Name: mitigation of blood borne cell attachment to metal implants through cd47-derived peptide immobilization
Uploaded: 2020-12-03T14:30:00
Description: Watch this Scientific Journal Video about Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization at JoVE.com

Protocol development and studies presented in this paper were supported by NIH (NBIB) R01 funding (# EB023921) to IF and SJS, and NIH (NHLBI) R01 funding (# HL137762) to IF and RJL.

All human samples for this experiment were obtained in accordance with the IRB of the Children&#8217;s Hospital of Philadelphia. All animal experiments were performed upon approval from IACUC of the Children&#8217;s Hospital of Philadelphia.
1. Coating bare metal surfaces with PEI-PDT
<ol>
	<li>Wash the stainless steel foil coupons (1 cm x 1 cm or 0.65 cm x 1 cm) or stainless steel mesh disks with 2-isopropanol &#160;in a shaker (60 &#176;C, speed of 200 rpm) for 5 min. Perf...

<ol>
	<li>Buccheri, D., Piraino, D., Andolina, G., Cortese, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+and+managing+in-stent+restenosis:+a+review+of+clinical+data,+from+pathogenesis+to+treatment.">Understanding and managing in-stent restenosis: a review of clinical data, from pathogenesis to treatment.</a> Journal Thoracic Disease. 8 (10), 1150-1162 (2016).</li><li>van Oeveren, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obstacles+in+haemocompatibility+testing.">Obstacles in haemocompatibility testing.</a> Scientifica. 2013, 392584 (2013).</li><li>Mitra, A. K., Agrawal, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+stent+restenosis:+bane+of+the+stent+era.">In stent restenosis: bane of the stent era.</a> Journal of Clinical Pathology. 59 (3), 232-239 (2006).</li><li>Iqbal, J., Gunn, J., Serruys, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+stents:+historical+development,+current+status+and+future+directions.">Coronary stents: historical development, current status and future directions.</a> British Medical Bulletin. 106, 193-211 (2013).</li><li>Hoffmann, 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=Patterns+and+mechanisms+of+in-stent+restenosis.+A+serial+intravascular+ultrasound+study.">Patterns and mechanisms of in-stent restenosis. A serial intravascular ultrasound study.</a> Circulation. 94 (6), 1247-1254 (1996).</li><li>Stefanini, G. G., Windecker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stent+thrombosis:+no+longer+an+issue+with+newer-generation+drug-eluting+stents.">Stent thrombosis: no longer an issue with newer-generation drug-eluting stents.</a> Circulation: Cardiovascular Interventions. 5 (3), 332-335 (2012).</li><li>Palmerini, T., 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=Clinical+outcomes+with+bioabsorbable+polymer-+versus+durable+polymer-based+drug-eluting+and+bare-metal+stents:+evidence+from+a+comprehensive+network+meta-analysis.">Clinical outcomes with bioabsorbable polymer- versus durable polymer-based drug-eluting and bare-metal stents: evidence from a comprehensive network meta-analysis.</a> Journal of the American College of Cardiology. 63 (4), 299-307 (2014).</li><li>Omar, W. A., Kumbhani, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Current+Literature+on+Bioabsorbable+Stents:+a+Review.">The Current Literature on Bioabsorbable Stents: a Review.</a> Current Atherosclerosis Reports. 21 (12), 54 (2019).</li><li>Slee, J. B., Christian, A. J., Levy, R. J., Stachelek, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addressing+the+Inflammatory+Response+to+Clinically+Relevant+Polymers+by+Manipulating+the+Host+Response+Using+ITIM+Domain-Containing+Receptors.">Addressing the Inflammatory Response to Clinically Relevant Polymers by Manipulating the Host Response Using ITIM Domain-Containing Receptors.</a> Polymers (Basel). 6 (10), 2526-2551 (2014).</li><li>Oldenborg, P. 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=Role+of+CD47+as+a+marker+of+self+on+red+blood+cells.">Role of CD47 as a marker of self on red blood cells.</a> Science. 288 (5473), 2051-2054 (2000).</li><li>vanden Berg, T. K., vander Schoot, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+'self'+recognition:+a+role+for+CD47-SIRPalpha+interactions+in+hematopoietic+stem+cell+transplantation.">Innate immune 'self' recognition: a role for CD47-SIRPalpha interactions in hematopoietic stem cell transplantation.</a> Trends in Immunology. 29 (5), 203-206 (2008).</li><li>Tengood, J. E., Levy, R. J., Stachelek, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+CD47-modified+biomaterials+to+mitigate+the+immune+response.">The use of CD47-modified biomaterials to mitigate the immune response.</a> Experimental Biology Medicine (Maywood). 241 (10), 1033-1041 (2016).</li><li>Tsai, R. K., Rodriguez, P. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self+inhibition+of+phagocytosis:+the+affinity+of+'marker+of+self'+CD47+for+SIRPalpha+dictates+potency+of+inhibition+but+only+at+low+expression+levels.">Self inhibition of phagocytosis: the affinity of 'marker of self' CD47 for SIRPalpha dictates potency of inhibition but only at low expression levels.</a> Blood Cells, Molecules and Diseases. 45 (1), 67-74 (2010).</li><li>Slee, J. B., 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=Enhanced+biocompatibility+of+CD47-functionalized+vascular+stents.">Enhanced biocompatibility of CD47-functionalized vascular stents.</a> Biomaterials. 87, 82-92 (2016).</li><li>Finley, M. J., 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=Diminished+adhesion+and+activation+of+platelets+and+neutrophils+with+CD47+functionalized+blood+contacting+surfaces.">Diminished adhesion and activation of platelets and neutrophils with CD47 functionalized blood contacting surfaces.</a> Biomaterials. 33 (24), 5803-5811 (2012).</li><li>Stachelek, S. J., 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=The+effect+of+CD47+modified+polymer+surfaces+on+inflammatory+cell+attachment+and+activation.">The effect of CD47 modified polymer surfaces on inflammatory cell attachment and activation.</a> Biomaterials. 32 (19), 4317-4326 (2011).</li><li>Inamdar, V. V., 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=Stability+and+bioactivity+of+pepCD47+attachment+on+stainless+steel+surfaces.">Stability and bioactivity of pepCD47 attachment on stainless steel surfaces.</a> Acta Biomaterialia. 104, 231-240 (2020).</li><li>Fishbein, I., 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=Local+delivery+of+gene+vectors+from+bare-metal+stents+by+use+of+a+biodegradable+synthetic+complex+inhibits+in-stent+restenosis+in+rat+carotid+arteries.">Local delivery of gene vectors from bare-metal stents by use of a biodegradable synthetic complex inhibits in-stent restenosis in rat carotid arteries.</a> Circulation. 117 (16), 2096-2103 (2008).</li><li>Moser, K. V., Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+rat+monocytes+migrate+through+a+BCEC-monolayer+and+express+microglia-markers+at+the+basolateral+side.">Primary rat monocytes migrate through a BCEC-monolayer and express microglia-markers at the basolateral side.</a> Brain Research Bulletin. 74 (5), 336-343 (2007).</li><li>vander Giessen, W. J., 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=Marked+inflammatory+sequelae+to+implantation+of+biodegradable+and+nonbiodegradable+polymers+in+porcine+coronary+arteries.">Marked inflammatory sequelae to implantation of biodegradable and nonbiodegradable polymers in porcine coronary arteries.</a> Circulation. 94 (7), 1690-1697 (1996).</li><li>Fishbein, I., 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=Bisphosphonate-mediated+gene+vector+delivery+from+the+metal+surfaces+of+stents.">Bisphosphonate-mediated gene vector delivery from the metal surfaces of stents.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (1), 159-164 (2006).</li><li>Mouro-Chanteloup, I., 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=Evidence+that+the+red+cell+skeleton+protein+4.2+interacts+with+the+Rh+membrane+complex+member+CD47.">Evidence that the red cell skeleton protein 4.2 interacts with the Rh membrane complex member CD47.</a> Blood. 101 (1), 338-344 (2003).</li><li>Finley, M. J., Clark, K. A., Alferiev, I. S., Levy, R. J., Stachelek, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+signaling+mechanisms+associated+with+CD47+modified+surfaces.">Intracellular signaling mechanisms associated with CD47 modified surfaces.</a> Biomaterials. 34 (34), 8640-8649 (2013).</li><li>Slee, J. B., Alferiev, I. S., Levy, R. J., Stachelek, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+ex+vivo+Chandler+Loop+Apparatus+to+assess+the+biocompatibility+of+modified+polymeric+blood+conduits.">The use of the ex vivo Chandler Loop Apparatus to assess the biocompatibility of modified polymeric blood conduits.</a> Journal of Visualized Experiments. (90), e51871 (2014).</li></ol>

We demonstrate and describe a relatively novel chemical strategy to append therapeutic peptide moieties to a stainless-steel surface with the overarching goal of reducing the surface&#8217;s reactivity with inflammatory cells found in blood. The bisphosphonate chemistry described herein involves co-ordinate bond formation between the metal oxides and bisphosphonate groups of PABT. The thickness of polybisphosphonate monolayer formed on the metal surface does not exceed 5 nm18, and, therefore, is i...

Percutaneous coronary intervention is the first line of therapy to treat coronary artery diseases (CAD) and primarily involves stenting the diseased arteries. However, in-stent restenosis (ISR) and stent thrombosis are common complications associated with stent deployment1. Blood interaction at the blood-stent interface is characterized by an almost immediate adsorption of plasma proteins on the metal surface, followed by platelet and inflammatory cell attachment and activation2. The release of the inflammatory cytokines and chemokines from activated inflammatory cells leads to the phenotypic modification of the vascular smooth muscle cells (VSMCs) in the tunica media and triggers their centripetal migration to the intimal compartment. Proliferation of activated VSMC in the intima results in intimal layer thickening, lumen narrowing and in-stent restenosis3. Drug eluting stents (DES) were developed to prevent VSMC proliferation; however, these drugs have an off-target cytotoxic effect on the endothelial cells4,5. Therefore, late stent thrombosis is a common complication associated with DES6,7. Stents made of biodegradable polymers, such as poly-L-lactide have shown much promise in the animal experiments and initial clinical trials, but were eventually recalled when the &#8220;real-life&#8221; clinical use demonstrated their inferiority to the 3rd generation DES8. Therefore, there is a need to improve the biocompatibility of bare metal stents for better patient outcomes.
CD47 is a ubiquitously expressed transmembrane protein that inhibits the innate immune response when bound to its cognate receptor Signal Regulatory Protein alpha (SIRP&#945;)9. The SIRP&#945; receptor has an immune cell tyrosine inhibitory motif (ITIM) domain and the signaling events upon SIRP&#945; - CD47 interaction ultimately result in the downregulation of inflammatory cell activation10,11,12,13. Research in our lab has shown that recombinant CD47 or its peptide derivative, corresponding to the 22 amino acid Ig domain of the extracellular region of CD47 (pepCD47), can reduce the host immune response to a range of clinically relevant biomaterials14,15,16. Recently, we have demonstrated that pepCD47 can be immobilized to stainless steel stent surfaces and significantly reduce the pathophysiological response associated with restenosis. Of note, the pepCD47 modified surfaces are amenable to relevant usage conditions such as long term storage and ethylene oxide sterilization17. To that end, pepCD47 may be a useful therapeutic target to address the clinical limitations of endovascular stents.
The strategy for the covalent attachment of pepCD47 to a metal surface involves a series of novel chemical modifications of the metal surface. The metal surfaces are first coated with polyallylamine bisphosphonate with latent thiol groups (PABT) followed by the deprotection of the thiols and attachment of polyethyleneimine (PEI) with installed pyridyldithio groups (PDT). PDT groups of PEI unconsumed in the reaction with deprotected PABT thiols are then reacted with pepCD47 incorporating thiols in the terminal cysteine residues, resulting in binding pepCD47 to the metal surface via a disulfide bond14,17,18. We used a fluorophore conjugated pepCD47 (TAMRA-pepCD47) to determine the input concentration of peptide that results in the maximum surface immobilization of the peptide. Finally, we evaluated the acute and chronic anti-inflammatory capacity of the pepCD47 coated metal surfaces, ex vivo, using the Chandler loop apparatus, and monocyte attachment/macrophage expansion assay, respectively.
This paper provides a systematic protocol for the attachment of thiolated peptides to the metal surface; determining the maximum immobilization density of the peptide; and assessing the anti-inflammatory properties of pepCD47 coated metal surfaces exposed to whole blood and isolated monocytes.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">1 M Tris-HCL</td>
			<td style="width:184px;">Invitrogen</td>
			<td style="width:129px;">15567-027</td>
			<td style="width:239px;">pH - 7.5</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">4% Glutaraldehyde</td>
			<td style="width:184px;">Electron Microscopy Sciences</td>
			<td style="width:129px;">16539-07</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">4% Sodium Citrate</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:129px;">S5770</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">ACK lysing buffer</td>
			<td style="width:184px;">Quality Biologicals</td>
			<td style="width:129px;">118-156-721</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">anti-CD45RA Ab (mouse anti-rat; clone OX-19)</td>
			<td style="width:184px;">Biolegend</td>
			<td style="width:129px;">202301</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">anti-CD5 Ab (mouse anti-rat; clone OX-19)</td>
			<td style="width:184px;">Biolegend</td>
			<td style="width:129px;">203501</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">anti-CD6 Ab (mouse anti-rat; clone OX-52)</td>
			<td style="width:184px;">BD Biosciences</td>
			<td style="width:129px;">550979</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">anti-CD68 Ab (mouse anti-rat; clone ED-1)</td>
			<td style="width:184px;">BioRad</td>
			<td style="width:129px;">MCA341</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">anti-CD8a Ab (mouse anti-rat; clone OX-8)</td>
			<td style="width:184px;">Biolegend</td>
			<td style="width:129px;">201701</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Chloroform Certified ACS</td>
			<td>Fisher Chemical</td>
			<td style="width:129px;">C298-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Dimethyl Formammide (DMF)</td>
			<td style="width:184px;">Alfa Aesar</td>
			<td style="width:129px;">39117</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Embra stainless steel grid</td>
			<td>Electron Microscopy Sciences</td>
			<td style="width:129px;">E200-SS</td>
			<td>stainless steel mesh mesh disks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Ficoll Hypaque</td>
			<td style="width:184px;">GE Healthcare</td>
			<td style="width:129px;">17-1440-02</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Glacial acetic acid</td>
			<td style="width:184px;">ACROS organic</td>
			<td style="width:129px;">148930025</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">goat anti-mouse IgG Alexa Fluor</td>
			<td style="width:184px;">ThermoFisher</td>
			<td style="width:129px;">A11030</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Heparin sodium</td>
			<td style="width:184px;">Sagent Pharmaceuticals</td>
			<td style="width:129px;">402-01</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human pepCD47</td>
			<td>Bachem</td>
			<td>4099101</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Isopropanol</td>
			<td>Fisher Chemical</td>
			<td style="width:129px;">A426P-4</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">Metal adapters</td>
			<td style="width:184px;">Leur Fitting</td>
			<td style="width:129px;">6515IND</td>
			<td style="width:239px;">1 way adapter 316 ss 1/4&#34;-5/16&#34; hoes end</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Methanol</td>
			<td style="width:184px;">RICCA chemical company</td>
			<td style="width:129px;">4829-32</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Microscope</td>
			<td style="width:184px;">Nikon Eclipse</td>
			<td style="width:129px;">TE300</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pottasium Bicarbonate (KHCO3)</td>
			<td>Fisher Chemical</td>
			<td style="width:129px;">P184-500</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">PVC tubes</td>
			<td style="width:184px;">Terumo-CVS</td>
			<td style="width:129px;">60050</td>
			<td style="width:239px;">1/4&#34; X 1/16 8&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">sodium cacodylate buffer with 0.1M sodium chloride</td>
			<td style="width:184px;">Electron Microscopy Sciences</td>
			<td style="width:129px;">11653</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td style="width:184px;">Bio-Rad laboratories</td>
			<td style="width:129px;">161-0302</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Sodum actetate (C2H3NaO2)</td>
			<td style="width:184px;">Alfa Aesar</td>
			<td style="width:129px;">A13184</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Src peptide</td>
			<td>Bachem</td>
			<td style="width:129px;">4092599</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">Stainless steel (AISI 304) cylinder-shaped samples with a lumen</td>
			<td>Microgroup, Medway, MA</td>
			<td style="width:129px;">20097328</td>
			<td>1 cm X 6 mm OD</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">Stainless steel foils (AISI 316L)</td>
			<td style="width:184px;">Goodfellow, Coraopolis, PA</td>
			<td style="width:129px;"></td>
			<td style="width:239px;">100 mm X 100 mm X 0.05 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">Tetramethylrhodamine-conjugated pepCD47 (TAMRA-pepCD47)</td>
			<td>Bachem</td>
			<td style="width:129px;">4100277</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:282px;">TMB (3,3&#8217; ,5,5&#8217; -tetramethylbenzidine) substrate and tris (2-carboxyethyl) phosphine hydrochloride (TCEP)</td>
			<td>Thermo Scientific</td>
			<td>PG82089</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Tween-20</td>
			<td style="width:184px;">Bio-Rad laboratories</td>
			<td style="width:129px;">170-6531</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Vybrant CFDA SE Cell Tracer Kit</td>
			<td>Invitrogen</td>
			<td style="width:129px;">V12883</td>
			<td style="width:239px;"></td>
		</tr>
	</tbody>
</table>

mitigation of blood borne cell attachment to metal implants through cd47-derived peptide immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

The metal surfaces are rendered thiol-reactive for peptide attachment via a series of chemical modifications, as illustrated in Figure 1. PABT incubation followed by PEI-PDT treatment makes the metal surface amenable for peptide attachment. Peptide CD47 (pepCD47) containing cysteine residue at C-terminus joined to the core pepCD47 sequence through a flexible dual AEEAc bridge is covalently attached to the thiol-reactive surfaces via disulfide bonds. Using this protocol, we have demonstrated ...

Watch this Scientific Journal Video about Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization at JoVE.com

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

Research

JoVE Journal

Bioengineering

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