The authors thank Tyra Witt, Cheri Mueske, Brant Newman and Dr. Peter J. Psaltis, MBBS, PhD for their valuable contributions. This study was financially supported by European Regional Development Fund &#8211; FNUSA-ICRC (No. CZ.1.05/1.100/02.0123), American Heart Association Scientist Development Grant (AHA #06-35185N), National Institutes of Health (T32HL007111) and The Grainger Innovation Fund &#8211; Grainger Foundation.

All animal studies were approved by the Institutional Animal Care and Utilization Committee (IACUC) at Mayo Clinic.
1. Design and Analysis of a 2205 Stainless Steel Stent
<ol>
	<li>Designing a bare metal stent using CAD
	<ol>
		<li>Make an extruded hollow cylinder by selecting on &#8216;extruded boss/base&#8217; feature with the wall thickness equal to the stent strut thickness.</li>
		<li>Design a stent pattern on a different sketch plane tangential to the extruded cylinder. Make the width of the flat pattern to match the circumference of the extruded hollow cylinder.</li>
		<li>Transfer the flat pattern design onto the hollow cylinder using the wrap feature.</li>
		<li>Save the part in its native format and also in ACIS format to be exported for FEA.</li>
	</ol>
	</li>
	<li>Finite element analysis for stent models
	<ol>
		<li>Import the solid geometry saved in ACIS format into the part module of the FEA software for further analysis.</li>
		<li>Model 2 analytical cylinders coaxially to the stent in the part modeler of the FEA software. The outer cylinder has an initial diameter larger than the diameter of the stent to simulate the crimper and the inner cylinder has an initial diameter of 1 mm to simulate a balloon for inflation.</li>
		<li>Double click on the &#8216;instances&#8217; tree item of the assembly modeler to assemble the above said parts in relative positions.</li>
		<li>Use the mesh module of the FEA software, specify the element type as 20-node hexahedral element with reduced integration, specify the element size, and mesh the stent.</li>
		<li>Specify frictionless rigid contact pairs between the stent and the two cylinders respectively in the &#8216;interaction properties&#8217; of the model tree.</li>
		<li>Assign elasto-plastic stress-strain behavior of 2205 stainless steel to the stent model.</li>
		<li>Define boundary conditions to firstly crimp the outer cylinder to 1 mm which simulates the crimping of the stent. Remove the outer cylinder to simulate the relaxation of the crimped stent. Expand the inner cylinder to 3 mm to simulate expansion and finally, remove the inner cylinder to simulate recoil of the stent.</li>
		<li>Define the simulation parameters including the number of processors and amount of RAM allocated in the &#8216;Analysis&#8217; model tree item and run the simulation.</li>
		<li>Once the simulation is complete, open the result file (filename.odb) and post-process the results to study the principal strains and iteratively improve the stent design to achieve a principal strain of 20% which is less than the failure limit of the material.</li>
	</ol>
	</li>
</ol>
2. Stent Fabrication and Testing for Crimping and Expansion
<ol>
	<li>Stent fabrication
	<ol>
		<li>Obtain the 2205 stainless steel tubes by gun drilling and precision grinding bar stock material at a precision machining company such as Action Precision Products in Pioneer, OH.</li>
		<li>Transfer the precision ground tubes and the flat pattern stent design to a stent cutting company such as Laserage Technology Corporation in Waukegan, IL for laser cutting and electropolishing.</li>
		<li>Passivate the surface of the electropolished stents by submersing them in a strong acid (50% HCl) for 10 min followed by a base (10% NaHCO3) for another 10 min. CAUTION: handle chemicals with proper protective equipment and under a fume hood. Finally, wash the stents with ethyl alcohol and deionized water. This process is called acid pickling.</li>
	</ol>
	</li>
	<li>Testing of manufactured stent for crimping and expansion
	<ol>
		<li>Crimp the stent onto a trifold balloon using a hand held crimping tool. Hold the stent and the trifold balloon in the crimping tool. Press the handle to radially deform the stent to be crimped on the balloon.</li>
		<li>Inspect the crimped stent using a microscope for uniform crimping and any signs of failure in the structure due to plastic deformation.</li>
		<li>Expand it to the designed diameter of 3 mm by pressurizing the trifold balloon with water. Examine the expanded stents for microscopic fractures and uniform expansion.</li>
	</ol>
	</li>
</ol>
3. Characterization of Stent for Retained Magnetic Field
NOTE: Cylindrical magnet of 2 inch diameter and 1 inch height was used in this study. The poles of the magnet are aligned along the axis. The surface magnetic flux density of the magnet is approximately 1 T.
<ol>
	<li>Magnetize the stents diametrically or axially using a strong neodymium magnet.&#160; Hold the stent close to the strong magnet for approximately 1 min for magnetization.</li>
	<li>Hold the stent on one of the flat faces with its diameter along the magnetic field lines to be magnetized diametrically or hold the stent next to the cylindrical surface with its axis along the magnetic field lines to magnetize it axially. Retained magnetic field of the stent was found to be stable for at least 24 hr, but use the stent as soon as possible after magnetization.</li>
	<li>Mount the stents individually onto glass mandrels and then mount the glass mandrels in the precision chuck of the magnetic probing fixture. Magnetic microsensor probe can be precisely positioned close to the stent without touching the surface using the XYZ stages assembly of the magnetic probing fixture (Figure 4).</li>
	<li>Measure the baseline reading of the magnetic microsensor far away from the stent and then measure the retained magnetic field at the stent&#39;s surface by positioning the probe using the XYZ stages of the magnetic probing fixture.</li>
</ol>
4. Magnetic Cell Capture Studies
<ol>
	<li>Obtaining cells, labeling with SPION and staining with fluorescent dye
	<ol>
		<li>Derive the endothelial outgrowth cells (EOC) from porcine peripheral blood as described in5,7. Culture in a T-75 flask until approximately 80% confluent (5x106 to 8x106 cells).</li>
		<li>Synthesize SPIONs as 10 nm diameter magnetite (Fe3O4) core surrounded by 50 nm thick poly(lactic-co-glycolic acid) (PLGA) shell as described in 8,9.</li>
		<li>Incubate the derived EOC with SPION at a concentration of 200 &#181;g/ml of cell culture medium for 16 hr at 37 oC.</li>
		<li>Aspirate the cell culture medium gently. Gently wash the cells by adding 10 ml of phosphate-buffered saline (PBS) to the flask, rocking, and aspirating the PBS.</li>
		<li>Stain the cells with fluorescent dye (CM-DiI) for visualizing during experiments. This is done per the manufacturer&#8217;s instructions by adding the dye to 10 ml of cell culture medium at a concentration of 5 &#181;l/ml and incubating with the cells for 30 min at 37 &#176;C.</li>
		<li>Wash the cells with PBS as in step 4.1.4 and incubate with 3 ml of 0.25% trypsin-EDTA solution for 5 min at 37 &#176;C to lift the cells from the flask.</li>
		<li>Transfer the cell suspension to a 15 ml conical tube, top off with PBS, and centrifuge at 500 x g for 5 min to form a cell pellet.</li>
		<li>Re-suspend the cell pellet in PBS at a concentration of 1-2x106 cells/ml and mix thoroughly by pipetting in and out of the conical tube several times.</li>
	</ol>
	</li>
	<li>In-vitro cell studies
	<ol>
		<li>Design and fabricate (e.g., 3D printing) a simple fixture to hold the stent just above the surface of a glass coverslip.</li>
		<li>Demagnetize a stent using an electromagnetic degausser or magnetize a stent diametrically or axially using a strong neodymium magnet.</li>
		<li>Pipette the SPION-labeled EOC suspended in PBS into the dish containing the axially magnetized or diametrically magnetized or non-magnetized control stents. Image the stents with EOC suspended in PBS immediately for fluorescence using an inverted fluorescence microscope.</li>
	</ol>
	</li>
</ol>
5. In-vivo Animal Studies
<ol>
	<li>Stent implantation
	<ol>
		<li>Draw peripheral blood from 4 healthy Yorkshire pigs &#8211; weighing approximately 50 kg &#8211; 3 weeks prior to stent implantation respectively and culture EOC as described in 5,7.</li>
		<li>Administer anti-platelet medication starting 3 days prior to surgery (aspirin 325 mg and clopidogrel 75 mg daily).</li>
		<li>On the stent implantation day, anesthetize the pigs with intramuscular Telazol, Xylazine, and Atropine (5/2-3/0.05 mg/kg respectively) as stated in the applicable institutional animal care and use guidelines.</li>
		<li>Intubate and place the pig on inhalation of 1-2.5% Isoflurane anesthesia.</li>
		<li>Shave the ventral neck region of the pig and conduct the procedure in general sterile conditions.</li>
		<li>Implant 1 magnetized and 1 non-magnetized stent into the right coronary artery (RCA) using standard cardiac catheterization technique.
		<ol>
			<li>Catheterization of animals should be performed by a trained interventional cardiologist. Access the right carotid artery with a 9 French sheath.</li>
			<li>Cannulate the target coronary artery and inject iodinated contrast dye to obtain fluoroscopic images.</li>
			<li>Place a 0.014 inch standard coronary guidewire in the artery. Advance the balloon and stent using this guide wire and deploy the stent in a 3-3.5 mm diameter vessel.</li>
		</ol>
		</li>
		<li>Occlude the blood flow within the RCA proximal to the implanted stents using an over the wire balloon and deliver approximately 2x106 autologous EOC labeled with SPION suspended in 4 ml of PBS via the central catheter over a 2 min period.</li>
		<li>Restore blood flow to the RCA after 2 min of additional occlusion.</li>
		<li>Transfer the animal to the recovery room and closely monitor the animal until it has regained consciousness.</li>
		<li>Continue to administer anti-platelet medication (aspirin 325 mg and clopidogrel 75 mg) post operatively until sacrifice.</li>
	</ol>
	</li>
	<li>Stent explant and histology
	<ol>
		<li>Euthanize the animals 7 days after surgery by first anesthetizing the animal as explained previously and then administer intravenously a lethal dose of sodium pentobarbital (100 mg/kg) as per applicable institutional animal care and use guidelines.</li>
		<li>Surgically harvest the stented arterial segments. Fix the explanted arteries in 10% formalin buffer for a minimum of 30 min. Leave the samples in formalin buffer for further histological analysis.</li>
		<li>Outsource the fixed sample to facilities capable of performing histology with metal stents. During this processing, the samples are embed in methylmethacrylate, cross-sectioned, and analyzed histologically using Mallory&#8217;s staining technique with Prussian blue stain for iron particles.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

We developed a magnetic stent which can function as a bare metal stent and can attract SPION-labeled endothelial cells. In previous studies involving magnetic stents, researchers have used nickel coated commercial stents and coils or meshes made of magnetic materials due to the unavailability of a ferromagnetic stent5,10-14. Other groups have also used the paramagnetic nature of commercially available 304-grade stainless steel stents for targeting nanoparticle loaded endothelial cells3. Nickel coati...

Patients implanted with vascular stents manufactured from thrombogenic materials like stainless steel, cobalt chromium, and platinum chromium &#8211; both bare metal stents (BMS) and drug eluting stents (DES) &#8211; need anti-platelet therapy to prevent thrombus formation. BMS heal rapidly, but are subject to late stage restenosis due to incomplete healing. DES require long term anti-platelet therapy due to delayed healing. Anti-platelet therapy administered to avoid thrombosis as a result of incomplete or delayed healing leads to increased bleeding risk and may not be suitable for certain patients1,2. An ideal stent will heal completely and quickly thus avoiding long-term anti-platelet therapy and late stage restenosis. This complete healing can only be achieved if the stent is rapidly coated with a monolayer of endothelial cells after implantation. Coating the stents with biocompatible materials such as gold or other biopolymers has been shown to improve thrombo-resistance, but none of these techniques achieved ideal blood compatibility as may be possible by coating with endothelial cells3,4.
A stent can be coated with endothelial cells post implantation by attracting circulating progenitor cells. This self-seeding technique can be achieved by utilizing ligands and antibodies. But this technique is limited by the low number of circulating endothelial progenitor cells. A promising strategy is to deliver cells directly to the stent immediately following implantation during a short period of blood flow occlusion3,5. This strategy requires a technique for rapidly capturing cells and retaining them on the stent even after restoring blood flow. We have developed a technique in which a magnetic stent is used to attract and retain magnetically-labeled endothelial cells delivered post implantation. To achieve this, a functional BMS with sufficient magnetic properties to capture and retain magnetically-labeled endothelial cells is required6.
In this paper, we discuss the methods for designing, manufacturing, and testing a 2205 stainless steel stent. The stents were designed using CAD and FEA. The manufactured stents were magnetized using a neodymium magnet and the retained magnetic field was measured using a magneto-resistance microsensor probe. We then tested the stents for magnetically-labeled cell capture in a culture dish during our in-vitro experiments. Finally, the stents were tested in-vivo by implanting magnetic and non-magnetic stents in 4 pigs and histologically analyzing the stented arteries.

<table><tbody><tr><td>2205 Stainless steel</td><td>Carpenter Technology Corporation</td><td></td><td>Round bar stock material</td></tr><tr><td>Abaqus</td><td>Dassault systems</td><td></td><td>Software</td></tr><tr><td>Atropine</td><td></td><td></td><td>Prescription drug.</td></tr><tr><td>Clopidogrel</td><td></td><td></td><td>Commercial name: Plavix. Prescription drug.</td></tr><tr><td>CM-DiI</td><td>Life Technologies</td><td>V-22888</td><td>Molecular Probes, Eugene, OR</td></tr><tr><td>Endothelial growth medium-2</td><td>Lonza</td><td>CC-3162</td><td></td></tr><tr><td>Hand Held Crimping tool</td><td>Blockwise engineering</td><td>M1-RMC</td><td></td></tr><tr><td>Hydrochloric acid (HCl)</td><td>Sigma Aldrich</td><td>MFCD00011324</td><td>CAUTION: wear proptective equipment and handle under fume hood</td></tr><tr><td>Isoflurane anesthesia</td><td>Piramal Critical Care, Inc.&amp;nbsp;</td><td></td><td></td></tr><tr><td>Ethyl alcohol</td><td>Sigma Aldrich</td><td>MFCD00003568</td><td></td></tr><tr><td>NdFeB magnet 2&quot; Dia x 1&quot; thick</td><td>Amazing magnets</td><td>D1000P</td><td>Axially magnetized disc magnet with poles on flat faces</td></tr><tr><td>Over-The-Wire trifold balloon</td><td></td><td></td><td>Any commercially available OTW trifold balloon can be used</td></tr><tr><td>Phosphate buffered saline</td><td>Life Technologies</td><td>10010-023</td><td>Commonly known as PBS</td></tr><tr><td>Sodium Bicarbonate (NaHCO3)</td><td>Sigma Aldrich</td><td>MFCD00003528</td><td></td></tr><tr><td>Sodium pentobarbital</td><td>Zoetis</td><td></td><td>Commercial Name: Sleepaway (26%), FatalPlus, Beuthanasi.&amp;nbsp; Controlled substance to be ordered only by licensed veternarian</td></tr><tr><td>SolidWorks</td><td>Dassault systems</td><td></td><td>Software</td></tr><tr><td>SpinTJ-020 micro sensor</td><td>MicroMagneitcs Sensible Solutions</td><td></td><td>Long probe STJ-020 microsensor</td></tr><tr><td>SPION</td><td>Mayo Clinic</td><td></td><td>Nanoparticles synthesized internally (Ref: Lee, S. J. et al. Nanoparticles of magnetic ferric oxides encapsulated with poly(D,L latide-co-glycolide) and their applications to magnetic resonance imaging contrast agent. J Magn Magn Mater 272, 2432-2433, doi:DOI 10.1016/j.jmmm.2003.12.416 (2004))</td></tr><tr><td>Telazol</td><td>Zoetis</td><td></td><td>Controlled substance to be ordered only by licensed veternarian</td></tr><tr><td>Trypsin EDTA</td><td>Life Technologies</td><td>25200-056</td><td>Gibco, Grand Island, NY</td></tr><tr><td>Xylazine</td><td>Bayer Animal Health</td><td></td><td>Commercial name: Rompun. Controlled sunstance to be ordered only by a licensed veternarian</td></tr></tbody></table>

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.

Iterative stent design based on FEA (Figure 1) showed a stent which can crimp and expand with a principal strain of 20% which is less than the 30% ultimate strain. Crimping and expansion test (Figure 2) showed no signs of fracture. Pictures of the deformed stent showed good agreement with FEA calculated deformations and also microscopy pictures showed no fractures (Figure 3). As expected from the retained magnetic field measurements (Figures 4 &#38; 5), ...

Watch this Scientific Journal Video about Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention at JoVE.com

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

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. ...

ferromagnetic-bare-metal-stent-for-endothelial-cell-capture

Research

JoVE Journal

Bioengineering

11.5K Views.  Mayo Clinic.  Onze doelen waren voor het ontwerpen, produceren en testen ferromagnetische stents voor endotheelcellen capture. Ten stents werden getest op breuk en 10 meer stents werden getest behouden magnetisme. Tenslotte werden 10 stents getest in-vitro en 8 meer stents werden geïmplanteerd in 4 varkens cell capture en retentie vertonen. 

Video: Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes.
A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo.
Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an &ldquo;off label&rdquo; use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

Medicine

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Fabrication of Small Caliber Stent-grafts Using Electrospinning and Balloon Expandable Bare Metal Stents

Stent-grafts are widely used for the treatment of various conditions such as aortic lesions, aneurysms, emboli due to coronary intervention procedures and perforations in vasculature. Such stent-grafts are manufactured by covering a stent with a polymer membrane. An ideal stent-graft should have a biocompatible stent covered by a porous, thromboresistant, and biocompatible polymer membrane which mimics the extracellular matrix thereby promoting injury site healing. The goal of this protocol is to manufacture a small caliber stent-graft by encapsulating a balloon expandable stent within two layers of electrospun polyurethane nanofibers. Electrospinning of polyurethane has been shown to assist in healing by mimicking native extracellular matrix, thereby promoting endothelialization. Electrospinning polyurethane nanofibers on a slowly rotating mandrel enabled us to precisely control the thickness of the nanofibrous membrane, which is essential to achieve a small caliber balloon expandable stent-graft. Mechanical validation by crimping and expansion of the stent-graft has shown that the nanofibrous polyurethane membrane is sufficiently flexible to crimp and expand while staying patent without showing any signs of tearing or delamination. Furthermore, stent-grafts fabricated using the methods described here are capable of being implanted using a coronary intervention procedure using standard size guide catheters.

In the protocol, we present a method to manufacture a small caliber stent-graft by sandwiching a balloon expandable stent between two electrospun nanofibrous polyurethane layers.

Stent-grafts are widely used for the treatment of various conditions such as aortic lesions, aneurysms, emboli due to coronary intervention procedures and ...

Biodegradable Magnesium Stent Treatment of Saccular Aneurysms in a Rat Model - Introduction of the Surgical Technique

The steady progess in the armamentarium of techniques available for endovascular treatment of intracranial aneurysms requires affordable and reproducable experimental animal models to test novel embolization materials such as stents and flow diverters. The aim of the present project was to design a safe, fast, and standardized surgical technique for stent assisted embolization of saccular aneurysms in a rat animal model.
Saccular aneurysms were created from an arterial graft from the descending aorta.The aneurysms were microsurgically transplanted through end-to-side anastomosis to the infrarenal abdominal aorta of a syngenic male Wistar rat weighing &#62;500 g. Following aneurysm anastomosis, aneurysm embolization was performed using balloon expandable magnesium stents (2.5 mm x 6 mm). The stent system was retrograde introduced from the lower abdominal aorta using a modified Seldinger technique.
Following a pilot series of 6 animals, a total of 67 rats were operated according to established standard operating procedures. Mean surgery time, mean anastomosis time, and mean suturing time of the artery puncture site were 167 &#177; 22 min, 26 &#177; 6 min and 11 &#177; 5 min, respectively. The mortality rate was 6% (n=4). The morbidity rate was 7.5% (n=5), and in-stent thrombosis was found in 4 cases (n=2 early, n=2 late in stent thrombosis).
The results demonstrate the feasibility of standardized stent occlusion of saccular sidewall aneurysms in rats - with low rates of morbidity and mortality. This stent embolization procedure combines the opportunity to study novel concepts of stent or flow diverter based devices as well as the molecular aspects of healing.

Reproducable experimental animal models are needed for the testing of novel embolization materials, which have been designed to treat endovascular occlusion of intracranial aneurysms (IA). The present study aims to develop a safe and standardized surgical technique for stent assisted embolization of saccular aneurysms in a rat animal model.

The steady progess in the armamentarium of techniques available for endovascular treatment of intracranial aneurysms requires affordable and reproducable ...

Neuroscience

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.

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, ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

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