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

Summary

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.

Abstract

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.

Introduction

Coronary intervention procedures cause significant vessel wall injury due to disruption of the plaque and vessel wall. This results in restenosis, peripheral embolism in vein grafts, and discontinuity of coronary lumen^1-4. To avoid these complications, a promising strategy will be to cover the vascular surface in the angioplasty site, which will potentially inhibit restenosis, mitigate risks from discontinuity of vessel lumen, and prevent peripheral embolism. Previous studies have compared bare metal stents to stent-grafts with positive outcomes for stent-grafts⁵. Researchers have used several materials to manufacture membranes to cover the stents. This includes synthetic materials like polyethylene tetraphthalate (PET), polytetrafluoroethylene (PTFE), polyurethane (PU), and silicon or autologous vessel tissue to manufacture covered stents^6-9. An ideal graft material used to cover the stent should be thromboresistant, non-biodegradable, and should integrate with native tissue without excessive proliferation and inflammation¹⁰. The graft material used to cover the stent should also promote healing of the stent-graft.

Stent-grafts are widely used for the treatment of aortic coarctation, pseudo-aneurysms of the carotid artery, arteriovenous fistulae, degenerated vein grafts, and large to giant cerebral aneurysms. But the development of small caliber stent-grafts is limited by the ability to maintain low profile and flexibility, which aids in deployment of the stent-grafts^11-14. PU is an elastomeric polymer with good mechanical strength which is a desired trait for achieving a low profile and good flexibility^15,16. In addition to having good deliverability, stent-grafts should also promote rapid healing and endothelialization. PU covered stent-grafts have demonstrated better biocompatibility and enhanced endothelialization¹⁷. Researchers have previously tried to endothelialize PU covered stent-grafts by seeding them with endothelial cells¹⁷. Electrospinning of PU to create nanofiber matrix has been shown to be a valuable technique for the production of vascular grafts^18,19. The existence of nanofibers that mimic the architecture of native extracellular matrix is also known to promote endothelial cell proliferation^20,21. Electrospinning also allows for control over the thickness of the material²². Small caliber vascular grafts made of PU have been studied to promote healing by using modifications such as surface coatings, anti-coagulants, and cell proliferation suppressants. All these modifications are designed to mediate host acceptance and promote graft healing²³.

Our group has developed a balloon expandable bare metal stent which can be deployed in animal models^24-26. The combination of an electrospun polyurethane mesh and a balloon expandable stent has enabled us to generate small caliber balloon expandable stent-grafts. Most of the currently available stent-grafts are introduced through the femoral artery during an interventional procedure, but only a few commercial covered stents can be introduced 1 French size larger than that required for an un-inflated balloon²⁷. In this study we have developed a small caliber vascular stent-graft by encapsulating a balloon expandable stent between two layers of electrospun PU which can be delivered to a coronary artery using a standard 8-9 French guide catheter in a percutaneous interventional procedure.

Protocol

1. Electrospinning of Polyurethane on Mandrel Collector

1. Prepare mandrel for electrospinning
   1. Melt approximately 8 ml of biocompatible, food-grade, water soluble support material in a graduated cylinder (approximately 9 mm diameter and 110 mm deep) at 155 °C using an oven.
   2. Dip a 3 mm diameter and 100 mm long stainless steel mandrel to obtain a coating of support material on the surface of the mandrel. Prior to dipping, place the mandrels in the oven at 155 °C for approximately 15 min to raise the temperature of the mandrel surface which helps in wetting the surface with the molten support material.
   3. Let the dipped mandrel cool to approximately 140 °C while the molten support material solidifies forming a uniform thin coating on the mandrel surface. During the cooling process, hang the mandrel vertically so that gravity causes excess support material to drip off. This coating enables easy removal of the finished stent-graft from the mandrel.
2. Setup of the mandrel collector of the electrospinning system (as shown in Figure 1)
   1. Align the laboratory mixer horizontally and connect a plastic rod which will hold the stainless steel mandrel at the opposite end inside the fume hood.
   2. Dissolve the support material from the tip of the mandrel by submerging only the tip of the mandrel in water to accommodate the plastic support rod at the end of the mandrel. Support the plastic support rod at the free end of the mandrel to assist in uniform rotation of the mandrel collector.
   3. Use set screws in the plastic support rods to secure the stainless steel mandrel and avoid slipping during electrospinning.
   4. Ground the mandrel collector by attaching a U-shaped ground wire to the stainless steel mandrel. Use rubber O-rings to hold the ground wire to the sides of the mandrel.
3. Setting up liquid polyurethane extrusion system of the electrospinning system
   1. Mix dimethylacetamide (DMA) with 25% (m/v) polyurethane (PU) stock solution to obtain 15% (m/v) PU in DMA solution (e.g., add 6 ml of DMA to 9 ml of 25% PU solution).
      CAUTION! Work inside a fume hood with proper personal protective equipment.
   2. Fill a 5 ml glass syringe with blunt end stainless steel needle (spinneret) with 15% PU solution.
   3. Program the syringe pump to extrude at 0.01 ml/min based on the inner diameter of the syringe.
   4. Mount the syringe with spinneret on the syringe pump horizontally with the needle tip approximately 20 cm from the mandrel collector. Insulate the syringe from the conductive parts of the syringe pump using rubber sheets to avoid electrical arcing.
   5. Connect the high voltage generator to the spinneret of the syringe using an alligator clip.
4. Run the syringe pump at 0.01 ml/min and rotate the mandrel with the laboratory mixer running at slow speed (e.g., 50 rpm).
5. Apply a voltage differential of 20 kV across the spinneret and the collector mandrel. PU nanofibers will start depositing on the rotating mandrel and a thin layer will be visible within several minutes. Ensure the fume hood is turned off and exhaust is closed to avoid loss of electrospun nanofibers.

2. Electrospinning a Stent-graft

1. Electrospin PU nanofibers on a rotating mandrel for 2 hr to create a uniform tube (as explained in step 1).
2. Remove the mandrel from the plastic rod connected to the laboratory mixer to install the bare metal stent. Turn on fume hood and open exhaust prior to removing the mandrel to ensure that remnant solvent fumes are removed.
3. Slide the balloon expandable stainless steel stent²⁶ onto the electrospun tube to a desired location. It may be necessary to slightly expand the stent so it slips on without damaging the electrospun tube.
4. Crimp the stent to make sure that the stent is tightly set on the tube material on the mandrel and not loose enough to slide. This will also help to prevent delamination of the inner and outer layers.
5. Load the mandrel with tube and stent again on the plastic rod of the laboratory mixer for electrospinning the outer layer of the stent-graft.
6. Electrospin nanofibers for 3 hr as explained in step 1 to fabricate the outer layer of the stent-graft.
7. After electrospinning the outer later, circumferentially cut the PU material approximately 1 mm from the ends of the stent using a scalpel.
8. Soak the mandrel with stent-graft in deionized water to dissolve the support material from the mandrel which will release the stent-graft from mandrel. Replace with fresh water as needed to dissolve the support material completely.
9. Once the support material is dissolved, gently remove the stent-graft from the mandrel and allow to dry. Consider soaking the removed stent-graft in deionized water to dissolve any remaining support material before allowing to air dry.

3. Testing of Manufactured Stent-grafts

1. Slide the stent-graft on a 3 mm trifold balloon.
2. Crimp the stent-graft onto the balloon using a hand held crimping tool.
3. Inspect the crimped stent-graft using a microscope for uniform crimping and any other signs of failure like delamination or puncture of the cover material due to stent deformation.
4. Expand the stent-graft to the designed diameter of 3 mm by pressurizing the trifold balloon with an inflation device and water. Again, examine the expanded stent-graft for uniform expansion and signs of failure.

Results

Our electrospinner setup (Figure 1) has resulted in high quality polyurethane nanofibers (Figure 2). A stent-graft is manufactured by electrospinning an inner layer of polyurethane onto a mandrel, slipping a bare metal stent over this layer, and electrospinning a second outer layer of polyurethane (Figure 3). Polyurethane nanofibers are electrospun at the rate of 50 µm/hr, which results in an inner layer of 100 µm and an outer layer of 150 µm on the stent-...

Discussion

We have developed a fabrication technique for a small caliber stent-graft which can be deployed using a standard percutaneous coronary intervention (PCI) procedure. Stent-grafts currently available are limited in their ability to maintain a low profile and flexibility for deployment. Bare metal stents developed by our group in our previous studies have proven to assist in rapid healing of the stented artery^24,26. Various polymers have been electrospun by other groups and polyurethane has been proven biostable ...

Materials

Name	Company	Catalog Number	Comments
Glass syringe	Air Tite	7.140-33	Syringe for spinneret
Graduated cylinder 5 mL	Fisher Scientific	08-552-4G	5 mL pyrex graduated cylinder about 9mm diameter and 11 cm long
High voltage generator	Bertan Accociates, Inc.	205A-30P	Used to apply voltage difference across spinneret and collector
Laboratory mixer with rpm control	Scilogex	SCI-84010201	Available from various laboratory equipment suppliers
Polyurethane	DSM	BioSpan SPU	Biospan Segmented Polyurethane
Rubber sheet	McMaster Carr	1370N11	Used to insulate syringe during electrospinning
Stainless steel mandrel	N/A	N/A	Manufactured
Stainless steel needle	Hamilton	91018	Used as spinneret in electrospinning
Support material	EnvisionTec	B04-HT-DEMOMAT	Biocompatible water soluble material
Syringe Pump	Harvard Apparatus	55-3333