in+situ+endothelialization:+a+review.">Biofunctionalization of biomaterials for accelerated in situ endothelialization: a review.</a> Biomacromolecules. 9, 2969-2979 (2008).</li><li>Zdrahala, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+caliber+vascular+grafts.+Part+I:+state+of+the+art.">Small caliber vascular grafts. Part I: state of the art.</a> J. Biomat. Appl. 10, 309-329 (1996).</li><li>Cleary, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+tissue+engineering:+the+next+generation.">Vascular tissue engineering: the next generation.</a> Trends Mol. Med. 18, 394-404 (2012).</li><li>Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGD+and+other+recognition+sequences+for+integrins.">RGD and other recognition sequences for integrins.</a> Annu. Rev. Dev. Bi. 12, 697-715 (1996).</li><li>Lei, Y., Remy, M., Labrugere, C., Durrieu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+immobilization+on+polyethylene+terephthalate+surfaces+to+study+specific+endothelial+cell+adhesion,+spreading+and+migration.">Peptide immobilization on polyethylene terephthalate surfaces to study specific endothelial cell adhesion, spreading and migration.</a> J. Mat. Sci. Mater. M. 23, 2761-2772 (2012).</li><li>Gabriel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalent+RGD+Modification+of+the+Inner+Pore+Surface+of+Polycaprolactone+Scaffolds.">Covalent RGD Modification of the Inner Pore Surface of Polycaprolactone Scaffolds.</a> J. Biomat. Sci.. Polym. E. 23, 941-953 (2012).</li><li>Ceylan, H., Tekinay, A. B., Guler, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+adhesion+and+growth+of+vascular+endothelial+cells+on+bioactive+peptide+nanofiber+functionalized+stainless+steel+surface.">Selective adhesion and growth of vascular endothelial cells on bioactive peptide nanofiber functionalized stainless steel surface.</a> Biomaterials. 32, 8797-8805 (2011).</li><li>Chlupac, J., Filova, E., Bacakova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessel+replacement:+50+years+of+development+and+tissue+engineering+paradigms+in+vascular+surgery.">Blood vessel replacement: 50 years of development and tissue engineering paradigms in vascular surgery.</a> Physiol. Res. / Academia Scientiarum Bohemoslovaca. 58, 119-139 (2009).</li><li>Wise, S. G., Waterhouse, A., Kondyurin, A., Bilek, M. M., Weiss, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma-based+biofunctionalization+of+vascular+implants.">Plasma-based biofunctionalization of vascular implants.</a> Nanomedicine UK. 7, 1907-1916 (2012).</li><li>Mikulikova, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+microarrays+on+photochemically+modified+polytetrafluoroethylene.">Cell microarrays on photochemically modified polytetrafluoroethylene.</a> Biomaterials. 26, 5572-5580 (2005).</li><li>Gabriel, M., Dahm, M., Vahl, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wet-chemical+approach+for+the+cell-adhesive+modification+of+polytetrafluoroethylene.">Wet-chemical approach for the cell-adhesive modification of polytetrafluoroethylene.</a> Biomed. Mater. 6, 035007 (2011).</li><li>Gabriel, M., van Nieuw Amerongen, G. P., Van Hinsbergh, V. W., Amerongen, A. V., Zentner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+grafting+of+RGD-motif-containing+peptide+on+the+surface+of+polycaprolactone+films.">Direct grafting of RGD-motif-containing peptide on the surface of polycaprolactone films.</a> J. Biomat. Sci.. Polym. E. 17, 567-577 (2006).</li><li>Larsen, C. C., Kligman, F., Kottke-Marchant, K., Marchant, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+RGD+fluorosurfactant+polymer+modification+of+ePTFE+on+endothelial+cell+adhesion,+growth,+and+function.">The effect of RGD fluorosurfactant polymer modification of ePTFE on endothelial cell adhesion, growth, and function.</a> Biomaterials. 27, 4846-4855 (2006).</li><li>Gabriel, M., Nazmi, K., Veerman, E. C., Nieuw Amerongen, A. V., Zentner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+LL-37-grafted+titanium+surfaces+with+bactericidal+activity.">Preparation of LL-37-grafted titanium surfaces with bactericidal activity.</a> Bioconjugate Chem. 17, 548-550 (2006).</li><li>Lotz, A., Heller, M., Brieger, J., Gabriel, M., F&#246;rch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivatization+of+Plasma+Polymerized+Thin+Films+and+Attachment+of+Biomolecules+to+Influence+HUVEC-Cell+Adhesion.">Derivatization of Plasma Polymerized Thin Films and Attachment of Biomolecules to Influence HUVEC-Cell Adhesion.</a> Plasma Process Polym. 9, 10-16 (2012).</li></ol>

The authors have nothing to disclose.

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 reflects the loss of double bonds. This process is clearly evidenced by IR spectroscopy.
In order to achieve a more generally applicable functionality, the pendant OH-groups were converted into amino-functionalities by simple treatment with a diisocyanate and subsequent hydrolysis of the NCO-moieties to primary amines. This is favorable, because amine-bearing material can be stored for prolonged periods whereas the highly reactive isocyanate is susceptible to the very presence of air humidity.
Care must be taken in the avoidance of even trace amounts of water in the initial activation step and the HMDI treatment because both sodium naphthalenide and the isocyanate are highly susceptible to hydrolysis. In contrast to standard procedures in which the sample is completely immersed in the solution, the present protocol necessitates only small amounts of the reagent. Therefore this method saves a lot of the costly peptide.
Amine-bearing surfaces are ideal starting points for the coupling of many biomolecules. The diepoxide employed in this work is interesting in several regards: it is comparatively cheap, hydrophilic, highly reactive towards amines and non-reacted epoxides are hydrolyzed in an aqueous environment into &#34;physiologically inert&#34; diols. Homobifunctional crosslinkers are suitable reagents where only one reactive group is present on the peptide to be immobilized. This applies for REDV containing only one single primary amino-group on the N-terminal (this is the same for the frequently used sequence RGDS). For peptides containing lysine residues (with side chain amines) the outcome of the conjugation is ambiguous, meaning that proper coupling does not occur in a defined way. In this case, a strategy using heterobifunctional crosslinkers (e.g., amine- and thiol-reactive) and cysteine (N- or C-terminal) containing peptides is helpful 18.
The results of the peptide immobilization in terms of cell adhesion is unambiguous (Figures 3 and 4). Whereas virtually no cells adhere on bare PTFE, REDV-modification renders the material an excellent cell-adhesive substrate. Complementary to the above mentioned protocol preliminary work show that this technique is also applicable on porous (expanded) PTFE with similar results (Figure 5). This is of special interest because this is the material vascular grafts are made of. In contrast to surface techniques that mostly make use of plasma treatment (plasma activation 13, plasma polymerization 19), this protocol provides a guideline for the covalent modification of PTFE by employing readily available chemicals and lab procedures and by avoiding highly sophisticated equipment. Plasma methodologies such as reactive plasma (O2, NH3, etc.) and plasma polymerization are not applicable within porous constructs or tubing because reactive species (e.g., radicals) are not able to penetrate these structures due to inactivation upon contact with the materials surface. For example small caliber vascular grafts (diameter &#60; 5 mm) cannot be treated in this way. In contrast to this, by means of wet chemistry, there is no restriction regarding the shape of the device. In this way, by enabling the formation of an endothelial layer on the luminal side of an ePTFE vascular graft, thrombogenicity and long term patency may be improved. In addition, a cell adhesive surface for surgical hernia meshes is supposed to result in better tissue integration.

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 agreement with the expected chemistry that is used: conjugated double bonding systems are expected to be brownish and its loss results in brightening (Figure 2). 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 (Figure 2).
Figures 3 and 4 show the outcome of REDV-immobilization on 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 (i.e., vascular grafts), the modification was identically performed on original material from a commercially available graft made of expanded PTFE with a similar results over a period of one week (Figure 5).
<img alt="Figure 1" src="/files/ftp_upload/54272/54272fig1.jpg" /> 
Figure 1. IR spectroscopy of PTFE. Treatment of pristine PTFE (A) results in the formation of double bonds and to a certain extent of hydroxy functions (B). Subsequently C=C bonds are reduced due to the oxidation (C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54272/54272fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54272/54272fig2.jpg" /> 
Figure 2. Optical appearance of bare and surface activated PTFE. (A) Whereas untreated PTFE (left) appears white, the activation using sodium naphthalenide yields a dark brownish color (middle) which is slightly brightened upon oxidation (right). Untreated (B) and oxidized PTFE (C) samples were additionally investigated using scanning electron microscopy (magnification: 2,000X). Discs are 12 mm in diameter. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54272/54272fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54272/54272fig3.jpg" /> 
Figure 3. Endothelial cell culture on pristine and peptide-modified PTFE. Untreated samples are not colonized by ECs (A, C, E after 24 hr, 1 w and 2 w respectively) whereas adhesion and growth on peptide-modified material is greatly enhanced (B, D and F after 24 hr, 1 w and 2 w respectively). Scale bar: 100 &#181;m, magnification 100X. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54272/54272fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54272/54272fig4.jpg" /> 
Figure 4. Quantification of cell coverage by ImageJ analysis. Cellular growth on bare PTFE (A, C, E) and on REDV-conjugated polymer surfaces (B, D, F) expressed as percentage coverage of total area. Immobilized peptide clearly enables initial adhesion (B) and supports colonization over a 2-week period (D, F). Triplicate determinations, mean &#177; standard deviation). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54272/54272fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54272/54272fig5.jpg" /> 
Figure 5. Endothelial cells grown for 1 week on expanded PTFE. The structure of ePTFE is shown using Scanning Electron Microscopy (A). The results obtained on porous material are in accordance with those obtained for flat PTFE specimen. In contrast to the few cells found on bare material (B) the modified surface (C) provides an excellent substrate for cell growth. Scale bars are 100 &#181;m for A, B and C respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54272/54272fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54272/54272fig6.jpg" /> 
Figure 6. Schematic representation of the chemical modification of PTFE using wet-chemistry. Double bonds generated by Na-naphthalenide treatment are oxidized resulting in OH-functionalities. A hydroxyl-reactive diisocyanate is then immobilized and hydrolyzed to an amine. Finally the bifunctional diepoxide is applied to conjugate the REDV peptide using the N-terminal amino group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54272/54272fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

wet-chemistry-peptide-immobilization-on-polytetrafluoroethylene-for

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.6K Views.  Sidra Cardiovascular Research. 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.

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

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the wet spinning method, gelatin fibers are produced by smooth extrusion in a suitable coagulation medium. To increase the functional surface of these gelatin fibers and their ability to mimic the features of tissues, gelatin can be molded into a tube form by referring to this concept. Examined by in vitro and in vivo tests, the gelatin tubes demonstrate a great potential for application in tissue engineering. Acting as a suitable filling gap material, gelatin tubes can be used to substitute the tissue in the damaged area (e.g., in the nervous or cardiovascular system), as well as to promote regeneration by providing a direct replacement of stem cells and neural circuitry. This protocol provides a detailed procedure for creating a biomaterial based on a natural polymer, and its implementation is expected to greatly benefit the development of correlative natural polymers, which help to realize tissue regeneration strategies.

We developed and describe a protocol based on the wet spinning concept, for the construction of gelatin-based biomaterials used for the application of tissue engineering.

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...