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

This protocol includes a novel chemical strategy to append therapeutic peptides to metallic surfaces, and has the potential to improve the biocompatibility of a wide range of metallic biomaterials. This is a universal and robust technique to append peptides and therapeutic proteins to the bare metal surfaces of bioimplants. We have used this technology to improve the biocompatibility of metallic stents by appending CD47 peptides in order to prevent the inflammation responses that are triggered by stenting.

Begin by washing the stainless steel foil coupons or mesh discs with 2-isopropanol in a shaker for five minutes. Perform this wash twice, then wash the samples twice with chloroform for 10 minutes per wash. Place the cleansed stainless steel samples in an oven at 220 degrees Celsius for 30 minutes.

Immerse the baked foils or mesh discs in 0.5%aqueous solution of PABT and incubate them in a shaker for one hour. Wash the PABT-modified samples with deionized distilled water five times. Transfer the specimens to a new vial and repeat the washes.

Treat the samples with TCEP for 15 minutes at room temperature on a shaker. Degas deionized distilled water in a round bottom flask with a vacuum generating device, such as a lyophilizer, and wash the foils or mesh discs with the degassed water five times. Transfer the samples to a new vial and repeat the washes.

Prepare five milliliters of 1%PEI-PDT solution by diluting 212.5 microliters of the stock PEI-PDT and 125 microliters of 0.4 molar sodium acetate in the degassed water. Incubate the washed stainless steel specimens with 1%PEI-PDT. Replace air with argon gas, seal the vials airtight, and mix them on a shaker for one hour.

Proceed immediately with the peptide conjugation or store the samples at four degrees Celsius for up to one week. Prepare one milligram per milliliter human peptide CD47 stock solution by dissolving human peptide CD47 powder in degassed 50%acetic acid, then prepare the working solution by dissolving 500 microliters of the stock in 4.5 milliliters of degassed PBS. Incubate the washed PEI-PDT coated samples with the working solution of peptide CD47 at room temperature with shaking for one hour.

When finished, wash the peptide CD47-coated samples as described in the text manuscript. After coating the metal foils with either peptide CD47 or scrambled peptide, cut quarter inch PVC tubes into three 38 centimeter long pieces and insert up to eight unmodified, scrambled peptide, or peptide CD47-modified metal foils into three different tubes. Collect 30 milliliters of blood from healthy human donors free of any anti-platelet medications, pre-loading the syringe with one milliliter of 4%sodium citrate to prevent coagulation of the collected blood.

Put 10 milliliters of blood into each tube using a 10 milliliter syringe and connect the ends with metal adapters. Place the blood-filled tubes on the wheels of the Chandler loop apparatus and pass the blood along the metal foils by wheel rotation at 37 degrees Celsius for four hours. Drain the blood from the tubes and dispose of it according to IRB requirements, then cut the tubes with a scalpel to retrieve the foils.

Prepare 2%glutaraldehyde solution by diluting the 70%solution in sodium cacodylate buffer with 0.1 molar sodium chloride. Incubate the foils in the 2%glutaraldehyde solution for 15 minutes and store them at four degrees Celsius overnight. Before analyzing the metal foils, wash them three times with PBS.

Warm eight milliliters of PBS in a 37 degrees Celsius water bath. Prepare 10 millimolar CFDA dye stock solution by adding 90 microliters of DMSO to one CFDA vial, then prepare the working solution by adding 75 microliters of the stock CFDA to the warm PBS. Invert the tube a few times to mix and cover it with aluminum foil.

Place each foil into one milliliter of CFDA dye in a 24-well plate. Cover the plate with aluminum foil and incubate the plate at 37 degrees Celsius for 15 minutes, then wash the foils three times in PBS and image them with a fluorescence microscope. The concentration of covalently-bound TAMRA conjugated peptide CD47 was quantified to determine maximal immobilization density of the peptide on the metal surface.

The maximum peptide retention was approximately 180 nanograms per centimeter squared, which was achieved with an input concentration of 100 micrograms per milliliter. Immobilization of TAMRA conjugated peptide CD47 on the modified metal surfaces was further corroborated by fluorescence microscopy, which showed a uniform fluorescence emitted from the surface of the TAMRA conjugated peptide CD47-treated mesh discs. Only minimal fluorescence was detected on the surface of the control meshes that lacked the modification, thereby excluding a non-specifically-bound TAMRA conjugated peptide CD47 as the main source of fluorescence.

It was also confirmed that the peptide CD47-coated surfaces show a drastic reduction in blood-borne cell attachment compared to the scrambled modified and unmodified peptide controls. Monocytes isolated from rat buffy coat cells were cultured on bare metal and peptide CD47-modified stainless steel foils. A 58%attenuation of acute monocyte attachment, their conversion to macrophages, and macrophage proliferation was observed on the functionalized surfaces.

While attempting this protocol, it is very important to perform the steps involving degassed water or reagents to be performed as quickly as possible and to minimize the exposure to oxygen. Our technology selectively modulates the cellular interaction with metal surfaces and a typical cell type identification assay, such as RT-PCR, flow cytometry, and immunofluorescence, are compatible with this technique. When developed for a range of bioactive peptide, this technology can be instrumental in increasing the biocompatibility at the tissue material interface.