Esophageal Stenture

Esophageal benign stricture is the pathological stricture caused by any disease in esophageal antrum. In addition, the external pressure of the mucosal ring and the mediastinal tissue can also cause esophageal stricture. Benign stricture of the esophagus (BSE) can severely reduce quality of life and cause major complications such as aspiration, weight loss and malnutrition [1]. Esophageal stent implantation has been widely used in clinical practice, and this surgical method effectively alleviates patients with dysphagia and has excellent treatment results. Clinical research [2-4] confirmed that esophageal stent implantation for esophageal benign stricture can achieve satisfactory results.
Metal and polymer stents are now the most widely used
stents in esophageal benign stricture. Metal stents have high strength and structural stability. Song et al. originally designed the stainless steel coated stent, and later the nickel titanium alloy stent which in a series of studies achieved good results [5-8]. However, due to the high strength and rigidity of metals, the stent is not easy to remove, and forcible removal causes pain, perforation, bleeding, and strong foreign body reaction and complications [9].

Polymer materials have good flexibility and high tensile strength, There is a lot of literature [11-15] that describe treatment of esophageal stricture, esophageal fistula, and postoperative anastomotic fistula.
Currently, polymer esophageal stent mainly consists of polymer membrane-coated metal stent, which takes advantage of the mechanical performance of metal stents, and covers a layer of polymer coating on the outside which improves the implantation and removal procedure. Poly(e-caprolactone) (PCL) and poly(trimethylene carbonate) (PTMC) are two aliphatic polyesters. They are both biodegradable and biocompatible but have different biodegradation rates and different biomedical applications. PCL is a semi-crystalline polymer and has been widely used in tissue engineering scaffolding, that being due to the properties including: nonimmunogenicity, slow biodegradability and good drug permeability[16]. PTMC is an amorphous biomaterial which holds elastic properties at ambient temperature. It exhibits good mechanical resistance and high chemical and thermal stability. In vivo biocompatibility and toxicity assays revealed that PTMC blend had no influence on heart, liver, and kidney tissues [17]. The synthetic copolymer of the two, P(CL-TMC), has been investigated as biopolymer to be used for surgery and nerve guide repairs because of its high biocompatibility and the advantage of controllable both the mechanical property and the degradation rates[18,19]. Hitherto, the more
promising coating membrane use of PCL/PTMC complex has not as yet been reported.

The Magnesium-(PCL-PTMC) stents used consisted of two parts: a bare magnesium stent and a PCL-PTMC copolymer membrane . The bare magnesium stent was knitted from a 0.20-mm-wide magnesium alloy wire (AZ31, Mg-3Al-1Zn). AZ31 is a commercial magnesium alloy (Sanming, Biomedical Company, Yangzhou, China) with the following chemical composition (in mass percentage): 3% Al, 1% Zn, and 0.43% Mn and Mg (balance). It is supplied in the form of cast ingots. The density of wire cross distribution, the height and the knit angle of the stent had designed.
The PCL-PTMC copolymer membrane (PCL (Mw～100,000) and PTMC (Mw～100,000) were provided by Minghe Functional Polymer Co., Ltd.
(Qingdao, China). was used to coat the magnesium stent via the dipping and spinning method: (a) Precision aluminum molds of the stent were used to fabricate the magnesium wires framework. (b) Equal portions of the two parts of the PCL and PTMC were thoroughly blended together（blend ratios of PCL and PTMC 5:5, w/w) were dissolved in a mixture of N-octane as a solvent prior to use. (c) The mixed (PCL-PTMC) copolymer was dipped on the magnesium stent mould repeated five times and cured for 6 h at 80°C for drying. The moulds were cooled for 3 h in ambient conditions; the stent prototypes were stripped from the moulds. Thus this process ensure the fabric shape and function will remain stable after withdraw from metal mandril. The stent body was not radio-opaque, so a mark was placed at its distal end to facilitate accurate positioning under
fluoroscopy.
All protocols were approved by the animal research committee of our institution and were conducted in accordance with the guidelines of the International Council on Animal Care (US National Institutes of Health and European Commission). forty healthy New Zealand rabbits of both sexes that were 5-8months old (weight, 2.3–3.8 kg) were randomly divided into a magnesium-(PCL-PTMC) stent group (n = 20) and a control group (n = 20). Rabbits in the magnesium-(PCL-PTMC) stent group received stent insertion into the lower 1/3 of the esophagus under fluoroscopic guidance. Control group didn`t receive intervention.

2.2 Magnesium-(PCL-PTMC) stents description
The stent consisted of a cylindrical, cross-linked mesh body made of the magnesium alloy, with a 14-mm cydariform and tubaeform at its head and distal end to prevent stent migration. The stent body and the tubiform tail were covered with a (PCL-PTMC) copolymer membrane. The diameter of the main body was 10 mm; the total stent length was 31 mm when fully expanded (Figure 1A). A trisected anti-reflux valve was added at the conjunction of the stent body and the tail. We compressed and deployed each stent using a 6-mm-wide (approximately 18F) delivery system, and the whole stent body was radiopaqued under the fluoroscope to facilitate accurate positioning.

2.3 Stent radial force test
The radial forces and compression–recovery characteristics of the magnesium-(PCL-PTMC) stent, detect the resist extrusion performance and radial direction braced force support. Measurements of the radial forces were obtained by direct measurement of stent compression between a pressure head (5 mm/in) width, with a 0.1 mm/s (downside) and flat plate using the Instron 5272 Advanced Materials Testing System (Instron Corporation, Norwood, Massachusetts, USA). The radial force curves represented the stability of the stent against potential outer radial forces. This test had been done 48 times.

2.4 Magnesium-(PCL-PTMC) stent evaluation in vitro
Stent degradation was evaluated by determining the magnesium and the (PCL-PTMC) copolymer membrane mass lost from the stent. Stents were sectioned into 1 × 1 mm2 squares. The pre-weighed pieces were incubated at 37°C in 20.0 ml of two phosphate-buffered saline (PBS) solutions with pH values of 7.4 and 4.0. At each experimental time point, triplicate stent samples were recovered, and rinsed with distilled water; the stents were dried to a constant weight in vacuum desiccators. Mass loss was determined gravimetrically by comparing the dry weight remaining at a specific time with the initial weight of the magnesium wires and the (PCL-PTMC) copolymer membrane.

2.5 Stent insertion
Rabbits in the Magnesium-(PCL-PTMC) stent groups underwent stent placement in the lower third of the esophagus. A 0.035-inch, 260-cm-long, stiff exchange wire (Terumo, Tokyo, Japan) was inserted through the mouth and into the stomach under fluoroscopic guidance. The stent-delivery system was introduced over the guidewire until it reached the lower third of the esophagus. The stent was then released, based on esophagographic images obtained under fluoroscopic guidance. A balloon catheter (10 × 40 mm) was inflated within the stent to achieve full stent expansion. Repeat esophagography was performed to confirm the degree of stent expansion and exclude esophageal perforation. Animals in the control group did not receive stent insertion.

2.6 Follow-up
Esophagography was performed under general anesthesia and in the upright position prior to stent placement and at 1, 2 and 4 weeks following stent insertion. Stent migration, stent patency and diameter of the stented esophagus were compared between the three groups.

2.7 Histological examination
Five animals in each group were euthanized at each time point to compare tissue reactions. The inserted stent was carefully removed from the resected esophageal sample. The framework of magnesium-(PCL-PTMC) stent was knitted using a magnesium line and composited of 420 mesh cells. Considering that the support provided by the stent was mainly attributable to the mesh cells, the stent-degradation rate was determined by calculating the percentage of degraded mesh cells. A mesh cell was considered to be degraded if one of its four sides appeared discontinuous under microscopic observation. Minor, moderate and severe degradation of the magnesium-(PCL-PTMC) stent were defined as 50% broken mesh cells in the total number of mesh cells.
The stented and control esophageal samples were fixed in 10% neutral-buffered formalin for a minimum of 48 h, passed through a series of graded ethanol solutions (70% to 100%) and embedded in paraffin. Serial paraffin-embedded, esophageal cross-sections were stained with hematoxylin and eosin (HE) to evaluate the inflammation reaction based on revised inflammation scores [20]. Masson trichrome staining was used to assess submucosal collagen deposition. Esophageal samples were immunostained with mouse anti–proliferating cell nuclear antigen (PCNA) antibody (1:100 dilution; NeoMarkers, Thermo Fisher Scientific Inc., Fremont, CA) and mouse monoclonal α-smooth muscle actin (α-SMA) antibody (1:50 dilution; Santa Cruz Biotechnology Inc., CA) via the Elivision immunohistochemical technique. Negative controls were prepared by omitting the primary antibodies. The pathologist who reviewed the specimens and performed the analysis was blinded to the animal randomization, treatment procedures and follow-up protocols.

2.8 Statistical analysis
GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA) were used for statistical analysis. The Fisher exact test was used to compare nonparametric data. Continuous variables were expressed as mean ± SD, and categorical variables as numbers or percentages. One-way and two-way ANOVA analysis of variance was used to compare the overall changes in esophageal diameter following stent insertion and at each follow-up time point, and to compare the PCNA proliferation index and collagen area at each follow-up time-point within or between the control and tested group. Before one-way ANOVA, the homogeneity of variance and normal distribution of the dependent variables were assessed using the Shapiro–Wilk test. Statistical significance was defined as P < 0.05.

The analysis of material structure and clinical performance (Fig.1) show perfect opened bare stents landscape and profile and related data parameters. The stent consists of a self-expanding, cross-linked polypropylene fiber cylindrical mesh body with a 15 mm cydariform and 5 mm tubiform at its head and distal ends to prevent stent migration at the gastroesophageal junction. The stent body and the tubiform tail were covered with a PCL-PTMC copolymer membrane. The diameter of the main body of the stent body is 10 mm, the diameter of the stent head and distal end is 15 mm and the total stent length is 25 mm when fully expanded. A trisected anti-reflux valve was added at the conjunction of the stent body and the tail.

Fig. 1 (a) Photographs of the perfecting opened PCL-PTMC copolymers coated magnesium- stent shape (b) the covered membrane stents different profile diagrammatic cross-section

3.2 Mg- PCL-PTMC stent and its mechanical evaluation
The tensile stress and strain for the magnesium (PCL-PTMC) stents were tested to determine the mechanical properties shown in Fig. 2a and 2b. The PCL-PTMC membrane had a uniform thickness of 100 μm, was tightly wrapped and fixed to the cross-linked, knitted, bare magnesium mesh tube. The tube was able to maintain its size and morphology due to its excellent flexibility and elasticity. The magnesium- PCL-PTMC stent displayed good elastic deformation properties with no tearing or ablations. The stent was flattened and modified enough to lose elasticity after 48 repeated compressions (compression distance: 0–8 mm). The curves shown in Fig. 2a show a radial force of 0.65 ± 0.23, 1.79 ± 0.17, and 5.91 ± 0.25 N when compressed at 4, 6 and 8 mm, respectively. Fig. 2a reveals that, at the same compression rate of each stent, a stronger compression load and a lower spring-back displacement are needed for the stent. Thus, the PCL-PTMC copolymer coated magnesium stents possess good flexibility and elasticity, and could provide enough support against lesion compression when used in vivo.

Fig. 2（a）Mechanical compression curve analysis of the stent;（b）compression–recovery curves of length and time in repeated compression tests (n = 48, constant pressure = 10 N).