Evaluation methods for intestinal anastomotic stents

Abstract

A method for evaluating an intestinal anastomosis stent is provided. By establishing an animal model of mechanical intestinal obstruction, an intestinal anastomosis stent implantation environment is simulated after intestinal tumor surgery in patients. It was observed that in the digestive tract anastomosis after intestinal severance under this intestinal obstruction animal model, the implantation of the anastomosis stent can promote wound healing at the intestinal anastomosis site, and the degree of abdominal adhesion is reduced during the healing process, and the intestinal bursting pressure strength is well restored; at the same time, according to the tissue section analysis of this site, it can be seen that its tissue healing process conforms to the physiological healing law and is better than the healing of the group without stent implantation. Moreover, from the expression of the degree of inflammation, the expression of new blood vessels, and the degree of fibrosis, it can be judged by comparing the stent implantation group and the group without stent implantation whether the stent to be tested has a significant advantage in promoting healing after intestinal anastomosis in this mechanical intestinal obstruction animal model. This provides an intuitive detection method for evaluating new materials for preparing intestinal anastomosis stents.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to an evaluation method for an intestinal anastomosis stent. Background Art

[0002] Digestive tract anastomotic leakage is a serious complication that affects intestinal tissue healing or repair during postoperative treatment of gastrointestinal diseases such as intestinal tumors and intestinal obstruction. High anastomotic tension, low blood supply or postoperative infection are the main reasons for the difficulty in healing the anastomosis and causing anastomotic leakage. In response to these problems, the implantation of biological tissue engineering scaffolds is considered to be a new way to solve intestinal anastomotic leakage. At the same time, this research field is of great significance for strengthening clinical treatment and improving the quality of life of patients.

[0003] Currently, self-expanding metal stents (SEMS) or self-expanding plastic stents (SEPS) are effective options for preventing and treating anastomotic leakage after digestive tract reconstruction due to advanced malignant tumors or other malignant lesions, but these stents still cause problems such as stricture formation, perforation, displacement, tissue embedding, and secondary surgical resection. In order to overcome these shortcomings, biodegradable stents have emerged.

[0004] Some rigid biodegradable polymer stents and flexible biodegradable stents have been developed and applied. Among them, polymers such as polylactic acid (PLA), polydioxane heterocycle (PPDO), polycaprolactone (PCL), and polytrimethylene carbonate (PTMC) have been studied the most as biodegradable stents. However, at the microscopic level, polymer stents have a heterogeneous structure and will collapse under the stress caused by curling and expansion even before chemical degradation. Therefore, it is necessary to develop biodegradable intestinal anastomosis stents with good tissue biocompatibility and structural stability.

[0005] However, after developing a new type of intestinal anastomosis stent made of biodegradable materials, it is necessary to evaluate its structural changes and specific efficacy in the in vivo environment to reflect whether the material is suitable for use as an intestinal anastomosis stent. However, the existing in vivo evaluation methods mostly use biodegradable intestinal anastomosis stents implanted in the intestines of healthy animal models for evaluation, but there are obvious differences in the intestinal environment and recovery status between healthy bodies and patients with intestinal tumor diseases, and it is impossible to make a more accurate judgment on the application performance of intestinal anastomosis stents in actual situations.

[0006] Moreover, it is difficult to obtain an animal model of malignant intestinal tumors among the existing animal models of diseases. Therefore, how to obtain an intestinal anastomosis stent implantation environment and a performance evaluation method for intestinal anastomosis stents that are closer to patients after intestinal tumor surgery is a technical difficulty that needs to be solved urgently. Summary of the invention

[0007] In order to solve the technical defects in the prior art, the present invention provides an evaluation method for an intestinal anastomosis stent.

[0008] The technical solution adopted by the present invention is: an evaluation method for an intestinal anastomotic stent, characterized in that it comprises the following steps:

[0009] (1) An intestinal obstruction animal model was used to simulate the intestinal anastomosis stent implantation of existing patients after intestinal tumor surgery;

[0010] (2) Establishment of an animal model of mechanical intestinal obstruction;

[0011] (3) Under this intestinal obstruction model, an intestinal resection surgery is performed at the obstruction site, and after the obstruction contents are released, an intestinal anastomosis is performed. Here, the anastomotic stent to be tested is implanted into the inner wall of the anastomosis site for application evaluation experiments;

[0012] (4) Experimental characterization and analysis of the postoperative recovery of the anastomotic site in an animal model of mechanical intestinal obstruction with implanted anastomotic stents.

[0013] The establishment of the intestinal obstruction animal model in step (2) is specifically as follows:

[0014] a) The experimental animals were randomly divided into several experimental groups. Before the experiment, they should be fasted for 24 hours and allowed to drink water freely to reduce intestinal contents and provide a relatively clean intestinal environment and clear surgical field for the subsequent intestinal surgery;

[0015] b) Induce anesthesia in experimental animals with xylazine hydrochloride injection (Sumenxin II). After the effect takes effect, use an inhalation anesthesia machine to maintain anesthesia by administering an oxygen mask mixed with isoflurane. After deep anesthesia, perform laparotomy;

[0016] c) Use exogenous compression to establish controllable intestinal obstruction, that is, place a soft rubber tube (infusion tube) in the rectum 5 cm distal to the anus to form a uniform live clamp obstruction, and thereby cause the experimental animals to form an obstruction state for 6h, 12h, 18h, 24h, 30h, and 36h respectively.

[0017] d) The clamp was released at different obstruction time points, and the physiological state of the experimental animals was subsequently observed. Based on the H&E and Masson results of tissue sections of the target intestinal segment at different obstruction time points, the critical point of irreversible intestinal damage was obtained through analysis, so as to select the most suitable obstruction time for subsequent intestinal anastomosis.

[0018] The animal is a small omnivorous mammal.

[0019] The step (3) of implanting the anastomotic stent to be tested into an intestinal obstruction animal model for application evaluation experiment is specifically as follows:

[0020] When the experimental animals after the above-mentioned obstruction experiment reach the predetermined obstruction time point, anesthesia is induced on the experimental animals with xylazine hydrochloride injection (Suminxin II). After the effect takes effect, an inhalation anesthesia machine is used to maintain anesthesia by administering an oxygen mask mixed with isoflurane. After deep anesthesia, a laparotomy is performed; after opening the abdominal cavity, the target intestinal segment in the obstructed state is exposed and isolated with gauze soaked in saline. Then, the hose clamp was released, and the blood vessels connected to the mesentery at the target intestinal segment to be removed were tied to prevent bleeding; the target site of obstruction was completely cut open to form intestinal severance, and the contents accumulated in the intestine due to obstruction were quickly cleaned up, and then cleaned and disinfected, and then the intestinal clamps were used to fix the intestinal tract on both sides after severance respectively; the traditional surgery, that is, the blank group directly used sutures to suture the anastomosis site of the severed intestine with interrupted full-thickness sutures; the control group implanted with anastomotic stents and the experimental group implanted with experimental stents were continued as follows: one side of the stent was embedded in the intestinal cavity at the severance, and then sutured and fixed it to the intestinal wall with No. 1 suture (6 stitches from the distal end of the anus and 3 stitches from the proximal end of the anus). After both sides were fixed, the anastomosis site of the severed intestine was sutured with No. 1 silk thread with interrupted full-thickness sutures; after the suturing was completed, the intestinal clamps were removed, the used gauze and instruments were replaced, and the intestinal tubes at both ends were gently squeezed by hand to observe whether there was leakage at the anastomosis, and finally, the surgical site was rinsed with saline and the abdominal cavity was cleaned. The characterization and analysis of the anastomotic healing condition include abdominal adhesion scoring, anastomotic bursting pressure analysis, histological analysis, in vivo degradation of the stent and observation of tissue physical images.

[0021] The abdominal adhesion score is as follows: at the selected time points of 3, 5, 7, and 14 days, the postoperative animals are subjected to another laparotomy, and the adhesion conditions in the abdominal cavity are observed and graded to obtain quantitative results.

[0022] The analysis of the anastomotic bursting pressure is specifically as follows: at the 3rd, 5th, 7th, and 14th day time points set after the implantation of the anastomotic stent, the in vitro pressure measurement method is used to perform a bursting pressure test on the intestinal segment at the anastomotic site after surgery. The test intestinal segment is taken from the intestinal tissue of the anastomotic site and the intestinal segments at the upper and lower ends of the anastomotic site, and the intestinal contents are rinsed clean with normal saline. One end of the intestinal tube is connected to a pressure gauge, and the other end is connected to a peristaltic pump, so that the intestinal tube and the pressure gauge are at the same level. The peristaltic pump uniformly injects 0.16 mg / mL of methylene blue dilution into the intestinal tube at a speed of 10mL / min. When blue liquid overflows from the anastomotic site or the pressure drops suddenly, the reading on the pressure gauge is recorded, which is the anastomotic bursting pressure value.

[0023] The histological analysis includes H&E staining analysis, Masson staining analysis and immunofluorescence staining analysis.

[0024] The H&E staining steps are:

[0025] On days 1, 3, 5, 7, and 14 after implantation, the anastomotic tissue was cut open and fixed with 4% paraformaldehyde solution. After dehydration with different concentrations of ethanol, the tissue was routinely embedded in paraffin and serially sectioned into paraffin blocks with a thickness of 4 μm.

[0026] Dehydration of paraffin sections: put the paraffin sections into xylene, anhydrous ethanol, 75% alcohol, and finally wash with deionized water; Hematoxylin staining: put the sections into hematoxylin staining solution for 3-5 minutes, rinse with deionized water, differentiate with differentiation solution, rinse with deionized water again, then blue with blueing solution, and finally rinse with running water;

[0027] Eosin staining: The sections were dehydrated in 85% and 95% gradient alcohol for 5 minutes each, and then stained in eosin staining solution for 5 minutes; the sections were dehydrated, sealed, and examined under a microscope. The Masson staining steps are as follows:

[0028] Dehydrate in the same way, then immerse the sections in Masson A solution overnight, wash with tap water; immerse the sections in a dye solution of Masson B solution and Masson C solution in equal proportions, dye for 1 min, rinse with deionized water, differentiate with 1% hydrochloric acid alcohol, rinse with deionized water, immerse the sections in Masson D solution for 6 min, rinse with deionized water, and dye with Masson E solution for 1 min;

[0029] Without washing, drain slightly and directly put into Masson F solution for staining for 2-30s, rinse and differentiate with 1% glacial acetic acid, and dehydrate with two cylinders of anhydrous ethanol;

[0030] The slides were transparently sealed and examined under a microscope. The immunofluorescence staining steps are as follows:

[0031] Wash tissue slices with TBS (Tris buffered saline) for 5 minutes × 2, which is conducive to antigen-antibody binding; block solution (10% serum + 1% BSA) + 0.3% triton-100* at room temperature for 2 hours; incubate primary antibodies (CD31, α-SMA, IL-6) at 4℃ overnight, with a concentration of approximately 1μg / mL; wash tissue slices with TBS solution for 5 minutes × 3; incubate secondary antibodies at room temperature for two hours or at 4℃ overnight; wash tissue slices on a shaker in TBS solution for 5 minutes × 3, stick tissue slices on slides in a dark room, dry at room temperature, and it is best that the slides are treated with formaldehyde gelatin to prevent adhesion and peeling; seal with 90% glycerol, pay attention to bubbles. Prepare 90% in advance, use a 20mL glass vial or test tube, add glycerol and PBS (or TBS OR saline) and mix well, and place to eliminate bubbles.

[0032] Fluorescence observation. Place the slide under an upright fluorescence microscope and observe under an oil immersion lens. The beneficial effect of the present invention is that the present invention provides an evaluation method for an intestinal anastomosis stent, by establishing an animal model of mechanical intestinal obstruction, simulating the postoperative intestinal environment of intestinal tumor patients with the intestinal obstruction animal model and implanting an anastomosis stent to evaluate the performance of the intestinal anastomosis stent under this environment. It was observed that in the digestive tract anastomosis after intestinal severance under the intestinal obstruction animal model, the implantation of the anastomosis stent can promote the healing of the wound at the intestinal anastomosis site, and the degree of abdominal adhesion is reduced during the healing process, and the intestinal bursting pressure strength is well restored; at the same time, according to the tissue section analysis of this site, it can be seen that its tissue healing process conforms to the physiological healing law and is better than the healing of the group without stent implantation. Moreover, from the expression of the degree of inflammation, the expression of new blood vessels and the degree of fibrosis, it can be seen by comparing the stent implantation group and the group without stent implantation, so as to see whether the stent to be tested has a significant advantage in promoting the healing effect after intestinal anastomosis in this mechanical intestinal obstruction animal model. This provides an intuitive detection method for evaluating the preparation of intestinal anastomosis stents with new materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The establishment of a mechanical intestinal obstruction model: (A) The process of establishing the intestinal obstruction model; (B) Actual pictures at different obstruction time points; (C) H&E and Masson staining at different obstruction times.

[0034] Figure 2 Stent implantation: (A) Schematic diagram of stent implantation; (B) Actual images of stents and tissues after intestinal surgery in the control group and experimental group.

[0035] Figure 3 Characterization methods for evaluating anastomotic healing: (A) intestinal adhesion score; (B) anastomotic bursting pressure; (C) SEM images of stent degradation in the control group and experimental group.

[0036] Figure 4 Actual pictures of intestinal healing at different times in the blank group, control group and experimental group.

[0037] Figure 5 H&E staining analysis of different groups: (A) blank group; (B) control group; (C) experimental group.

[0038] Figure 6 Masson staining analysis of different groups: (A) blank group; (B) control group; (C) experimental group.

[0039] Figure 7 Immunofluorescence expression of IL-6 in different groups: (A) Immunofluorescence images of different groups; (B) Statistical graph of IL-6 expression.

[0040] Figure 8 Immunofluorescence expression of CD31 in different groups: (A) Immunofluorescence images of different groups; (B) Statistical graph of CD31 expression.

[0041] Fig. 9 Immunofluorescence expression of α-SMA: (A) Immunofluorescence images of different groups; (B) Statistical graph of α-SMA expression. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Synthesis of PTMC / PHA scaffold

[0044] Synthesis of PTMC

[0045] We synthesized PTMC by ring-opening polymerization of TMC monomer. The details are as follows:

[0046] 1) In a vacuum glove box, weigh a certain amount of monomer TMC and add 1-5wt% of catalyst Sn(Oct)2

[0047] (dissolved in anhydrous toluene), add it to the reaction tube, put in a magnetic particle and seal the tube mouth with silicone grease.

[0048] Then seal the tube opening with sealing film to ensure that no oxygen and moisture can enter;

[0049] 2) Take the reaction tube out of the glove box and place it in an oil bath to heat the reaction at 130~160℃.

[0050] Time: 48-60 hours;

[0051] 3) After the reaction is completed, take out and add chloroform solvent, wait for complete dissolution, purify the polymer solution with excess n-hexane, repeat 3 times. Dry the purified precipitated polymer in a vacuum drying oven for 48 h to obtain PTMC for standby use.

[0052] Blending of PTMC and PHA

[0053] 1) Weigh a certain amount of prepared PTMC, add different amounts of PHA to make PTMC / PHA form a certain ratio (9 / 1, 8 / 2, 7 / 3, 6 / 4, 5 / 5), dissolve the above PTMC and PHA mixtures in different ratios in HFIP to form a spinning solution with a solute concentration of 3.5%, and finally perform electrospinning.

[0054] 2) After molding, the tensile properties were tested to explore the PTMC / PHA spinning scaffold with the best mechanical properties.

[0055] In order to explore its in vitro degradation performance, in vitro degradation experiments were carried out on each ratio of the scaffold, and a group of ratios with the best morphology during the degradation process and a degradation time of about three weeks were selected.

[0056] 3) Select the appropriate ratios mentioned above and perform the same tensile and degradation tests on different thicknesses under the same ratio conditions. Select the set of thicknesses with the best performance.

[0057] 4) Select the scaffold samples with the above thickness to conduct cell attachment experiments to determine the optimal blending ratio and optimal thickness of the main scaffold.

[0058] Synthesis of antibacterial surface layer PVA / OHA / GS

[0059] Synthesis of OHA

[0060] HA is an important component of the natural extracellular matrix and plays a vital role in various functions of cells and tissues. Using sodium periodate (NaIO4) as an oxidant to oxidize HA is an effective method for hydroxyl modification. The details are as follows:

[0061] 1) Weigh 1 g of HA and dissolve it in 99 mL of distilled water. Stir at 50°C for 3 h to allow it to dissolve naturally. Adjust the pH to 4 and shake it overnight at room temperature.

[0062] 2) Dissolve 0.42 g of NaIO4 in 10 mL of deionized water to prepare a 0.5 mol / L NaIO4 solution. Place in a syringe away from light.

[0063] 3) At room temperature and in the dark, inject NaIO4 solution into the dissolved HA solution at a rate of 2.5 mL / h and stir for 6 hours in the dark. Then add 1 mL of ethylene glycol to remove the unreacted sodium periodate.

[0064] 4) Place the solution in a 3000 Da dialysis bag and dialyze for 3 days, then freeze-dry and store.

[0065] Determination of OHA hydroformylation degree

[0066] The degree of hydroformylation of OHA was determined by using 2,4-dinitrophenylhydrazine reagent (DNPH).

[0067] 1) First, prepare the DNPH reagent solution: dissolve 40 mg of DNPH in 0.3 mL of concentrated sulfuric acid. Add 3 mL of ethanol to the slurry under continuous stirring at 25°C. Allow the mixture to react for 10 minutes to achieve homogenization. Then make up the volume to 10 mL with distilled water.

[0068] 2) Add 0.3% OHA to 10 mL of freshly prepared DNPH solution. Incubate the reaction mixture at 25°C for 1 hour and then centrifuge at 10,000 rpm for 10 minutes.

[0069] 3) Use a UV spectrophotometer to measure the absorbance of unreacted DNPH in the supernatant at a wavelength of 357 nm. The amount of aldehyde produced is calculated according to the following formula:

[0070]

[0071] Blending of PVA / OHA / GS 1) PVA was swelled with deionized water, then heated and dissolved at 90°C for 2-3h to prepare a 10wt% PVA solution. 2) Then, the prepared OHA and PVA were taken to form water-soluble spinning solutions with different ratios (9 / 1, 8 / 2, 7 / 3, 6 / 4, 5 / 5). The composite spinning solutions with different ratios were spun.

[0072] 3) For the antibacterial layer that has been spun, the swelling performance test and degradation performance test are carried out to select the appropriate ratio of PVA / OHA spinning solution.

[0073] 4) Then add different contents of GS (0.1%, 0.25%, 0.50%, 0.75%, 1.00%) to the spinning solution in appropriate proportions and shake at room temperature until a transparent solution is formed. Then spin and test the cytotoxicity and antibacterial properties of different contents. Select the proportion with good antibacterial properties and low cytotoxicity as the final antibacterial surface layer.

[0074] Spinning of anastomotic stents

[0075] According to the parameters in Table 1, the anastomotic stent was prepared by electrospinning technology.

[0076]

[0077] Establishment of rabbit intestinal obstruction model

[0078] According to the references, we improved the mechanical intestinal obstruction model in rabbits and established this model to evaluate the effect of anastomotic stents on anastomotic healing after intestinal obstruction.

[0079] 1) In brief, 18 rabbits were randomly divided into 6 experimental groups (3 rabbits in each group). Before the experiment, the rabbits were fasted for 24 h and had free access to water to empty the intestinal contents and provide a clear view for the subsequent intestinal surgery.

[0080] 2) Induce anesthesia in the rabbit with Su Mian Xin. After the effect is achieved, administer isoflurane and oxygen mask anesthesia. After the rabbit is deeply anesthetized, perform laparotomy.

[0081] 3) A soft rubber tube was placed in the non-vascular area of ​​the mesentery 5 cm distal to the anus to form a uniform loop obstruction. The rabbits were subjected to obstruction for different periods of time (6h, 12h, 18h, 24h, 30h, 36h). Sham-operated rabbits received simulated intestinal manipulation without the locking clamp device.

[0082] 4) Obtain the rabbit's obstruction status at the most appropriate time point based on H&E and Masson analysis results.

[0083] Application evaluation experiment of anastomotic stent implantation in animal model

[0084] 1) The rabbits were fixed with wooden fixtures after the above obstruction experiment and injected intramuscularly with Sumixin (0.1-0.2

[0085] mL / kg) for induction of anesthesia, and after the onset of anesthesia, isoflurane and oxygen were administered via mask anesthesia;

[0086] 2) After opening the abdominal cavity, pull the intestinal tract scheduled for surgery out of the wound, protect and isolate it with gauze soaked in saline, select the target intestinal tract, gently squeeze the chyle in the intestinal segment to both sides, and clamp a handful of

[0087] Intestinal clamp, used to tie the blood vessels at the target intestinal segment;

[0088] 3) Make an incision at the part of the contralateral mesentery where there are fewer blood vessels. After the incision, the aseptic surgery is transferred to the aseptic surgery. Hold the intestinal clamp handle to bring the two ends of the intestine together. Pay attention to aligning the two intestinal cavities without twisting them. Separate them with saline gauze pads. Use No. 1 silk thread to suture the seromuscular layer at the mesentery and its contralateral edge, about 0.5 cm away from the intestinal end. Do not tie a knot for the time being. Clamp the end with a vascular clamp for positioning and traction;

[0089] 4) The traditional surgery blank group sutured the anastomosis site with silk thread in an end-to-end suture manner;

[0090] 5) The control group implanted with PTMC stents and the experimental group implanted with experimental stents continued with the following procedures.

[0091] The stent was inserted into the intestinal cavity, and only four diagonal sutures were made with No. 1 silk thread. After that, the anterior and posterior walls of the anastomosis were sutured with No. 1 silk thread in an interrupted full-thickness manner (6 stitches were made at the anastomosis site);

[0092] After suturing, remove the intestinal clamp, replace the gauze and instruments used for anastomosis, rinse the gloves with saline, and wipe them dry with iodine cotton balls. Gently squeeze the intestinal tubes at both ends by hand to observe whether there is leakage at the anastomosis, and then rinse with saline. Abdominal Adhesion Scoring

[0093] At selected time points (3, 5, 7, and 14 days), the rabbits were reoperated and the adhesions in the abdominal cavity were observed and graded to obtain quantitative results. The scoring criteria are as follows:

[0094]

[0095] Anastomotic burst pressure

[0096] The anastomotic tissue bursting pressure is an important mechanical index for detecting the healing strength of the anastomosis. It reflects the pressure that the intestine can withstand and is generally used to detect the healing strength of the anastomosis.

[0097] The bursting pressure test of the anastomotic intestinal segment at different recovery time points after surgery was performed using an in vitro pressure measurement method. The details are as follows: Take the intestinal tube of about 5 cm around the anastomosis and rinse the intestinal contents with normal saline. Connect one end of the intestinal tube to the pressure gauge (YB-150A precision pressure gauge) and fix it with silk thread. The other end is also tied to the peristaltic pump with silk thread so that the intestinal tube and the pressure gauge are at the same horizontal plane. Use a peristaltic pump to uniformly inject methylene blue dilution (0.16 mg / mL) into the intestinal tube at a speed of 10 mL / min, and record the reading on the pressure gauge when blue liquid overflows from the anastomosis (or the pressure drops suddenly), which is the bursting pressure value of the anastomosis.

[0098] Histological analysis

[0099] H&E staining

[0100] 1) On the 1st, 3rd, 5th, 7th and 14th day after implantation, the anastomotic tissue was cut open and fixed with 4% paraformaldehyde solution. After dehydration with different concentrations of ethanol, the tissue was routinely embedded in paraffin and sliced ​​into paraffin blocks with a thickness of 4 μm;

[0101] 2) Dehydration of paraffin sections: Place the paraffin sections in xylene, anhydrous ethanol, and 75% alcohol, and finally wash with deionized water;

[0102] 3) Hematoxylin staining: Stain the sections with hematoxylin stain for 3-5 min, rinse with deionized water, differentiate with differentiation solution, rinse again with deionized water, then blue with bluing solution, and finally rinse with running water;

[0103] 4) Eosin staining: Dehydrate the sections in 85% and 95% graded alcohol for 5 min each, and then stain them in eosin staining solution for 5 min.

[0104] 5) Dehydrate, seal and examine under a microscope.

[0105] Masson staining

[0106] 1) Dehydrate in the same way, then soak the sections in Masson A solution overnight and wash with tap water;

[0107] 2) Slices were immersed in a dye solution of Masson B and Masson C in equal proportions for 1 min, rinsed with deionized water, differentiated with 1% hydrochloric acid alcohol, rinsed with deionized water, immersed in Masson D for 6 min, rinsed with deionized water, and immersed in Masson E for 1 min;

[0108] 3) Without washing, drain slightly and directly put into Masson F solution for 2-30 seconds, rinse and differentiate the slices with 1% glacial acetic acid, and dehydrate with two cylinders of anhydrous ethanol;

[0109] 4) Seal the slides and examine them under a microscope.

[0110] Immunofluorescence staining

[0111] 1) Wash the tissue slices with TBS (Tris buffered saline) for 5 minutes × 2 to facilitate antigen-antibody binding;

[0112] 2) Blocking solution (10% serum + 1% BSA) + 0.3% triton-100* at room temperature for 2 hours;

[0113] 3) Incubate with primary antibodies (CD31, α-SMA, IL-6) at 4°C overnight, with a concentration of approximately 1 μg / mL;

[0114] 4) Wash tissue slices with TBS solution for 5 minutes × 3;

[0115] 5) Incubate with secondary antibody at room temperature for two hours or at 4°C overnight;

[0116] 6) Wash the tissue slices on a shaker in TBS solution for 5 minutes x 3, stick the tissue slices on a glass slide in a dark room, and dry them at room temperature. It is best to treat the glass slide with formaldehyde gelatin to prevent it from sticking and falling off;

[0117] 7) Seal the slides with 90% glycerol, paying attention to bubbles. Prepare 90% glycerol in advance, use a 20 mL glass vial or test tube, add glycerol and PBS (or TBS OR saline), mix well, and let it stand to eliminate bubbles.

[0118] 8) Fluorescence observation. Place the slide under an upright fluorescence microscope and observe under an oil immersion lens.

[0119] Statistical analysis

[0120] The experimental data are expressed as mean ± standard deviation (SD) (n = 3). Graph pad prism one-way analysis of variance was used for statistical analysis, and p value < 0.05 was considered significant.

[0121] Results Analysis

[0122] Analysis of mechanical intestinal obstruction model in rabbits

[0123] The experimental model of intestinal obstruction in rabbits was designed, and the mechanical intestinal obstruction model of rabbits was established by extraintestinal compression. The infusion set widely used in clinical settings is converted into a locking clip in vivo ( Figure 1 A) Due to the high probability of colorectal cancer, we chose a mechanical obstruction model in the colorectal area. After laparotomy, a soft infusion tube was used to form a uniform ring obstruction in the non-vascular area of ​​the mesentery 5 cm distal to the anus. Photos were taken to record the intestinal conditions at different time points after the obstruction ( Figure 1 B). It can be observed that within 12 hours of intestinal clamping, a small amount of feces can be seen moving to the obstruction site, and no intestinal dilatation occurs. As time goes on, gas and feces accumulate in the intestine that has been clamped for 18 hours, causing intestinal dilatation and obstruction. As time goes on, gas and feces gradually increase, and the intestine continues to dilate, resulting in severe obstruction after 24 hours. Subsequently, the obstruction continues to worsen, causing intestinal ischemia, which is irreversible. At the same time, we combined the H&E and Masson staining analysis of intestinal obstruction pathology ( Figure 1 C). H&E observations showed that the pathology within 12 hours of intestinal clamping was not significantly different from that of normal intestines. As the obstruction time prolonged, the thickness of the intestinal mucosal layer gradually decreased and fell off at 30 hours, eventually leading to severe damage to the entire mucosal layer. After that, the integrity of the intestinal epithelium was completely lost and the intestinal mechanical barrier was severely damaged. Masson staining also showed that its collagen fibers gradually decreased as the obstruction worsened, and the morphology was gradually severely damaged. At the same time, blood vessels and vascular systems gradually expanded, eventually leading to rupture, causing congestion and edema. Too mild or too severe is not conducive to the conduct of our experiment, so we selected the time point of the obstruction model for the next experiment when the intestine was clamped for 18 hours (the time point when the obstruction began).

[0124] Matching experiment

[0125] The rabbits were relieved of obstruction after 18 hours of obstruction, and then the wound was cut open with surgical scissors to empty the contents. A blank group (traditional manual suture group), a control group (PTMC stent implanted), and an experimental group (stent implanted) were set up. The blank group was sutured by conventional surgical suture, and the control group and the experimental group were implanted with stents according to our schematic diagram ( Figure 2 A). The postoperative recovery process was heavy. We opened the abdomens of the three groups to observe their internal conditions ( Figure 2 B). It was found that some contents in the intestine of the blank group were attached to the anastomosis, and there was a possibility of overflow on the outside. Observation of the control group and the experimental group found that the stent in the control group collapsed due to shrinkage, causing secondary obstruction. At the same time, it could not fit closely with the intestine, resulting in the accumulation of intestinal contents at the stent and the anastomosis, causing extreme expansion of the upper intestine, incomplete healing, and blood congestion. The stent in the experimental group fit closely with the intestine, the intestinal anastomosis was restored intact, and no other pathological changes observable to the naked eye, such as tissue edema and hypertrophy, occurred. In addition, the overall structure of the stent did not shrink, which fully demonstrated that the stent we prepared had good biocompatibility and protective effects on the healing process of the intestinal anastomosis, which provided a guarantee for indirectly promoting the healing of the anastomotic wound.

[0126] Relevant characterization of the matching experiment

[0127] Abdominal adhesion score

[0128] When the abdominal cavity is stimulated or damaged and infected, the local fibrin gelatinous liquid is converted into fibrin coagulation, which covers the wound mucosal surface and plays a repair and protection role. However, the highly adhesive fibrin can cause abdominal adhesion and affect the normal physiological activity of the intestine. Therefore, we use the anastomotic adhesion score to determine the effect of stent implantation on the normal physiological activity of the intestine within 1 week ( Figure 3 A). We found that the adhesion scores caused by fecal leakage in the blank group and the control group were significantly higher than those in the experimental group. In the control group, the PTMC scaffold was severely deformed, causing the body to have severe adhesions in order to protect the intestine. In contrast, in the experimental group, we found that the scaffold we prepared had little effect on the intestine, and the adhesion level was also the lowest. This played a positive role in promoting intestinal healing.

[0129] Anastomotic burst pressure

[0130] The bursting pressure of the tissue anastomosis after stent implantation can effectively reflect the firmness of the healing of the anastomosis after a period of time. This mechanical index can quantitatively reveal the tension that the anastomosis can withstand ( Figure 3B). It can be clearly observed that the anastomotic bursting pressure of the experimental group gradually recovered to the level before anastomosis as the healing time prolonged, while the bursting pressure of the blank group and the control group was lower than the normal intestinal bursting pressure due to incomplete wound healing. This further illustrates that our stent protects the healing of the anastomotic site and is conducive to the smooth recovery of the intestine to a normal state.

[0131] Degradation of stents in vivo

[0132] The morphological changes after stent implantation can better explain the morphological maintenance ability of our stents ( Figure 3 C). Therefore, it provides a good cell-like matrix structure for cell adhesion and proliferation at the anastomosis. It can be found that the fiber filaments of the control group were quickly destroyed and dissolved about 3 days after implantation in the body, causing its structure to collapse. However, the stents of the experimental group can still maintain the interlaced support of fibers of different thicknesses in the intestine for a week, proving the good mechanical properties and structural stability of the experimental stents.

[0133] Organization physical map observation

[0134] The intestinal recovery of rabbits in each group was observed and photographed on the 1st, 3rd, 5th, 7th and 14th day after stent implantation. Figure 4 ). It can be observed that under the same suturing method and number of stitches, the wound of the rabbits in the blank group was infected at the wound anastomosis due to contact with intestinal contents, resulting in the inability of the wound to heal smoothly. The PTMC scaffold of the rabbits in the control group contracted, and the contents contacted the wound, resulting in the inability of the anastomosis to heal. The scaffold in the intestine of the rabbits in the experimental group played a barrier role in intestinal healing, and at the same time had a certain antibacterial effect in the early stage of intestinal healing. The anastomosis healed within 7 days and no leakage was found. It was observed that the tissue had significant progress in wound healing (i.e., from incomplete healing at the beginning to complete recovery of the wound at the later stage). This once again illustrates the advantages of our scaffold in intestinal healing.

[0135] H&E staining

[0136] To further illustrate the healing of the intestine, we further verified the healing of the intestine through HE pathological sections ( Figure 5). Through the observation of the three groups of HE, we found that although the intestinal tissues of the blank group and the control group showed a general healing trend, careful observation showed that the infiltration of inflammatory cells was throughout the entire healing process. The continuous inflammatory damage caused obvious obstacles to their healing, causing excessive or insufficient tissue proliferation, and the mucosa was never able to cover the anastomosis. In contrast, in the experimental group, we can find that its inflammatory infiltration began to decrease around the 5th day, and at the same time, the anastomosis gradually healed, the damaged muscle layer was gradually repaired, the leakage was reduced, and the mucosa was covered. On the 7th day, the mucosal apposition was nearly completed, the degree of inflammatory infiltration was significantly reduced, and the healing was initially completed in about two weeks.

[0137] Masson staining

[0138] Combined with Masson staining ( Figure 6 ), we can also observe that: the formation of collagen fibers in the blank group was significantly delayed, and significant collagen formation began around the fifth day, which further prolonged its healing cycle; while the collagen fibers in the control group were generated in the early stage, but the collagen was greatly reduced on the fifth day, which may be due to the shrinkage and deformation of the PTMC scaffold, which made the tissue healing environment worse and hindered the healing process; the collagen fiber proliferation in the experimental group gradually increased, and the large-scale generation of collagen fibers after two weeks provided strong evidence for strengthening the strength of the anastomotic site. In summary, although the trend of the blank group was similar to that of the experimental group, the degree of healing was not as good as that of the experimental group in the same cycle. The healing of the control group was more obviously inferior. The mucosa was not apposed, and the inflammatory infiltration was obvious throughout the cycle, and the collagen fiber formation was insufficient, which led to the unsatisfactory strength of the anastomotic site. Therefore, the scaffold prepared by us has a good protective effect and a positive promoting effect on the healing of the anastomosis.

[0139] Immunofluorescence

[0140] We further analyzed the tissue at the anastomotic site by immunofluorescence for IL-6, CD31, and α-SMA. IL-6 immunofluorescence was performed to characterize the level of infection-induced inflammation during wound healing ( Figure 7 ). We can clearly observe that the expression of IL-6 in the experimental group showed a significant downward trend as the healing time went by, and the expression of IL-6 tended to disappear around the 7th day, indicating that the inflammation basically disappeared during the healing process. Although the degree of inflammation in the blank group and the experimental group was generally similar, the inflammatory expression in the later stage was higher, which may be due to the increase in the amount of feces adhering to the anastomosis site in the later stage, which slowed down the inflammation in the later stage. The overall inflammatory expression in the control group was significantly too high, which may be due to the shrinkage of the stent leading to obstruction, so that feces and the like came into contact with the anastomosis, thereby significantly increasing the expression of IL-6. At the same time, vascularization is also one of the important indicators for measuring wound healing and is the most important process in tissue repair ( Figure 8 ). CD31 immunofluorescence staining can be used to observe angiogenesis during tissue treatment. The expression of CD31 in the experimental group reached a peak at about 3 days, and then gradually decreased slowly after 5 days. This is because in the early stage of healing, the scaffold in the experimental group created a relatively clean healing environment for it, the body quickly started repair, and a large number of new blood vessels were generated, and the expression increased accordingly. In the process of gradual recovery of healing, the number of new blood vessels gradually decreased. The initial angiogenesis in the blank group was worse than that in the experimental group. This may be because the initial recovery of the blank group was affected by intestinal feces. The obvious lack of CD31 expression in the control group once again illustrates the damage to healing caused by the shrinkage of the scaffold. α-SMA is the most common marker of myofibroblasts in tissues and can be used to identify myofibroblasts. Myofibroblasts are involved in the contraction of the wound surface and the process of tissue fibrosis. Therefore, α-SMA plays an important role in the fibrosis of tissue healing ( Fig. 9 ). We can find that the degree of fibrosis in the experimental group reached a peak on the 5th day and then decreased. It can be seen that the fibrosis showed a trend of first rising and then falling, which prevented excessive fibrosis and adhesion formation while the tissue healed in the early stage. The initial fibrosis degree of the blank group and the control group was higher than that of the experimental group, which made their fibrosis excessive, resulting in the aggravation of the adhesion mentioned above, making it difficult for the intestine to function, and further making it difficult to recover the anastomosis. Therefore, through a series of in vivo characterizations, it can be found that the scaffold we prepared has a positive significance for the recovery of the anastomosis after obstruction.

[0141] Summarize

[0142] We placed the stent in the intestine and observed that the adhesion level was low and the burst pressure recovery strength was good during the intestinal healing process after the stent was implanted. At the same time, we observed the tissue sections and found that the tissue healing process was in line with the physiological healing law. From the expression of the degree of inflammation, the expression of new blood vessels and the degree of fibrosis, we can see that the flexible and degradable stent with antibacterial coating we prepared provides good conditions for the healing of the obstructed intestine after anastomosis and promotes the healing of the intestine. All these prove that it provides a new way for the healing of intestinal anastomosis after obstruction. Of course, this still requires a lot of research to promote the further development of intestinal stents.

Claims

1. A method for evaluating an intestinal anastomotic stent, characterized in that: The following steps are involved: (1) An animal model of intestinal obstruction with 18-hour obstruction was used to simulate the intestinal anastomosis stent implantation after intestinal tumor surgery in existing patients; (2) Experimental characterization and analysis of the postoperative recovery of the anastomotic site in an animal model of mechanical intestinal obstruction with an anastomotic stent implanted. The characterization and analysis of the anastomotic healing included abdominal adhesion scores at 3, 5, 7, and 14 days after the implantation of the anastomotic stent, anastomotic bursting pressure analysis at 3, 5, 7, and 14 days after the implantation of the anastomotic stent, histological analysis, in vivo degradation of the stent, and observation of actual tissue images.

2. The method for evaluating an intestinal anastomotic stent according to claim 1, characterized in that: The experimental animals were small omnivorous mammals.

3. The method for evaluating an intestinal anastomotic stent according to claim 1, characterized in that: The histological analysis includes H&E staining analysis, Masson staining analysis and immunofluorescence staining analysis.

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