Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

The overall goal of the following experiment is to develop a ferromagnetic bare metal stent capable of capturing and retaining magnetic nanoparticle labeled endothelial cells. This is achieved by designing a 2205 stainless steel stent using computer aided design or CAD and finite element analysis or FEA softwares to properly understand the mechanical behavior of the stent material and structure during crimping and expansion. Next, the fabricated stent is mechanically crimped and expanded to demonstrate the mechanical integrity and the magnetic field retention of the stent is studied, which validates the magnetic behavior.

Then the stents are implanted in pigs and magnetic nanoparticle labeled endothelial cells are delivered in order to verify cell capture and retention in the coronary circulation. The results show that the ferromagnetic stent developed in this study is able to capture and retain magnetically labeled endothelial cells based on histological analysis of explanted blood vessels from in vivo experiments. The implications of this technique extend toward development of various cardiovascular devices because rapid endothelialization can promote healing and improve blood compatibility.

Generally, individuals new to this method will struggle because of the complexity of the CAD and FEA procedures involved in stent designing cell culture can also be a challenge for beginners. Due to the importance of maintaining a strict aseptic environment, We wanted to improve the healing of stents and came up with the idea of using magnetic forces to attach living cells to stents. Visual demonstration of this method is critical as a stent development and cell heating steps are difficult to learn because of the complexity of the material behavior during crimping and expansion and the novelty of the magnetic cell capture of the stent.

To begin, use CAD software to make an extruded hollow cylinder by selecting an extruded boss base feature with the wall thickness equal to the stent strut thickness. 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.

Using the wrap feature, transfer the flat pattern design onto the hollow cylinder. Save the part in its native format and also in ACEs format to be exported for FEA To carry out finite element analysis, import the solid geometry saved in ACEs format into the part module of the FEA software for further analysis in the part modeler of the software model Two analytical cylinders coaxially to the stent. Double click on the instances tree item of the assembly modeler to assemble the parts into relative positions with a mesh module of the FEA software.

Specify the element, type as 20 node hexed element with reduced integration. Specify the element size and mesh the stent within the interaction properties of the model tree. Specify frictionless rigid contact pairs between the stent and the two cylinders respectively.

Assign ELAs opl stress strain behavior of 2205 stainless steel to the stent model. Define boundary conditions to firstly crimp the outer cylinder to one millimeter, which simulates the crimping. Remove the outer cylinder to simulate the relaxation of the crimped stent.

Expand the inner cylinder to three millimeters to simulate expansion, and finally remove the inner cylinder to simulate recoil of the stent. Once the simulation is complete, open the result, file and post process the results to study the principle strains and iteratively. Improve the stent design to achieve a principle strain of 20%which is less than the failure limit of the material.

After obtaining the 2205 stainless steel tubes and cutting the stents according to the text protocol under a fume hood, using proper protective equipment acid pickle the stent for passivating the surface of the electro polished stents by submersing them in 50%hydrochloric acid for 10 minutes, followed by 10%sodium bicarbonate for another 10 minutes to complete acid pickling. Use ethanol followed by deionized water to wash the stents. Then to test the stents for crimping, hold a stent and a trifold balloon.

In a handheld crimping tool, press the handle to deform the stent to be crimped on the balloon. After inspecting the crimped stent under the microscope for uniform crimping and any signs of failure, expand the stent to the design diameter of three millimeters by using water to pressurize the trifold balloon. Examine the expanded stents for microscopic fractures and uniform expansion using a strong neodymium magnet.

Magnetize the stents diametrically by holding the stent on one of the flat faces with its diameter along the magnetic field lines. Alternatively, magnetize the stents axially by holding the stent next to the cylindrical surface with its axis along the magnetic field lines. Hold the stent close to the strong magnet for approximately one minute.

For magnetization, mount the stents individually onto glass mandrels and then mount the glass mandrels into the precision chuck of the magnetic probing fixture. With the X, Y, Z stages, assembly of the magnetic probing fixture precisely position the magnetic microsensor probe close to the stent without touching the surface. Measure the baseline reading of the magnetic microsensor far away from the stent, and then use the x, y, Z stages to position the probe and measure the retained magnetic field at the stem surface.

After deriving endothelial outgrowth cells or EOCs from porcine peripheral blood as described in the text protocol culture, the cells in a T 75 flask until they are approximately 80%con fluent. Following the synthesizing of super paramagnetic iron oxide nanoparticles or pyon, add pyon to the EOCs in cell culture medium at a concentration of 200 micrograms per milliliter. Incubate a 37 degree Celsius for 16 hours.

Gently aspirate the cell culture medium and add 10 milliliters of PBS to the flask. Then gently rock and aspirate. The PBS Add five micrograms per milliliter of fluorescent dye such as CM DAI to 10 milliliters of culture medium to stain the cells per the manufacturer's instructions and incubated 37 degrees Celsius for 30 minutes.

Use PBS to gently wash the cells as before then add 0.25%tripsin EDTA solution and incubate a 37 degree Celsius for five minutes to lift the cells from the flask. Transfer the cell suspension to a 15 milliliter conical tube. Use PBS to top it off and centrifuge at 500 G for five minutes to pellet the cells after decanting.

Use PBS to resuspend the cells at a concentration of one to 2 million cells per milliliter. For in vitro cell studies design and fabricate a simple fixture to hold the stent just above the surface of a glass cover slip. Use an electromagnetic dauer to demagnetize a stent or with a strong neodymium magnet, magnetize a stent diametrically or axially pipette the spy on labeled e ooc suspended in PBS into the dish containing the axially magnetized or diametrically magnetized or non magnetized control stents.

Then with an inverted fluorescence microscope image, the stents with EOC suspended in PBS to carry out stent implantation after administering anti-platelet medication to four healthy Yorkshire pigs and anesthetizing according to the text protocol, use a standard cardiac catheterization technique to implant one magnetized and one non magnetized stent into the right coronary artery or RCA After using a guidewire to advance the balloon and stent with an over the wire balloon, occlude the blood flow within the RCA proximal to the implanted stents. Then over a period of two minutes, deliver approximately 2 million autologous EOCs labeled with s scions suspended in four milliliters of PBS via the central catheter, following an additional two minutes of occlusion, restore blood flow to the RCA, harvest the stent, and carry out histology. According to the text protocol as shown here, iterative stent design based on FEA indicated a stent which can crimp and expand with a principle strain of 20%which is less than the 30%ultimate strain.

A laser cut, an electro polished stent crimped onto a trifle balloon catheter and expanded to three millimeters using a trifle balloon showed no signs of fracture. Pictures of the deformed stent showed good agreement with FEA calculated deformation and also microscopy pictures showed no fractures as expected from the retained magnetic field measurements. SPY on labeled cells were preferentially attracted to Ben segments in axially magnetized stents and more uniformly attracted to straight segments.

In diametrically magnetized stents, histology images showed iron staining near the stent struts confirming EOC attraction and retention to the stent during the seven day implantation period. While attempting this procedure, it's important to understand the concepts of FEA and CAD with improper input parameters. One would obtain erroneous stent design, which is either too stiff or with a propensity for stent fracture.

Following this procedure. Other methods like accelerated fatigue testing and toxicity testing can be performed in order to answer additional questions like What does the long-term durability and biocompatibility of the stents? After several years of development, we now have a functional technology that has been validated in a porcine model.

After watching this video, you should have a good understanding of how to develop a ferromagnetic stent and test for magnetically labeled and arterial cell capture and retention.