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. 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.
Patients implanted with vascular stents manufactured from thrombogenic materials like stainless steel, cobalt chromium, and platinum chromium – both bare metal stents (BMS) and drug eluting stents (DES) – 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.
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
2. Stent Fabrication and Testing for Crimping and Expansion
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.
4. Magnetic Cell Capture Studies
5. In-vivo Animal Studies
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 & 5), SPION-labeled cells were preferentially attracted to bent segments in axially magnetized stents and more uniformly attracted to straight segments in diametrically magnetized stents (Figure 6). Histology images showed iron staining near the stent struts proving EOC attraction and retention to the stent during the 7 day implantation period (Figure 7).
Figure 1. Stent modeling and analysis flow chart. The schematic shows the computer aided modeling and finite element analysis showing a step-by-step process applied to a 2205 stainless steel stent. Modified from Uthamaraj et al. 20146 with re-print permission.
Figure 2. Stainless steel stent crimping and expansion. Laser cut and electropolished stent a) as-cut, b) crimped onto a trifold balloon catheter, and c) expanded to 3 mm using the trifold balloon. Modified from Uthamaraj et al. 20146 with re-print permission.
Figure 3. Microscopic inspection of stent. Light microscopy was used to image the expanded stent which was compared to FEA simulation.
Figure 4. Magnetic probe measurement stage setup. The XYZ stages and rotational stages were assembled for positioning the stents and magnetic probe during magnetic field measurements.
Figure 5. Magnetic field measurement regions on a stent and the measurement values. The image shows the measured retained magnetic fields of 2205 stents in axially magnetized and diametrically magnetized configurations. Modified from Uthamaraj et al. 20146 with re-print permission.
Figure 6. In-vitro cell capture studies. Fluorescence microscopy images of 2205 stainless steel stents showing cell capture in (A) non-magnetized stent, (B) diametrically magnetized stent and (C) axially magnetized stent. Modified from Uthamaraj et al. 20146 with re-print permission.
Figure 7. Images of histological cross sections of stented coronary artery segments of (A) magnetic stent with blue iron staining near the strut and (B) non-magnetic control stent showing no blue staining near the strut. The samples were stained using Mallory’s staining technique with iron particles stained with Prussian blue stain. The “*” symbol indicates stent strut locations. Modified from Uthamaraj et al. 20146 with re-print permission.
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 coatings may be allergenic to the patients receiving the stents and the paramagnetic stents need an external magnetic field to attract and retain magnetic nanoparticles3,5. Hence, designing and developing a functional ferromagnetic stent is important for cell delivery applications as well as other clinical applications10,15-20. The duplex nature of the material chosen for this study – 2205 stainless steel – makes it weakly ferromagnetic. In addition, 2205 stainless steel has a lower ultimate strain of 30% when compared to other stainless steels used to make stents such as 316L stainless steel (70%)6,21,22.
Based on this novel application of 2205 stainless steel, the protocol presented in this study explains the methods to design, manufacture, and test a weakly magnetic stent. First, a simple stent design pattern was developed using an existing stent pattern as a guide. Results from the FEA simulations suggested that material needed to be added to the bent segments of the stent to achieve a maximum principal strain of 20% which is less than the ultimate strain of the material. The final stent design had a strut thickness of 90 µm. Second, the manufactured stents were magnetized and their retained magnetic fields were measured. The retained magnetic field strength of the 2205 stainless steel stent depends upon the orientation of the applied magnetic field6. Axially magnetized stents showed preferential cell capture at the bent segments of the stent while diametrically magnetized stents showed a more uniform cell capture along the struts. This was also confirmed by the magnetic measurement experiments conducted on the stent23. Magnetized stents showed a retained magnetic field in the range of 100-750 mG compared to a maximum of 10 mG for control, non-magnetized stents. Finally, the large animal implantation studies showed that the BMS manufactured from 2205 stainless steel can be used to attract and retain SPION-labeled endothelial cells even when blood flow is restored post-implantation. Histology showed the presence of blue iron staining near the stent struts of the magnetized stent, thus proving cell capture and retention after 7 days of implantation.
CAD and FEA used in our study can be applied for proper design and analysis of similar balloon expandable stents. In the current protocol, steps 1.2.5, 1.2.6, and 1.2.7 are critical for setting up the boundary conditions and material property assignment and are required for properly designing a stent. Resulting magnetized 2205 stainless steel stents implanted in large animals showed cell capture and retention. Steps 5.1.7 and 5.1.8 are also critical to achieve proper cell seeding on magnetized stents. In addition, the introduction of cells to the magnetic stent implant site during a 2 min occlusion is unique to our presented study.
The stents developed in the current study were able to rapidly endothelialize and withstand short term implantation, but it is unclear if the stents can withstand long-term implantation. To date, ferromagnetic materials have not been extensively studied to understand their limitations for clinical applications. However, our 7 day pig implantation data showed that 2205 stainless steel had good blood and tissue compatibility. The methods presented in this study do not address the techniques for advanced mechanical testing of the stents such as fatigue testing or long-term interaction of the magnetic material with the blood24-28. In addition, the weak ferromagnetic nature of 2205 stainless steel was able to capture magnetically-labeled cells, but a novel material with stronger magnetic properties may improve cell capture. Further research is also needed to study the biocompatibility and long term safety of ferromagnetic materials. The endothelial outgrowth cells used in this study were obtained by following a previously published protocol which showed how to isolate and characterize the endothelial outgrowth cells5,7. The current study was also limited by the small number of animals.
In summary, rapid endothelialization of stents has been limited to date because of the unavailability of optimal delivery devices and poor adhesion of endothelial cells. The ferromagnetic stents developed in this study have the advantage of functioning as a BMS while also providing enough retained magnetic field to capture magnetically-labeled endothelial cells. As a part of our continuing studies of long term implantation effects, the stents need to undergo more rigorous mechanical and biocompatible testing. The stent developed in this study shows great promise as a functional ferromagnetic stent capable of endothelial cell capture and retention and the methods presented in this study can be used for future stent development and testing.
The authors have nothing to disclose.
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 – 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 – Grainger Foundation.
2205 Stainless steel | Carpenter Technology Corporation | N/A | Round bar stock material |
Abaqus | Dassault systems | N/A | Software |
Atropine | Prescription drug. | ||
Clopidogrel | Commercial name: Plavix. Prescription drug. | ||
CM-DiI | Life Technologies | V-22888 | Molecular Probes, Eugene, OR |
Endothelial growth medium-2 | Lonza | CC-3162 | |
Hand Held Crimping tool | Blockwise engineering | M1-RMC | |
Hydrochloric acid (HCl) | Sigma Aldrich | MFCD00011324 | CAUTION: wear proptective equipment and handle under fume hood |
Isoflurane anesthesia | Piramal Critical Care, Inc. | ||
Isopropyl alcohol | Sigma Aldrich | MFCD00011674 | |
NdFeB magnet 2" Dia x 1" thick | Amazing magnets | D1000P | Axially magnetized disc magnet with poles on flat faces |
Over-The-Wire trifold balloon | N/A | N/A | Any commercially available OTW trifold balloon can be used |
Phosphate buffered saline | Life Technologies | 10010-023 | Commonly known as PBS |
Sodium Bicarbonate (NaHCO3) | Sigma Aldrich | MFCD00003528 | |
Sodium pentobarbital | Zoetis | Commercial Name: Sleepaway (26%), FatalPlus, Beuthanasi. Controlled substance to be ordered only by licensed veternarian | |
SolidWorks | Dassault systems | N/A | Software |
SpinTJ-020 micro sensor | MicroMagneitcs Sensible Solutions | N/A | Long probe STJ-020 microsensor |
SPION | Mayo Clinic | N/A | 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)) |
Telazol | Zoetis | Controlled substance to be ordered only by licensed veternarian | |
Trypsin EDTA | Life Technologies | 25200-056 | Gibco, Grand Island, NY |
Xylazine | Bayer Animal Health | Commercial name: Rompun. Controlled sunstance to be ordered only by a licensed veternarian |