Fabrication of nitrided-iron tubes and LBS stentsA pure iron tube (Φ6.0 mm, wall thickness 0.5 mm) was cut into segments with 25–35 cm length. The tube segments were put into an in-house-design nitriding furnace to be nitrided with a gas method. We carried out the initial nitriding at 500 °C with nitrogen for 30 min, then started vacuuming. The samples were heated at 950 °C for 30 min, and finally annealed at 500 °C for 30 min. The semi-finished nitride tube was drawn into nitride iron tubes of desired sizes (Φ1.6 mm, wall thickness 0.11 mm).The finished nitride-iron tube was laser-cut into multiple stents. All the stents were polished to accurate dimensions. A nitride-iron stent was eletroplated with a pure zinc layer with 600 nm thickness. We then used a Sono-Tek system (2012 Route 9W, Milton, N.Y. 12547 USA) to spray a solution of PLA (Evonik Industries, Germany) on the surface of stent. The injection flow rate of spraying was 0.05 mL/min, and the rotation speed of the stent was 500 rpm/s. The ethyl acetate solution was used to dissolve PLA and sirolimus.To maintain sample consistency, all LBS stents used in in vitro and in vivo experiments were EO sterilized prior to characterization.Characterization of nitrogen content and PLA coatingNitrogen content was measured with an ONH analyzer (ONH-2000, ELTRA Inc., Germany). Nitrogen-permeable semi-finished iron pipe (500 mg) was used to determine the nitrogen content of the stent (wt. %).The PLA coating thickness was measured by spectroscopic reflectometry with a 3D optical profilometer (Q six, Sensofar Medical, Spain).Mechanical measurementsThe tensile strength of the finished iron tube of 100 mm was measured with a universal test machine (C43.504, MTS Inc., USA) at room temperature. The measurement standard follows ASTM E8.A radial strength tester (RX550-100, Machine Solution Inc., USA) was applied to detect the radial force of LBS with a 0.1 mm/s of compression rate. The strength at 10% compression of the outer diameter of the initial stent was defined as the radial strength in units of kilopascal. The measurement standard follows ASTM 3067-14.Crush resistance was evaluated by compression between two parallel plates perpendicular to the longitudinal axis of the stent following ISO 25539-1. The measurement was done in an electromechanical universal testing machine (C43.504, MTS, USA) with a 0.1 mm/s of compression rate. The force per unit length at 50% compression of outer diameter of the original stent was defined as crush resistance in units of N/mm according to ISO 25539-1.We also determined local compression resistance (CR), which is important to deal with the case of calcified plaque. As such, we implanted the stent with nominal pressure into a mock vessel (inner diameter 2.7 ± 0.2 mm, radial compliance 5-7% per 100 mmHg@72 bpm, Dynatec Labs, Inc., Galena, MO, USA) with a simulated plaque and record the resist distance. The height, width and length of the simulated plaque in the experiment read 6.8 mm, 2.8 mm and 1.7 mm, respectively. CR is defined as$${{{\rm{CR}}}}=d/D{{\times }}100\%$$
(1)
Here, d presents the diameter of the LBS under a simulated plaque, and D represents the diameter of the normally expanded stent. The measurement standard follows ISO 25539-1.Animal models and stent implantationThe preclinical study was approved by the Ethics Committee of Shenzhen Advanced Medical Services Corporation in China. Small animals were used to evaluate the in vivo drug release and individual content degradation profile of the LBS. New Zealand rabbits of an average weight of 2.5 kg ranging from 1.9 to 3.2 kg experienced a standard diet without cholesterol or lipid supplements. The implantation sites were abdominal aorta and iliac arteries. We first punctured the right femoral artery of the rabbit and introduced a 5F guide catheter over a 0.014-inch guidewire. Then, a Φ3 × 8 mm LBS was introduced and positioned in the vessel segments avoiding the main branch of aorta under the fluoroscopic control. The stent was deployed under 8–10 inflation pressure at a target balloon to artery ratio of 1.1∼1.2 to 1.0 over 30 s. Then we deflated the balloon, withdrew the guidewire, and sutured the puncture site.Large animals were used to evaluate the operability, safety and effectiveness of the BTK stent. Labrador dogs were chosen with weight between 20-35 kg. The preclinical study of LBS was made in canine BTK arteries. The control device was Xience Prime™ stents (Abbott Vascular, Santa Clara, CA, USA), which has obtained CE mark for additional infrapopliteal indication. Imaging of infrapopliteal artery OCT (C7 XR Fourier-Domain System, Light Lab Imaging, Westford, Massachusetts) was performed before and after implantation, at 1, 3, and 6 months follow ups.The recovery of a damaged vessel lasts usually for 3-6 months, and later stenting may no longer profit the recover vessel but cage it. Additionally, 3-6 months is a crucial period, because majority of the drug sirolimus was released in 3 months and LBS started to degrade significantly. Hence, we set follow ups of the LBS at 1 month, 3 months and 6 months. Our follow ups to 6 months well evaluated the capability of our LBS to provide a sufficient support to the vessel and meanwhile evaluated the potential risk along with biodegradation.Qualitative characterization of in vivo degradation of LBS through micro-CT analysisWe implanted LBSs into rabbit iliac arteries. At given follow-up periods, animals were sacrificed, and the stented artery segments were dissected. We then scanned the stents with vessel tissues through high-resolution micro-CT (Skyscan1172, Bruker, Germany) to acquire images and conducted 3D reconstruction to analyze the degradation extent of the LBS.Quantitative characterization of in vivo material degradation and drug release of nitrided iron, Zn, PLA, and sirolimus in LBSAt given follow up periods, rabbits were sacrificed and the stented artery segments were dissected. We quantified the in vivo degradation of LBS via atomic absorption spectroscopy and the mass loss method. The stents were carefully separated from the vessel tissues and dissolved through microwave nitrification. The Zn concentration in the tissues was determined with an atomic absorption spectroscope (AA240FS, Agilent, USA).We first used tweezers to remove the vessel tissues, and then immersed the stents in ethyl acetate (CH3COOC2H5) under ultrasound for 20 min to separate the PLA coating from the matrix. The PLA-CH3COOC2H5 solution was used to identify MW of the polymer via gel permeation chromatography coupled with multiangle laser light scattering (GPC-MALLS) combined with the molecular weight testing system of Wyatt Company. The test system included a liquid phase pump and injector from Agilent Company, an Agilent PL MIXED-C GPC column (Agilent, United States of American), a multi-angle laser light scattering instrument and a differential detector (Wyatt Corporation of America). The mobile phase during testing was tetrahydrofuran; the pump flow rate was set as 1 mL/min; the injection volume was 100 μL. The GPC data were processed using the software ASTRA 6.1.The stents were immersed in tartaric acid (3 wt. %) under ultrasound for 20 minutes to remove the biodegradation products. The remaining stent struts were cleaned with NaOH, deionized water, and absolute ethyl alcohol, in sequence. Then we weighed the dried metal and calculated the biodegradation rate via the mass loss method.After removing the tissues, we also immersed the LBS into a bottom of acetonitrile to quantify the drug content. The drug-eluted LBS was ultrasonically treated for 20 minutes to extract the residual sirolimus, which was further measured with high-performance liquid chromatography using Agilent 1260 with C18 column and a flow rate of 1 mL/min at room temperature. Sirolimus was analyzed at 278 nm with the mixture of acetonitrile and purified water (65:35 v/v) as the mobile phase. The cumulative drug release of each LBS was calculated from the residual drug amount on the LBS over the initial total drug amount.Histological analysisSamples fixed with 10% formalin for 48 hours were trimmed to remove excess tissues and rinsed with running water for over 10 minutes, followed by gradient dehydration with alcohol at concentration of 70%, 80%, 90% and 100%. The samples then underwent vitrification with xylol and were placed into a glass tube containing a proper amount of solution I (60 mL methyl methacrylate, 35 mL methyl butyl acrylate, 4.5 mL methyl benzoate, and 0.5 mL poly(glycolic diol) per 100 mL), vacuumed for 1 hour, and soaked for 2–3 d at 4 °C to make the polymer solution fully penetrate into deep tissues and to remove the gas in the tissue and polymer solution. After that, the samples were transferred to the polymer solution II (100 ml polymer solution I + 0.4 g/100 ml benzoyl peroxide) to have samples impregnated and were then placed in solution III (100 mL solution II + 150 µL N, N-dimethyl-p-toluidine and 150 µL decane-1-thiol), vacuumed for 1 hour, and preserved at 4 °C till solidification. The glass tube was broken to take out the embedded specimen, which was then sectioned with the precise cutting machine (BUEHLER Lsomet5000) into about 150 μm thickness, and was ground into slices of 10–20 μm thickness with the polish-grinding machine (BUEHLER Ecomet250). The slices were stained with hematoxylin and eosin (H&E) prior to optical observations. The local tissue response and the biodegradation products were observed with an optical microscope (DM2500, Leica, Germany).FIM implantationThe FIM study of the LBS implantation for infrapopliteal lesions was approved by the Institutional Review Board of Chinese PLA General Hospital with approval number S2020-184-01. All procedures in this article were performed at the First Medical Center of Chinese PLA General Hospital (Beijing, China). Patients received dual antiplatelet therapy (100 mg aspirin and 75 mg clopidogrel once daily) for at least 3 days in advance. During the procedures, 5000 IU (50 IU/kg) of unfractionated heparin was administrated after 6 French sheath was placed. Written informed consent was obtained from all of patients. An 80-year-old man presented with left foot rest pain was first enrolled in this study. The target lesion in the first case was TPT of the left leg. The lesion was pre-dilated by plain old balloon angioplasty (Φ2 × 40 mm), and then LBS (Φ3 × 38 mm) was implanted to cover the lesion.In the second case, the targeted lesion was situated in the PTA of the left lower extremity. A balloon catheter with dimensions Φ2 × 80 mm was deployed for pre-dilatory measures, followed by the implantation of LBS (Φ2.75 × 78 mm). In the third case, the lesion was localized in the left PA. A pre-dilation procedure employed a Φ2.5 × 60 mm balloon catheter, and subsequently, an LBS (Φ2.75 × 58 mm) was implanted. Both interventions serve to augment the cumulative evidence regarding the operability and effectiveness of the LBS technology in the management of lower extremity arterial occlusions.Prior to stent implantation, peripheral arterial assessments were conducted via CT imaging systems (GE Company, USA). Digital subtraction angiography based on X-ray imaging (Angiostar, Siemens, Germany) was performed both pre- and post-implantation to evaluate vascular patency. Follow-up ultrasonography evaluations were carried out using an EPIQ 7 system (Philips, Netherlands) at immediate post-procedure intervals, and then at 6- or 13-month time point. Subsequent to the interventional procedures, patients were prescribed a daily regimen of 100 mg aspirin and 75 mg clopidogrel, to be maintained for a duration of 6- or 13-month. These comprehensive diagnostic and therapeutic protocols serve to reinforce the evidentiary basis for the effectiveness and safety of the LBS technology in the treatment of PAD.Statistical analysisMinitab 17 software was used for data analysis. Results are expressed as mean ± standard deviation. The experimental data were examined first for normality using the Anderson-Darling test; then, Student’s t-test was applied to evaluate the extent of stent restenosis, and p < 0.05 was considered statistically significant.The percent luminal area stenosis in the canine BTK artery was defined as the ratio of the lumen area at the follow-up time to the reference stent area at the same time. A total of 15 dogs were implanted with 15 LBS (Φ2.5 × 18 mm/Φ2.5 × 8 mm) and 15 Xience Prime (Φ2.5 × 18 mm/Φ2.25 × 8 mm) and divided into three groups. In each group, 5 dogs were chosen for analysis, and the sample size was set considering that animals for histological analysis should contain at least 3-4 individuals. Every dog received one LBS and one Xience in each of the two hind legs and followed up by X-ray imaging & OCT immediately after implantation. Five dogs followed up at 1 month, 3 months, and 6 months.We carried out in vivo degradation of LBS in rabbit abdominal aorta/iliac model. Since the drug content of a single LBS was too small to detect, we combined the stents collected from each rabbit as one sample. The mass loss of The PLA coating, nitrided-iron, zinc and sirolimus were analyzed by pooled date collecting from follow-up date. We denote the number of animals as N and the number of stents for each group as n. In tests of the mass loss of the PLA coating, we examined 3 months (N = 3, n = 9), 6 months (N = 3, n = 9), 12 months (N = 1, n = 3), and 18 months (N = 1, n = 3) after implantation of the stents; in tests of mass loss of the Zn sacrificial layer, we examined 1 month (N = 10, n = 11), 2 months (N = 10, n = 11), and 3 months (N = 7, n = 11); in test of mass loss of the nitride Fe, we examined 2 months (N = 9, n = 11), 3 months (N = 7, n = 11), 6 months (N = 9, n = 11), 9 months (N = 6, n = 11), 12 months (N = 9, n = 11), and 24 months (N = 7, n = 11); in tests of release of the sirolimus, we examined 7 days (N = 3, n = 6), 14 days (N = 3, n = 6), 1 month (N = 3, n = 6), 2 months (N = 3, n = 6), 3 months (N = 3, n = 6), 6 months (N = 5, n = 10), and 12 months (N = 1, n = 2).Reporting summaryFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Maglev-fabricated long and biodegradable stent for interventional treatment of peripheral vessels
