Prospective study to assess the tissue response to HPC-coated p48 flow diverter stents compared to uncoated devices in the rabbit carotid artery model

Eight New Zealand white rabbits, of similar age (20 ± 4 weeks, mean ± standard deviation) and weight (4.125 ± 0.45 kg, mean ± standard deviation; range 3.4–4.8 kg), were given acetylsalicylic acid (10 mg/kg/day) and clopidogrel (10 mg/kg/day), via their drinking water. All animals received the DAPT starting 3 days prior to the procedures.

The supra-aortic vessels of the rabbit were of a suitable calibre for the deployment of the p48 FD as well as having a similar calibre to typical intracranial vessels treated with flow diversion in humans. In addition, this animal model has been extensively used in the assessment of neurovascular devices previously and is a widely accepted model [11]. Previous studies have tested HPC in different animal species [12].

All procedures were performed with the animals under general anaesthesia with Ketamin (60 mg/kg)/Rompun 2% (6 mg/kg) IM and maintenance with Ketamin (60 mg/kg)/Rompun 2% (6 mg/kg) in 10 mL NaCl at a flowrate of 2.5 mL/h via the ear vein. After surgical exposure of the right common femoral artery a 4Fr introducer sheath was inserted. Digital subtraction angiography of the common carotid arteries (CCAs) was performed with a 4Fr vertebral catheter (Glidecath, Terumo Europe, Leuven, Belgium). All the procedures were conducted under fluoroscopy and DSA by means of a single-arm angiographic system in posterior-anterior projection at two frames per second (Ziehm Vision imaging, Nuremberg, Germany). Prior to selective catheterisation and stent implantation the animals were fully heparinised to achieve an activated clotting time (2–2.5 times normal). A combination of 0.014 in. pORTAL microwire (Phenox GmbH, Bochum, Germany) and 0.021-in. Trevo Pro 18 microcatheter (Stryker, Kalamazoo, USA) or Prowler Select plus (Codman Neurovascular, West Chester, USA) was used to access the CCAs where deployment of the p48 MW and p48 MW HPC devices was performed. Acetylsalicylic acid and clopidogrel were continuously provided for the time until final angiography and sacrifice and are applied by the drinking water.

The p48 MW FD is constructed from 48 braided drawn filled tubes (DFT) with each DFT strand constructed from a platinum filled nitinol tube. For p48 MW HPC, each DFT strand is coated with pHPC. The delivery system has a central, independently moveable wire and is compatible with 0.021-in. inner diameter (ID) microcatheters. The movable inner wire is made of nitinol with an atraumatic distal tip to prevent rupture. The p48 MW and p48 MW HPC is available in different sizes and is designed to treat vessels between 1.75 and 3 mm.

The pHPC surface coating is a hydrophilic surface modification with antithrombogenic properties. Recently, two stent devices with this coating received the CE mark (pCONUS HPC and p48 MW HPC, phenox GmbH, Bochum, Germany). The coating is less than 12-nm thin, dense (as determined by x-ray photoelectron spectroscopy analysis) and covalently bound to the surface.

The thin nature of the coating does not affect the mechanical properties of the DFT strands or the device as a whole.

In vitro testing showed a significant reduction in the adherence of immunofluorescent CD61+ platelets on pHPC coated electropolished nitinol test plates and braided devices when incubated with whole blood from healthy volunteers compared with uncoated ones. Scanning electron microscopy demonstrated minimal adherent platelets on the coated test plates and FDs whereas a thick layer of adherent platelets was seen on uncoated specimen [13].

Previous in vivo studies have shown that the HPC does not affect the neoendothelialisation of the pCONUS neck bridging device in rabbits nor does it elicit either an acute or chronic inflammatory reaction within the vessel wall nor intima hyperplasia was observed when assessed at 30 and 180 days, respectively [14].

In each animal the uncoated p48 MW was implanted into the left CCA and the coated p48 MW HPC was implanted into the right CCA. The size of the implanted device was based upon the maximum diameter of the CCA at the site of the planned implantation on either side. The size of each device was calculated based on the two-dimensional angiographic imaging. The same size p48 MW HPC and p48 MW was implanted in each animal. In total, four p48 MW HPC 300-15, four p48 MW 300-15, four p48 MW HPC 200-12, and four p48 MW 200-12 were implanted.

Control angiography was performed following implantation of the devices and 30 days after implantation. Selective angiography at final follow-up, using a 4Fr vertebral catheter, was performed via arteriotomy of the left common femoral artery.

Euthanasia by pentobarbital (Narcoren, Merial GmbH, Hallbergmoos, Germany) overdose (6–8 mL) was performed at 30 days whilst the animals were under anaesthesia with Ketamin (60 mg/kg)/Rompun 2% (6 mg/kg) in 10 mL NaCl at a flow rate of 2.5 mL/h via the ear vein. Each of the arterial segments in which either a p48 MW or p48 MW HPC were implanted were surgically resected and then fixed in 10% formaldehyde following which they were photographed and radiographed using a LX-60 cabinet radiography system (Faxitron, AZ, USA).

After gross imaging, the excised arterial segments were dehydrated in a graded series of ethanol following which they were embedded in Spurr’s epoxy resin. After polymerisation, transverse sections from the proximal, middle, and distal third of each device shaft were removed and the cross sections adhered to plastic slides. The slides were prepared to a thickness of < 100 μ (Exakt, Oklahoma City, USA). After polishing of the slides they were stained with haematoxylin and eosin.

An independent, experienced observer (CV Path, Gaithersburg, MD, USA) used digital planimetry with a calibrated camera to perform the morphometric analysis. The morphometric analysis included an assessment of the luminal area of the vessel, the area of the internal elastic laminae (IEL) and external elastic lamina (EEL) and the neointimal thickness which was measured as the distance from the IEL to luminal border. Semiquantitative data including medial fibrin and calcification deposition, inflammatory cell invasion into the media and adventitia, and the thickness of the adventitia were recorded (Table 1). The inflammation score was based on both the extent and the significance of inflammatory cell infiltration within each layer of the vessel wall (adventitia, media, and neointima). Final inflammatory scores were determined using the scale in Table 2. The giant cell reaction was recorded using the same scale.

An independent pathologist (CV Path, Gaithersburg, MD, USA), blinded to the coating status of the p48 MW, performed the histopathological analysis. All slides and prepared stent sections were examined by light microscopy (× 20 as the maximum magnification). The presence of giant cells and granulomas on the stent struts was assessed and calculated as a percentage.

The media area (EEL area minus IEL area), neointimal area (IEL minus luminal are) and percent luminal stenosis (1 − [luminal area/IEL area] × 100) were also calculated.

Normal or near-normal data distributions were expressed as mean ± standard deviation. Paired t test comparisons were used to calculate the significance of differences between continuous variables of all parametric data using JMP software (version 13.0, Cary, NC). Nonparametric score data, including injury, fibrin (Table 1), and neointimal and adventitial inflammation were compared using a Wilcoxon test (JMP software version 13.0). A p value lower than 0.05 was considered significant.

Implantation of all devices was successful; all implants were fully expanded, with no in-stent stenosis and no thrombus formation visible. There was no angiographic evidence of impaired blood flow. All animals survived until the end of the study (30 days). Angiography at 30-day follow-up showed both implants fully expanded with no in-stent stenosis, no visible thrombus formation and the unimpeded blood flow in all vessels.

Figure 1 shows the angiographic results of one animal, with p48 MW 200-12 in the left CCA and p48 MW HPC 200-12 in the right CCA, initially after implantation and at 30-day follow-up. In total, 16 stents and associated CCAs were available for morphological and histological analysis. On gross evaluation, there was no evidence of obvious abnormality (Fig. 2). Examples of Faxitron imaging of implanted p48 MW and p48 MW HPC are shown in Fig. 3.

The morphometric findings between the two groups were similar. There was no statistically significant difference between the luminal area of the vessel, IEL, or EEL area, neointimal area or neointimal thickness. Importantly, there was no evidence of a statistically significant difference in the degree of stenosis between the p48 MW and p48 MW HPC (Fig. 4). The results are summarised in Table 3.

There was no evidence of greater inflammation within the intima, media or adventitia between the two groups. There was no evidence of a statistically different inflammation score between the two groups or of a greater number of giant cells. Neointimal coverage appeared to be complete in both cohorts. The results are summarised in Table 4.

At histological analysis, all devices were well expanded without evidence of luminal thrombosis, although some sections showed postmortem clots, likely from incomplete washout of blood. All devices were fully incorporated by a smooth-muscle cell rich neointima and stenosis values were minimal and similar between test (p48 MW HPC, 13.76 ± 2.79, mean ± standard deviation) and control (uncoated p48 MW = 14.31 ± 4.30, p = 0.766). There was no/minimal fibrin and no evidence of haemorrhage or calcification. The majority of devices, irrespective of the coating showed circumferential to near circumferential mild to moderate chronic inflammation nearest the mesh, including giant cells (inflammation scores: p48 MW HPC, 2.12 ± 0.75; uncoated p48 MW, 14.31 ± 4.30, p = 0.707). Endothelial coverage over luminal surfaces was mostly complete (Fig. 3).