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. Perform this step 2x. Then wash 2x with chloroform (60 &#176;C, speed of 200 rpm) for 10 min each.</li>
	<li>Place the cleansed stainless-steel samples in an oven at 220 &#176;C for 30 min.</li>
	<li>Prepare 5 mL of 0.5% of PABT solution by dissolving 25 mg of polyallylamine bisphosphonate with latent thiol groups (PABT) and 5 mg of potassium bicarbonate (KHCO3) in 5 mL of DDW and incubate at 72 &#176;C in a shaker at 200 rpm for 30 min. 
	NOTE: For PABT synthesis refer to previously published literature18.</li>
	<li>Immerse the baked foils or mesh disks in 0.5% aqueous solution of PABT and incubate in a shaker (72 &#176;C, speed of 200 rpm) for 1 h.</li>
	<li>Wash the PABT-modified samples with deionized distilled water (DDW) for 5x, transfer the specimens in a new vial and wash again with DDW for 5x.</li>
	<li>Prepare a total of 5 mL of TCEP solution (12 mg/mL) by dissolving 60 mg of Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) in 5 mL of 0.1 M acetic buffer (0.57 mL of glacial acetic acid, 820 mg of sodium acetate in 100 mL of DDW).</li>
	<li>Treat the PABT-modified samples with TCEP for 15 min at room temperature (RT) on a shaker. &#160; 
	NOTE: TCEP is used to deprotect the thiol groups.</li>
	<li>Degas DDW in a round bottom flask by using a vacuum generating device, such as a lyophilizer and wash the TCEP treated foils or mesh disks with degassed DDW for 5x. Transfer the samples in a new vial, and additionally wash 5x with degassed DDW. 
	NOTE: It is paramount to work fast to prevent oxidation of thiols on the metal surface by atmospheric oxygen.</li>
	<li>Prepare 5 mL of 1% PEI-PDT solution by diluting 212.5 &#181;L of stock PEI-PDT and 125 &#181;L of 0.4 M sodium acetate in degassed DDW. Carry out step 1.9 simultaneously with step 1.8 to minimize exposure of the samples to atmospheric air. 
	NOTE: The synthesis of PEI-PDT is described in previously published literature18.</li>
	<li>Incubate the washed stainless-steel specimens with 1% PEI-PDT. Replace air with argon gas, seal the vials air-tight and mix on a shaker at RT for 1 h. Either proceed immediately with the peptide conjugation or store at 4 &#176;C up to 1 week.</li>
</ol>
2. Attachment and qualitative/quantitative assessment of fluorophore conjugated pepCD47 retention on metal surface using fluorescence microscopy and fluorimetry
<ol>
	<li>Wash foil coupons or mesh disks prepared as described in the section 1, steps 1.1-1.10 above with DDW 5x. Transfer the samples to a new vial and wash with DDW for additional 5x. Finally wash 2x with degassed ethanol and 2x with degassed dimethyl formamide (DMF).</li>
	<li>Prepare tetramethylrhodamine (TAMRA)-conjugated pepCD47 stock solution by dissolving TAMRA-conjugated pepCD47 powder in degassed dimethylformamide (DMF) to a final concentration of 1 mg/mL. Aliquot the stock solution in 1 mL allotments. Store in well-sealed tubes under argon atmosphere at -20 &#176;C.</li>
	<li>Dilute 1 mg/mL of the stock solution of TAMRA-conjugated pepCD47 using degassed DMF to prepare the following concentrations of fluorophore conjugated pepCD47 - 10, 30, 100, and 200 &#181;g/mL. 
	NOTE: If TAMRA-conjugated pepCD47 appears to be precipitated, reduce the stock TAMRA-conjugated pepCD47 solution using TCEP beads in ratio 1:1, at RT for 20 min. Note that before adding the TAMRA-conjugated pepCD47 to the TCEP beads, spin the TCEP beads solution, remove the supernatant and then proceed with addition of the TAMRA-conjugated pepCD47.</li>
	<li>Incubate the PEI-PDT modified foil coupons with 10, 30, 100 or 200 &#181;g/mL of TAMRA-conjugated pepCD47 in triplicates for each condition on a shaker at RT under argon atmosphere for 1 hour. Incubate PEI-PDT modified mesh disks, as well as mesh disk not modified beyond step 1.2 (bare metal controls) with 100 &#181;g/mL of TAMRA-conjugated pepCD47 in triplicates at RT under argon atmosphere on a shaker for 1 h. 
	NOTE: From this step onwards, the vials are wrapped in aluminum foil to protect the contents from light.</li>
	<li>Wash the fluorophore conjugated pepCD47-coated surfaces to remove the non-covalently attached peptide in the following order: DMF (3x), DMF/DDW at 1:1, DDW (3x), 0.3% SDS in 20 mM Tris pH 7.4 &#160;&#160;(3x, 5 min each at 70 &#176;C on a shaker), DDW (3x), change vials, and a final DDW wash.</li>
	<li>Place control and covalently conjugated mesh disks on a microscope glass, add 50 &#181;L of PBS and place a coverslip. Image mesh disks using an inverted fluorescence microscope equipped with a rhodamine filter set. Take representative images at 100x magnification.</li>
	<li>Prepare 15 mL of 12 mg/mL TCEP solution by dissolving 180 mg of TCEP in 1:1 v/v mixture of methanol and 0.1 M acetic buffer.</li>
	<li>Incubate each washed foil with 1 mL of TCEP solution on a shaker at RT for 15 min.</li>
	<li>Prepare the following standards by serially diluting the TAMRA-conjugated pepCD47 stock (1 mg/mL) &#8211; 100 &#181;g/mL, 10 &#181;g/mL, 1 &#181;g/mL, 0.1 &#181;g/mL, and 0.01 &#181;g/mL. Use the TCEP solution as the diluent.</li>
	<li>Analyze the TAMRA-conjugated pepCD47 released from the metal surface against the calibration curve generated using the standards by fluorimetry at 544/590 nm excitation and emission wavelengths.</li>
</ol>
3. Attaching human pepCD47 to PEI-PDT modified surfaces
<ol>
	<li>Wash PEI-PDT coated samples formulated as described in the section 1, steps 1.1-1.10 above, with degassed DDW 5x, change the vial and wash with degassed DDW 5x.</li>
	<li>Prepare human pepCD47 stock solution (1 mg/mL) by dissolving human pepCD47 powder in degassed 50% acetic acid to achieve a concentration 1 mg/mL.</li>
	<li>Prepare working concentration of human pepCD47 (100 &#181;g/mL) by dissolving 500 &#181;L of the stock of human pepCD47 in 4,500 &#181;L of degassed 1x phosphate buffered saline (PBS).</li>
	<li>Incubate the washed PEI-PDT coated samples with 100 &#181;g/mL of pepCD47 at RT with shaking for 1 h.</li>
	<li>Wash the human pepCD47-coated samples to remove excess peptide in the following order PBS (3x), DDW (3x), 0.2% Tween-20 (3x, 5 min each), DDW (3x), change vials and final DDW wash. 
	NOTE: Human pepCD47 coated surfaces can be stored dry at 4 &#176;C for up to 6 months.</li>
</ol>
4. Coating the PEI-PDT modified surfaces with scrambled sequence (Scr)
<ol>
	<li>Dissolve the scrambled sequence powder in degassed 0.1% acetic acid to prepare a stock solution of 1 mg/mL.</li>
	<li>Prepare a solution of 100 &#181;g/mL of scrambled peptide by dissolving 500 &#181;L of the in 4,500 &#181;L of degassed 0.1% acetic acid.</li>
	<li>Coat the washed PEI-PDT specimens with 100 &#181;g/mL of scrambled peptide at RT with shaking for 1 h.</li>
	<li>To remove unattached scrambled peptide, wash the surfaces in the following order 0.01% acetic acid (3x), DDW (3x), 0.2% Tween-20 (3x, 5 min), DDW (3x), change vials and one DDW wash.</li>
</ol>
5. Chandler loop for analyzing cellular attachment to metal surfaces
<ol>
	<li>Coat the metal foils (0.65 cm x 1cm) with either human pepCD47 or scrambled peptide as per the description on section 1 followed by 3 or 4.</li>
	<li>Cut &#188;&#8221; PVC tubes into three 38 cm long pieces.</li>
	<li>Insert up to 8 unmodified, scrambled peptide or pepCD47 modified metal foils in three different tubes.</li>
	<li>Collect 30 mL of blood from healthy human donors free of any anti-platelet medications as per institutional IRB protocol. Preload syringe with 1 mL of 4% sodium citrate to prevent coagulation of collected blood.</li>
	<li>Put 10 mL of blood into each tube using a 10 mL syringe and connect the ends with metal adapters. Place the blood-filled tubes on the wheels of the Chandler loop apparatus.</li>
	<li>Pass the blood along the metal foils by wheel rotation at 37 &#176;C at the speed calculated to produce the shear of 25 dyns/cm2 for 4 h.</li>
	<li>Drain the blood from the tubes and dispose of the blood according to the IRB requirements.</li>
	<li>Cut the tubes using the scalpel to retrieve the foils from each tube.</li>
	<li>Prepare 2% glutaraldehyde solution by diluting the 10 ml of 4% glutaraldehyde solution using 10 mL of sodium cacodylate buffer with 0.1 M sodium chloride.</li>
	<li>Incubate the foils in 2% glutaraldehyde solution for 15 min and store at 4 &#176;C overnight. Before analyzing, wash the metal foils 3x with PBS.</li>
</ol>
6. Analyzing cellular attachment to metal surfaces using CFDA dye
<ol>
	<li>Warm 8 mL of PBS in 15 mL tube in a water bath set to 37 &#176;C.</li>
	<li>Prepare the CFDA (carboxy-fluorescein diacetate, succinimidyl ester) dye solution as follows - add 90 &#181;L of DMSO to one CFDA dye vial to achieve a stock concentration of 10 mM. Next, prepare the working concentration of 93.75 &#181;M by adding 75 &#181;L of the stock CFDA to 8 mL of warm PBS. Mix by inverting the tubes a few times and cover the tube with aluminium foil. 
	NOTE: It is advisable to freshly prepare the CFDA dye before every use.</li>
	<li>Incubate each foil with 1 mL of CFDA dye in a 24 well plate. Cover the plate with aluminum foil and incubate the plate at 37 &#176;C for 15 min.</li>
	<li>Wash the metal foils 3x with PBS to remove excess dye. Image using the inverted fluorescence microscope.</li>
</ol>
7. Monocyte attachment and macrophage expansion on the pepCD47-modified and bare metal surfaces
<ol>
	<li>Collect 10 mL of peripheral blood via the vena cava access during the sacrifice of a 400-450 g male Sprague-Dawley rat. Mix immediately with 1,000 IU of sodium heparin to prevent coagulation.</li>
	<li>Pipette 10 mL of density gradient medium into a 50 mL conical tube. Mix the blood with 5 mL of PBS, and carefully layer diluted blood over the density gradient medium using a Pasteur pipette. Centrifuge in a swinging bucket rotor at 800 x g and 18-25 &#176;C for 20 min with minimal settings of acceleration and deceleration.</li>
	<li>Using a Pasteur pipette, collect an opaque layer of buffy coat on the interface between plasma and Ficoll. Dilute buffy coat 1:3 with PBS and centrifuge at 550 x g and 4 &#176;C for 10 min to buffy coat cells.</li>
	<li>Re-suspend buffy coat cells in 3 mL of ACK lysis buffer to lyse contaminating erythrocytes. Incubate on ice for 4 min. Add 12 mL of cell separation buffer (CSB; 0.5% BSA, 0.5% FBS, 2 mM EDTA/PBS).</li>
	<li>Centrifuge at 550 x g and 4 &#176;C for 10 min. Re-suspend the pellet in 10 mL of CSB.</li>
	<li>Centrifuge at 200 x g and 4 &#176;C for 10 min to eliminate platelets. Repeat twice.</li>
	<li>Re-suspend the pellet in 500 &#181;L of CSB. Add 10 &#181;g of each of the following mouse anti-rat antibodies: CD8a (clone OX-8), anti-CD5 (clone OX-19), anti-CD45RA (clone OX-33), and anti-CD6 (clone OX-52).</li>
	<li>Incubate at 4 &#176;C on a vertical tube rotator for 1 h. Add 9.5 mL of CSB. Centrifuge at 300 x g and 4 &#176;C for 10 min. Discard supernatant, re-suspend the pellet in 10 mL of CSB and repeat centrifugation.</li>
	<li>Re-suspend the pellet in 500 &#181;L of CSB. Add 150 &#181;L of goat anti-mouse IgG microbeads. Incubate at 4 &#176;C on a vertical tube rotator for 20 min. Add 9.5 mL of CSB. Centrifuge at 300 x g and 4 &#176;C for 10 min.</li>
	<li>Discard the supernatant. Re-suspend the pellet in 1 mL of CSB.</li>
	<li>Place a LS column in a magnetic separator.&#160; Prime the LS column with 3 mL of CSB. Discard the column throughput. Add 1 mL of re-suspended cell pellet from the step 7.10. Start collecting the throughput. After flow stops, add 5 mL of CSB and keep collecting the column throughput until the flow stops.</li>
	<li>Centrifuge the column throughput (6 mL) containing negatively selected monocytes at 300 x g and 4 &#176;C for 10 min. Re-suspend the resulting small pellet in 2 mL of RPMI-1640 medium supplemented with 10% FCS, 1% pen/strep and 100 ng/mL rat macrophage colony stimulating factor (M-CSF).</li>
	<li>Count the monocytes using hemocytometer. Adjust monocyte concentration to 5 x 105 cells/mL.</li>
	<li>Add 1 mL volumes of monocyte suspension to the individual wells of a 12 well plate with the bare stainless steel foil samples (N=3) or stainless steel samples modified with rat pepCD47 (N=3) as per 1.1-1.10 and 3.1-3.6.</li>
	<li>Change the medium on days 3 and 5 post-seeding. On day 6 post-seeding wash the cells with PBS and fix with 4% paraformaldehyde at room temperature for 15 min. Wash twice with PBS for 5 min.</li>
	<li>Remove the stainless-steel foils and place them individually into a new 12 well plate. Do not flip the foils.</li>
	<li>Incubate in 0.5% Tween-20/PBS for 15 min to permeabilize the cells. Wash twice with PBS for 5 min.</li>
	<li>Block in 10% goat serum/PBS for 20 min. Aspirate the serum. Do not wash. Add mouse anti-rat CD68 antibody (diluted 1:100 in 1%BSA/PBS). Incubate at room temperature for 1 h. Wash 3x in PBS for 5 min each.</li>
	<li>Add goat anti-mouse IgG Alexa Fluor 546 (diluted 1:200 in 1% BSA/PBS). Incubate in dark at room temperature for 45 min. Wash in PBS for 5 min. Counterstain with 1 &#181;g/mL Hoechst 33342 dye in dark at room temperature for 10 min. Wash 3x in PBS for 5 min each.</li>
	<li>Flip the foils and image using a fluorescent microscope with the inverted optics. Capture images at 200x magnification with blue and red filter settings.</li>
	<li>Count the attached monocytes in the individual images and calculated the group averages and standard deviations.</li>
</ol>

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The authors have nothing to disclose.

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.

<table><tbody><tr><td>1 M Tris-HCL</td><td>Invitrogen</td><td>15567-027</td><td>pH - 7.5</td></tr><tr><td>4% Glutaraldehyde</td><td>Electron Microscopy Sciences</td><td>16539-07</td><td></td></tr><tr><td>4% Sodium Citrate</td><td>Sigma</td><td>S5770</td><td></td></tr><tr><td>ACK lysing buffer</td><td>Quality Biologicals</td><td>118-156-721</td><td></td></tr><tr><td>anti-CD45RA Ab (mouse anti-rat; clone OX-19)</td><td>Biolegend</td><td>202301</td><td></td></tr><tr><td>anti-CD5 Ab (mouse anti-rat; clone OX-19)</td><td>Biolegend</td><td>203501</td><td></td></tr><tr><td>anti-CD6 Ab (mouse anti-rat; clone OX-52)</td><td>BD Biosciences</td><td>550979</td><td></td></tr><tr><td>anti-CD68 Ab (mouse anti-rat; clone ED-1)</td><td>BioRad</td><td>MCA341</td><td></td></tr><tr><td>anti-CD8a Ab (mouse anti-rat; clone OX-8)</td><td>Biolegend</td><td>201701</td><td></td></tr><tr><td>Chloroform Certified ACS</td><td>Fisher Chemical</td><td>C298-500</td><td></td></tr><tr><td>Dimethyl Formammide (DMF)</td><td>Alfa Aesar</td><td>39117</td><td></td></tr><tr><td>Embra stainless steel grid</td><td>Electron Microscopy Sciences</td><td>E200-SS</td><td>stainless steel mesh mesh disks</td></tr><tr><td>Ficoll Hypaque</td><td>GE Healthcare</td><td>17-1440-02</td><td></td></tr><tr><td>Glacial acetic acid</td><td>ACROS organic</td><td>148930025</td><td></td></tr><tr><td>goat anti-mouse IgG Alexa Fluor</td><td>ThermoFisher</td><td>A11030</td><td></td></tr><tr><td>Heparin sodium</td><td>Sagent Pharmaceuticals</td><td>402-01</td><td></td></tr><tr><td>Human pepCD47</td><td>Bachem</td><td>4099101</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Chemical</td><td>A426P-4</td><td></td></tr><tr><td>Metal adapters</td><td>Leur Fitting</td><td>6515IND</td><td>1 way adapter 316 ss 1/4&quot;-5/16&quot; hoes end</td></tr><tr><td>Methanol</td><td>RICCA chemical company</td><td>4829-32</td><td></td></tr><tr><td>Microscope</td><td>Nikon Eclipse</td><td>TE300</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Gibco</td><td>14190-136</td><td></td></tr><tr><td>Pottasium Bicarbonate (KHCO3)</td><td>Fisher Chemical</td><td>P184-500</td><td></td></tr><tr><td>PVC tubes</td><td>Terumo-CVS</td><td>60050</td><td>1/4&quot; X 1/16 8&#039;</td></tr><tr><td>sodium cacodylate buffer with 0.1M sodium chloride</td><td>Electron Microscopy Sciences</td><td>11653</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Bio-Rad laboratories</td><td>161-0302</td><td></td></tr><tr><td>Sodum actetate (C2H3NaO2)</td><td>Alfa Aesar</td><td>A13184</td><td></td></tr><tr><td>Src peptide</td><td>Bachem</td><td>4092599</td><td></td></tr><tr><td>Stainless steel (AISI 304) cylinder-shaped samples with a lumen</td><td>Microgroup, Medway, MA</td><td>20097328</td><td>1 cm X 6 mm OD</td></tr><tr><td>Stainless steel foils (AISI 316L)</td><td>Goodfellow, Coraopolis, PA</td><td></td><td>100 mm X 100 mm X 0.05 mm</td></tr><tr><td>Tetramethylrhodamine-conjugated pepCD47 (TAMRA-pepCD47)</td><td>Bachem</td><td>4100277</td><td></td></tr><tr><td>TMB (3,3&amp;rsquo; ,5,5&amp;rsquo; -tetramethylbenzidine) substrate and tris (2-carboxyethyl) phosphine hydrochloride (TCEP)</td><td>Thermo Scientific</td><td>PG82089</td><td></td></tr><tr><td>Tween-20</td><td>Bio-Rad laboratories</td><td>170-6531</td><td></td></tr><tr><td>Vybrant CFDA SE Cell Tracer Kit</td><td>Invitrogen</td><td>V12883</td><td></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, ...

mitigation-blood-borne-cell-attachment-to-metal-implants-through-cd47

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

4.6K Views.  The Children's Hospital of Philadelphia. 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.

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

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Chemistry

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

Immunology and Infection

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the subsequent attachment and activation of platelets, monocyte/macrophage adhesion, and inflammatory cell signaling events, leading to post-procedural complications. The Chandler Loop Apparatus is an experimental system that allows researchers to study the molecular and cellular interactions that occur when large volumes of blood are perfused over polymeric conduits. To that end, this apparatus has been used as an ex vivo model allowing the assessment of the anti-inflammatory properties of various polymer surface modifications. Our laboratory has shown that blood conduits, covalently modified via photoactivation chemistry with recombinant CD47, can confer biocompatibility to polymeric surfaces. Appending CD47 to polymeric surfaces could be an effective means to promote the efficacy of polymeric blood conduits. Herein is the methodology detailing the photoactivation chemistry used to append recombinant CD47 to clinically relevant polymeric blood conduits and the use of the Chandler Loop as an ex vivo experimental model to examine blood interactions with the CD47 modified and control conduits.

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits.

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the ...

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.

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 ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

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 ...

Identifying PD-1/PD-L1 Inhibitors with Surface Plasmon Resonance Technology

The disruption of the PD-1/PD-L1 interaction is a promising strategy for cancer immunotherapy. Reliable screening platforms are essential for evaluating the efficacy of PD-1/PD-L1 inhibitors. A previously established human PD-1/PD-L1 blockade assay utilizing Surface Plasmon Resonance (SPR) technology (first-generation PD-1/PD-L1 inhibitor SPR screening platform) demonstrated results comparable to those obtained through Homogeneous Time-Resolved Fluorescence (HTRF) and cell-based assays, with potential for large-scale screening. Herein, an optimized version of this assay (second-generation PD-1/PD-L1 inhibitor SPR screening platform) is presented, featuring a dual-step coupling process that combines amine and bio-streptavidin coupling to enhance PD-1 orientation control on the chip and reduce PD-1 protein consumption. The updated platform was successfully validated using the PD-1/PD-L1 inhibitor BMS-1166, showing blockade effects comparable to the previous SPR-based method and other established techniques such as ELISA. These results confirm the reliability of the approach. This optimized SPR screening platform offers a high-throughput and reliable tool for identifying novel PD-1/PD-L1 inhibitors, advancing cancer immunotherapy research, and highlighting the potential of SPR in immune checkpoint inhibitor screening.

This protocol describes a blockade assay for PD-1/PD-L1 inhibitors using surface plasmon resonance technology. It employs a dual-step immobilization strategy and a tailored buffer system to accurately measure response units, facilitating the assessment of blockade rates for compounds or biologics. Additionally, it supports the high-throughput identification of PD-1/PD-L1 inhibitors.

The disruption of the PD-1/PD-L1 interaction is a promising strategy for cancer immunotherapy. Reliable screening platforms are essential for evaluating ...

Cancer Research

Platelet-Derived Extracellular Vesicle Functionalization of Ti Implants

Extracellular Vesicles (EVs) are biological nanovesicles that play a key role in cell communication. Their content includes active biomolecules such as proteins and nucleic acids, which present great potential in regenerative medicine. More recently, EVs derived from Platelet Lysate (PL) have shown an osteogenic capability comparable to PL. Besides, biomaterials are frequently used in orthopedics or dental restoration. Here, we provide a method to functionalize Ti surfaces with PL-derived EVs in order to improve their osteogenic properties.
EVs are isolated from PL by size exclusion chromatography, and afterward Ti surfaces are functionalized with PL-EVs by drop casting. Functionalization is proven by EVs release and its biocompatibility by the lactate dehydrogenase (LDH) release assay.

Here, we present a method for the isolation of Extracellular Vesicles (EVs) derived from the platelet lysates (PL) and their use for coating titanium (Ti) implant surfaces. We describe the drop casting coating method, the EVs release profile from the surfaces, and in vitro biocompatibility of EVs coated Ti surfaces.

Extracellular Vesicles (EVs) are biological nanovesicles that play a key role in cell communication. Their content includes active biomolecules such as ...

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

Summary

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Chapters in this video

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

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

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Biomimetic Materials to Characterize Bacteria-host Interactions

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

Identifying PD-1/PD-L1 Inhibitors with Surface Plasmon Resonance Technology

Platelet-Derived Extracellular Vesicle Functionalization of Ti Implants