Treatment of a canine carotid artery aneurysm model with a biodegradable nanofiber-covered stent: A prospective pilot study
Aim: To evaluate the use of a biodegradable nanofiber-covered stent (BDNCS) in the treatment of a canine carotid artery aneurysm. Materials and Methods: Seventeen beagle dogs, each with one lateral saccular aneurysm created using a venous pouch, were selected to test the BDNCS. The BDNCS consists of three parts: A bare stent, a biodegradable nanofiber membrane, and a balloon catheter. The bare stent was sculpted by a laser from a cobalt chromium superalloy, and the biodegradable nanofiber membrane was constructed from polylactic acid (PLA) and polycaprolactone [PCL, P (LLA-CL)] by the electro-spinning method. The biodegradable nanofiber stent was premounted on a balloon catheter to form a BDNCS. Angiographic assessments were categorized as complete or incomplete occlusion. Data regarding technical success, initial and final angiographic results, mortality and morbidity were collected, and follow-up was performed at 1 and 3 months after the procedure. Results: BDNCS placement was successful in 17 canines with 17 aneurysms. The initial angiographies showed that a complete occlusion was achieved in 13 canines (76.5%) and an incomplete occlusion in 4 (23.5%). One canine died 1 week later. The angiographies obtained at 3-month follow-up exhibited complete occlusion in 14 canines (87.5%) and an incomplete occlusion in 2 canines, with mild in-stent stenosis in 5 canines. Conclusions: Our results suggest that BDNCS may be a feasible approach for aneurysm occlusion, although the occurrence of mild in-stent stenosis was relatively high. Longer-term follow-up investigations are needed to validate these findings.
Keywords: Aneurysm, animal models, covered stent, nanofibers
Intracranial aneurysms are among the most common cerebrovascular diseases, with an incidence of 8-16 per 100,000 persons per year. Subarachnoid hemorrhage (SAH), caused by the rupture of an intracranial aneurysm,  is a catastrophic event with a mortality rate of 40% and a disability rate of 33%. , Active treatment of the aneurysm can effectively reduce the risk of aneurysm rupture.
Currently, coil embolization is the most commonly used approach for the treatment of intracranial aneurysms. However, shortcomings remain for the use of coil embolization in aneurysms, and these include a low rate of dense packing, ,,,, a high rate of recurrence, ,,,,,, rupture during surgery, and aggravated mass effect, amongst others. Furthermore, surgical approaches are associated with high rates of morbidity and mortality. , Recently, the newly developed pipeline stent has demonstrated excellent flexibility and can be delivered through a micro-catheter, but it is not suited for the treatment of acute aneurysmal SAH as a result of of its low initial complete occlusion rate and delayed endothelialization. ,
Covered stent implantation could eliminate unfavorable blood flow and reconstruct the parent artery by blocking the aneurysm neck without touching the aneurysm wall. Therefore, covered stent implantation is theoretically a good method for the treatment of aneurysms. Although the Willis covered stent has demonstrated great potential for the treatment of aneurysms in animal experiments and clinical trials, ,,,,,, there have been several problems with this method, including endoleak and in-stent stenosis. ,
To overcome these disadvantages, a novel biodegradable nanofiber-covered stent (BDNCS) has been developed by the MicroPort Medical Company and Donghua University for use in the treatment of aneurysms. The purpose of this study was to evaluate the use of BDNCS in the treatment of a canine model of carotid artery aneurysm.
All experimental procedures were performed in accordance with the National Institutes of Health guidelines for the humane handling of animals and were approved by the committee of animal research at our institute. Between June 2010 and November 2011, a total of 20 male beagle dogs, weighing 15-25 kg, were included in the present study. The aneurysm models were established using the venous sac method, and the covered stent was placed 2 weeks after the formation of the aneurysm.
The biodegradable nanofiber-covered stent
The BDNCS was developed by the MicroPort Medical Company (Shanghai, China) (and Donghua University and consists of three parts: a bare stent, a biodegradable nanofiber membrane, and a balloon catheter [Figure 1]. The bare stent was sculpted with a laser from a cobalt chromium superalloy (0.06 mm in diameter) into a segmented design with an open cell structure. The biodegradable nanofiber membrane was constructed from polylactic acid (PLA) and polycaprolactone (PCL), which degrades equably within 3 months. The two polymers were initially mixed in a specific proportion to form the polymer spinning solution. Degradable nanofibers were prepared by the electro-spinning method and they were then spun directly onto the bare stent. The balloon is made from a high-molecular-weight FANTAX® material with three valves. The biodegradable nanofiber stent is premounted onto the balloon catheter to form a novel BDNCS [Figure 1].
Placement of the BDNCS
All procedures were performed under general anesthesia. After positioning a 6-F Envoy (Cordis, Miami Lakes, FL, USA) guiding catheter to the internal carotid artery (ICA), a micro-guidewire (Transcend Floppy, Boston Scientific, Natick, MA, USA) was navigated into the distal branch of the middle cerebral artery. We measured the aneurysm diameter, width, and the aneurysm neck width, and the diameter of the parent artery in tangential positions proximal and distal to the aneurysm. According to these measurements, we selected a stent size that was 0.5 mm larger in diameter than the proximal diameter of the parent artery, with the stent ending about 2 mm beyond the aneurysm neck. Under roadmap guidance, the BDNCS was navigated over the micro-guidewire to a point bridging the aneurysm orifice. The stent was then deployed with the recommended pressure. Angiography was performed immediately after the balloon deflation to confirm the correct placement of the stent and satisfactory occlusion of the aneurysm. If an endoleak was observed, post-procedural dilation was performed to correct the stent position and eliminate the endoleak. If the endoleak persisted or worsened after re-inflation, an additional covered stent was deployed opposite to the proximal or distal end of the first stent to eliminate the endoleak. Immediately after the procedure, angiography was performed to evaluate possible complications.
The animals were fasted for 12 h before the procedure. Low molecular heparin calcium (100 U/kg/12 h) was administered for 3 days. Antibiotics (penicillin 4,000,000 U/d or cefuroxime 1 g/d, streptomycin 0.5 g/d) were administered for 3 days following the procedure. The animals were administered aspirin (25 mg/d) and ticlopidine (62.5 mg/d) orally for 6 months to avoid thrombosis and in-stent stenosis.
Follow-up and postoperative outcome evaluation
The angiographic follow-ups were performed at 1 and 3 months after the stent placement by one of the two authors (Y. D. L. or B. X. G.)). Data on the technical success, initial and final angiographic results, mortality and morbidity were prospectively collected and analyzed.
Angiographic data were categorized into complete occlusion, no residual cavity, and no endoleak, or incomplete occlusion, a residual cavity, or an endoleak. The in-stent stenoses were categorized as follows: normal, mild stenosis (≤29% closure), moderate stenosis (30%-69% closure), severe stenosis (70%-99% closure), or occlusion (99%-100% closure).
Primary procedural results
Twenty experimental sidewall aneurysm models in 20 canines were created, of which three aneurysm models were not included in this study because of massive thrombosis in the aneurysm sac. The baseline characteristics of the 17 remaining canines are summarized in [Table 1]. The BDNCS placement was technically successful in the remaining 17 canines. There were no adverse events related to the navigation of the BDNCS in all canines that underwent stent placement, and no obvious acute thrombus formation was observed. The deployment of the BDNCS was successful without any instance of migration or collapse of the covered stent in the 17 canines.
Complete occlusion of the aneurysms without endoleak was achieved in 13 canines with 13 aneurysms [Figure 2]. Transient endoleaks into the aneurysm sac were observed in four canines with four aneurysms immediately after the deployment of the BDNCS [Figure 3]. The severity of the endoleak was dramatically reduced to a minimum in three of the four aneurysms after balloon re-inflation; however, the last one remained a medium endoleak. One canine with a minimal endoleak died of extensive subcutaneous and visceral hemorrhage 1 week after stent placement because of excessive anticoagulation.
The navigation and deployment of the covered stents were technically successful in all attempted animals, giving a technical success rate of 100%. One covered stent was placed in all of the 17 canines. The angiographies obtained at the end of the initial procedure showed that a complete occlusion was achieved in 13 canines [76.5% (95% CI: 54%, 99%)] and an incomplete occlusion in four (23.5%).
Follow-up angiographic results
At the time of manuscript preparation, all of the surviving 16 canines underwent a 1- and 3-month follow-up angiography. On the 1-month follow-up angiography, the 12 canines with initial complete occlusion were observed to have complete occlusion of the 12 aneurysms with reconstruction of the ICA, and a new endoleak was found in 1 aneurysm. In-stent stenosis (<30%) in the middle of the stent was observed in three canines with three aneurysms. In three canines with three aneurysms with an initial endoleak, the endoleaks persisted without obvious change.
At the 3-month follow-up angiography, of the four canines with persistent endoleaks at the 1-month follow-up, two aneurysms demonstrated a spontaneous resolution of the endoleak with reconstruction of the ICA [Figure 3] and the remaining two aneurysms had a persistent minimal endoleak. The 12 dogs with 12 aneurysms with initial complete occlusion of the aneurysm were stable without recurrence, but two new in-stent stenoses were observed (<30%).
The final follow-up angiographs at 3-month follow-up showed complete occlusion in 14 canines [87.5% (95% CI: 69%, 106%)] with 14 aneurysms and an incomplete occlusion in 2 canines with a mild in-stent stenosis in 5 canines [29.4% (95% CI: 5%, 53%)].
In the past decade, vascular reconstruction techniques have become the preferred endovascular treatment of aneurysms. The Willis covered stent and pipeline low-gap brackets are significant technical advances in vascular remodeling that have been developed in recent years. Although the pipeline low-gap stent has good flexibility and curative clinical effects, it is not suited for patients with acute aneurysmal intracranial SAH. Our recent animal experiments and clinical studies suggest that the Willis covered stent is a good treatment method for the endovascular treatment of cranial aneurysm, which maintains the patency of the parent artery and has an occlusion rate as high as 95%. ,,,,,, However, the Willis covered stent also exhibits some defects and weaknesses: (1) the flexibility and adherent performance of a full covered stent for intracranial applications are unsatisfactory; (2) the covered stent blocks intracranial collateral vessels at the site of stent implantation, and it may cause severe neurological dysfunction with occlusion; , and (3) the Willis covered stent might delay endothelialization when used in tortuous ICAs because the length is unequal between the inside and outside walls of the parent vessel, leading to a rough and uneven membrane in the vessel. Therefore, interventional neuroradiologists hope to develop a covered stent that is made of new composite material for the treatment of intracranial ICA aneurysms.
BDNCS can be spun directly onto the metal stent using the electro-spinning method. This solves the problem of the membrane that adhering to the bracket, and the thickness of the membrane could be controlled according to our requirements. The membrane is uniformly spread when dilated with a balloon because of its multilayer structure, without the occurrence of local dehiscence. The experimental results show that the micro-gap between the fibers does not cause an acute fistula. Nakayama et al.  have produced a microporous drug-covered stent, which placed a polyurethane membrane onto a Palmaz bracket. On the membrane, there were 100-μm-diameter holes created by excimer laser hits. They found that the stent surface was completely covered with endothelial cells 1 month after stent placement.
In addition, another major characteristic of BDNCS was that the membrane was biodegradable. In vitro testing showed that the nanofiber membrane can completely degrade within 6-48 months. The stent occluded the aneurysm from its parent artery after placement without in-stent thrombus formation in the acute phase. Therefore, the BDNCS might solve the problem of delayed endothelialization with the membrane degradation, while completely occluding the aneurysm without the creation of an endoleak.
The results of this study demonstrated a high complete occlusion rate at the 3-month follow-up, which agrees with our previous studies using the Willis covered stent. ,,,,,, However, mild in-stent stenosis was found in five cases (29.4%) in this study, which was higher than that of our previous reports. ,,,,,, Whether the high in-stent stenosis was attributed to the characteristics of the membrane or not is still to be determined and longer-term follow-ups are needed. In addition, we observed one new slight endoleak at 1-month follow-up, probably caused by early degradation of the membrane.
There are several problems that arise when using a covered stent in the treatment of intracranial aneurysms. The first is how to avoid blocking collateral vessels. The second is the poor performance of balloon-expandable covered stents when more serious vascular tortuosity is involved, thus preventing proper positioning of the stent. Much research has been performed to solve this problem. Krings  used a dense mesh stent and stent-graft placement in a rabbit model of carotid aneurysm. They found that in five rabbits, two aneurysms were occluded in January and four aneurysms in March. They also found that the covered stent can produce a stable aneurysm occlusion and only slight changes to the vessel wall. Subsequently, Rudin et al.  designed an asymmetric bracket, which was characterized by the same bracket having both high- and low-porosity segments. They used the asymmetric stent in both in vitro and in animal experiments, and found that the asymmetric stent produces obvious hemodynamic changes to occlude the aneurysm. Perhaps, in the near future, the self-expanding covered stent may offer an effective approach for the treatment of intracranial aneurysms.
In conclusion, our results suggest that BDNCS may be a useful approach for aneurysm occlusion. However, because the occurrence of mild in-stent stenosis was relatively high in our experiments, we recommend future studies with longer-term follow-up durations.
[Figure 1], [Figure 2], [Figure 3]