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>
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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>
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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, ...

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.

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

Research

JoVE Journal

Bioengineering

Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To this end, our laboratory has applied the principles of nanomedicine to simplify ART regimens and as such reduce toxicities while improving compliance and drug pharmacokinetics. Simple and reliable methods for manufacturing nanoformulated ART (nanoART) are shown. Particles of pure drug are encapsulated by a thin layer of surfactant lipid coating and produced by fractionating larger drug crystals into smaller ones by either wet milling or high-pressure homogenization. In an alternative method free drug is suspended in a droplet of a polymer. Herein, drug is dissolved within a polymer then agitated by ultrasonication until individual nanosized droplets are formed. Dynamic light scattering and microscopic examination characterize the physical properties of the particles (particle size, charge and shape). Their biologic properties (cell uptake and retention, cytotoxicity and antiretroviral efficacy) are determined with human monocyte-derived macrophages (MDM). MDM are derived from human peripheral blood monocytes isolated from leukopacks using centrifugal elutriation for purification. Such blood-borne macrophages may be used as cellular transporters for nanoART distribution to human immunodeficiency virus (HIV) infected organs. We posit that the repackaging of clinically available antiretroviral medications into nanoparticles for HIV-1 treatments may improve compliance and positively affect disease outcomes.

Nanoparticles of indinavir, ritonavir, efavirenz and atazanavir were manufactured using wet milling, homogenization and ultrasonication. These nanoformulations, collectively termed nanoformulated antiretroviral therapy (nanoART), assessed macrophage-based drug delivery. Monocyte-derived macrophage nanoART uptake, retention and sustained release were determined. These preliminary studies suggest the potential of nanoART for clinical use.

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To ...

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 ...

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &amp;#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

Graphene Coatings for Biomedical Implants

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with nanometer thick layers of graphene for applications as a stent material. Graphene was grown on copper substrates via chemical vapor deposition and then transferred onto nitinol substrates. In order to understand how the graphene coating could change biological response, cell viability of rat aortic endothelial cells and rat aortic smooth muscle cells was investigated. Moreover, the effect of graphene-coatings on cell adhesion and morphology was examined with fluorescent confocal microscopy. Cells were stained for actin and nuclei, and there were noticeable differences between pristine nitinol samples compared to graphene-coated samples. Total actin expression from rat aortic smooth muscle cells was found using western blot. Protein adsorption characteristics, an indicator for potential thrombogenicity, were determined for serum albumin and fibrinogen with gel electrophoresis. Moreover, the transfer of charge from fibrinogen to substrate was deduced using Raman spectroscopy. It was found that graphene coating on nitinol substrates met the functional requirements for a stent material and improved the biological response compared to uncoated nitinol. Thus, graphene-coated nitinol is a viable candidate for a stent material.

Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with ...

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing.

Our goals were to design, manufacture and test ferromagnetic stents for endothelial cell capture. Ten stents were tested for fracture and 10 more stents were tested for retained magnetism. Finally, 10 stents were tested in-vitro and 8 more stents were implanted in 4 pigs to show cell capture and retention.

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

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 ...

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 ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...