WO2022228356A1 - 基于ptmc的生物可吸收柔性弹性体的肠吻合支架及其制备方法 - Google Patents

基于ptmc的生物可吸收柔性弹性体的肠吻合支架及其制备方法 Download PDF

Info

Publication number
WO2022228356A1
WO2022228356A1 PCT/CN2022/088873 CN2022088873W WO2022228356A1 WO 2022228356 A1 WO2022228356 A1 WO 2022228356A1 CN 2022088873 W CN2022088873 W CN 2022088873W WO 2022228356 A1 WO2022228356 A1 WO 2022228356A1
Authority
WO
WIPO (PCT)
Prior art keywords
ptmc
stent
intestinal anastomosis
intestinal
bioabsorbable
Prior art date
Application number
PCT/CN2022/088873
Other languages
English (en)
French (fr)
Inventor
石长灿
李徐坚
季志孝
潘璐琪
杨啸
姜大伟
Original Assignee
国科温州研究院(温州生物材料与工程研究所)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国科温州研究院(温州生物材料与工程研究所) filed Critical 国科温州研究院(温州生物材料与工程研究所)
Priority to JP2023565596A priority Critical patent/JP2024515141A/ja
Publication of WO2022228356A1 publication Critical patent/WO2022228356A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
    • A61B17/11—Surgical instruments, devices or methods, e.g. tourniquets for performing anastomosis; Buttons for anastomosis
    • A61B17/1114—Surgical instruments, devices or methods, e.g. tourniquets for performing anastomosis; Buttons for anastomosis of the digestive tract, e.g. bowels or oesophagus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/06—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/148—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/16—Biologically active materials, e.g. therapeutic substances
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
    • D01D5/00—Formation of filaments, threads, or the like
    • D01D5/0007—Electro-spinning
    • D01D5/0015—Electro-spinning characterised by the initial state of the material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
    • A61F2002/045—Stomach, intestines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/202—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with halogen atoms, e.g. triclosan, povidone-iodine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents

Definitions

  • the invention specifically relates to the technical field of polymer materials, in particular to a PTMC-based bioabsorbable flexible elastomer intestinal anastomosis stent and a preparation method thereof.
  • Gastrointestinal reconstruction anastomosis is one of the most common surgical operations in abdominal surgery.
  • the incidence of anastomotic leakage has not decreased significantly, and it has always been a problem that plagues the success rate of gastrointestinal surgery.
  • Benign and malignant tumors of the digestive tract, perforation of the digestive tract, obstruction of the digestive tract, bleeding, ischemia and other intestinal diseases often require resection of part of the diseased intestine before anastomosis.
  • Traditional methods are mostly manual suture anastomosis. Staplers for end-to-end or end-to-side anastomosis, or straight-cut closures for side-to-side anastomosis. No matter what kind of anastomosis is used, there is no way to prevent anastomotic leakage, a fatal complication.
  • the purpose of intestinal anastomosis is to restore the physical, histological and physiological functions of the intestines at both ends of the anastomosis.
  • the main problems of traditional staplers include: (1) Metal staplers are not biodegradable, resulting in permanent retention in the body; (2) Degradable polymer staplers lack mechanical matching with wound tissue; (3) The stapler does not have the function of tissue repair and regulation, and cannot reasonably regulate the recovery of normal intestinal function.
  • the invention patent CN 111449707A proposes an anorectal anastomosis device, which includes a handle base, a transmission assembly, a firing assembly and a kiss cutting assembly; An adjustment mechanism connected to the tail end; the front end of the screw rod is fixedly installed with a nail abutment seat; the firing assembly includes a movable handle set on the handle base and a straight push rod sleeved on the screw rod; the kiss cutting assembly includes a nail push piece, a nail cartridge Sets, cartridges and ring knives.
  • the nail pusher, the nail cartridge cover and the nail cartridge are all made of metal materials, and the parts cannot be degraded in the body, and can only be permanently retained in the body or taken out by a second operation.
  • Patent CN109480943A is made of degradable material, adopts nail body perforation fixing mode, and designs a support frame at the rear end of nail body, but the anastomotic ring has high hardness and inelasticity, cannot well adapt to intestinal peristalsis, and has obvious foreign body sensation.
  • Patent CN103230265A which uses degradable materials polyglycolide and polylactide as raw materials, and is applied to gastrointestinal anastomosis.
  • the stapler has a fragile function, but also lacks the mechanical matching with the intestinal tissue.
  • An ideal stapler should have the following characteristics: (1) effectively isolate the intestinal contents; (2) the implantation of the stapler has little damage to the intestinal wall of the anastomosis; (3) the operation is simple and easy to operate. None of the anastomotic devices currently on the market can meet the above requirements at the same time.
  • stents must be easy to manufacture in a variety of lengths and diameters to suit different individuals, and not require any complicated storage procedures. All of these had to be met while keeping the brackets affordable and affordable.
  • the present invention provides a PTMC-based bioabsorbable flexible elastomer intestinal anastomosis stent and a preparation method thereof. Significantly reduce the incidence of intestinal anastomotic leakage and other complications.
  • the technical solution adopted in the present invention is: an intestinal anastomosis stent based on PTMC bioabsorbable flexible elastomer, the intestinal anastomosis stent is made of PTMC homopolymer material as a whole, and the PTMC homopolymer is high
  • the molecular medical material PTMC monomer adopts the method of ring-opening polymerization to synthesize the polymer material, and the thickness of the intestinal anastomosis stent is 0.05-0.3 mm.
  • the PTMC homopolymer material of the bioflexible elastomeric intestinal anastomosis stent is loaded with triclosan (TCS).
  • the intestinal anastomosis bracket is also provided with a plant cellulose sleeve, and the intestinal anastomosis bracket is a gapless nested structure, the inner part is a tube of plant cellulose material, and the outer part is a PTMC homopolymer material.
  • a preparation method of a bioabsorbable flexible elastomer intestinal anastomosis stent characterized in that the preparation is carried out through the following steps:
  • the dissolving condition of the product is to dissolve with CHCl 3 or DMF or THF, and place it on a shaker, and the temperature of the shaker is set to 37°C.
  • n-hexane or ethanol is used for purification, and a glass rod is used for constant stirring.
  • the present invention provides an intestinal anastomosis stent based on PTMC bioabsorbable flexible elastomer and a preparation method thereof, and an intestinal anastomosis stent with PTMC as the base material is prepared by an electrospinning method, and According to the degradation rate and mechanical properties, the suitable range for implantation in vivo is screened.
  • the TCS with bactericidal effect is loaded to make it have the function of tissue repair regulation, so that the wound can achieve faster healing in the harsh multi-bacterial intestinal environment, and it is used to regulate postoperative tissue healing and functional repair.
  • An in vivo animal intestinal anastomosis experiment was performed to verify the actual effect and the feasibility of the method.
  • Figure 1 shows the experimental process in vivo; (1) cecal incision; (2) implantation of anastomotic stent; (3) interrupted full-thickness suture.
  • Fig. 2 is a schematic diagram of the PTMC synthesis process of the present invention.
  • Figure 3 is an infrared spectrum of PTMC.
  • FIG. 4 is a 1 H-NMR spectrum of homopolymer PTMC.
  • Figure 5 is a SEM micrograph of an electrospinning sample.
  • Figure 6 shows (A) the degradation time of PTMC membranes with different molecular weights in enzyme solution, (B) the pH curves of lipase solution after enzymatic degradation of PTMC membranes with different molecular weights, (C) the degradation time of PTMC membranes with different thicknesses, (D) SEM of the anastomotic scaffold after implantation, (E) physical map of the degradation of the anastomotic scaffold, (F) weight loss of the anastomotic scaffold from the rat at the corresponding time, (G) weight loss of the anastomotic scaffold from the rat at the corresponding time Weight Loss Length Loss.
  • FIG. 7 shows the antibacterial effects of samples without triclosan (upper panel) and samples containing triclosan (lower panel), A is Staphylococcus aureus, and B is Escherichia coli.
  • Figure 8 shows the hemolysis rates of different samples.
  • Figure 9 shows the cytotoxicity of the samples.
  • Figure 10 shows the abdominal adhesion scores at different times after intestinal anastomosis.
  • Figure 11 shows the anastomotic rupture pressure of different sample groups 7 days after surgery.
  • Figure 12 shows the results of H&E staining and Masson staining.
  • TMC 1,3-trimethylene carbonate
  • the mouse fibroblast cell line L929 was provided by the Type Culture Collection, Chinese Academy of Sciences (Shanghai, China). Petri dishes were purchased from Corning Incorporated (New York, USA). Cultures were performed in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 IU/ml penicillin and 100 mg/ml streptomycin sulfate. All cells were cultured in an incubator at 37°C, 5% CO2 , in complete humidification.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • mice Male Sprague-Dawley rats (200 ⁇ 20 g) provided by the Experimental Animal Center of Wenzhou Medical University (Wenzhou, China) were bred at 25°C and 55% humidity. All animal experiments were performed in accordance with ethics committee-assessed and approved guidelines.
  • PTMC homopolymers were synthesized by ring-opening polymerization. Briefly, the metered TMC monomer was transferred into a completely dry glass reactor with a magnetic stir bar. Sn(Oct) 2 was dissolved in anhydrous toluene solution under N 2 atmosphere, and 100 ppm was added to the reaction vessel with a pipette. The copolymerization reaction was carried out at 130 ⁇ 2°C for 24h to ensure that the whole process was free of water and oxygen. After 24 hours, the product was dissolved in chloroform, and when it was completely dissolved, the polymer solution was purified by excess n-hexane, and the procedure was repeated three times. The purified homopolymer was dried in a vacuum drying oven at 40 °C for 48 h, and then stored in a drying cabinet.
  • the dried sample was dissolved in a mixed solution of CHCl 3 /DMF (9/1, V/V), the concentration of the prepared solution was 5.5%, and the sample was placed on a shaker at 37°C for 36h until the sample was fully dissolved to obtain uniform co-dissolution spinning dope.
  • the specific electrospinning conditions are shown in Table 1 [30] .
  • the thickness of the sample after spinning was 0.2 ⁇ 0.01 mm.
  • the resulting fibers were further dried in a vacuum oven at room temperature for 24 hours to remove residual organic solvent and moisture. Mechanical property tests as well as in vitro degradation tests were performed using the spun samples.
  • the FTIR-ATR spectrum of the polymer PTMC was measured with a Nicolet Magna-560 spectrometer equipped with an ATR accessory.
  • the 1 H-NMR spectrum of homopolymer PTMC was measured by Bruker spectrometer. All 1 H-NMR used tetramethylsilane (TMS) as the internal reference, deuterated chloroform (CDCl 3 ) as the solvent, and ppm as the internal reference. Units record the chemical shift (D) of the sample.
  • TMS tetramethylsilane
  • CDCl 3 deuterated chloroform
  • Units record the chemical shift (D) of the sample.
  • Hitachi cold field emission electron microscope SU8010 field emission scanning electron microscope was used to photograph the microscopic topography of the samples after electrospinning and the microscopic topography after implantation in animals.
  • DSC analysis was performed using a DSC8000 (PerkinElmer, USA), and the thermal properties of the polymer PTMC were recorded at a heating rate of 10°C/min.
  • the intrinsic viscosity of PTMC was measured using an Ubbelohde viscometer in a constant temperature water bath at 25°C, and the test results were the average stress and strain of three experiments. It was carried out on an electronic universal material testing machine (Instron 5944), and the samples after electrospinning were processed to size It is a 45.0mm ⁇ 25.0mm sheet material, and the size of SD rat cecum is 45.0mm ⁇ 25.0mm ⁇ 0.3mm. Rinse with normal saline and wipe off excess water on the surface.
  • a 10.0 ⁇ 10.0mm PTMC membrane was taken, placed in 1 mL of lipase solution, placed in an air bath at 37°C, and shaken for 8 hours a day with an amplitude of 65 times/min.
  • the enzyme solution was replaced every 3 days to maintain the activity of the enzyme, and samples were taken out after the 1st, 5th, 10th, 15th, 20th, 30th, 40th, and 50th days, and three parallel splines were randomly selected.
  • the surface water was absorbed by the filter paper, and then dried under vacuum at 37°C for 12h to constant mass. Record the mass of the dry sample and the pH of the medium containing the degradation products.
  • the in vivo biodegradation behavior was obtained by recording the mass and size of the anastomotic stent before and after implantation. After the anastomotic stent was taken out, it was cleaned with distilled water, and the surface moisture was absorbed by filter paper. The weight loss rate is calculated by the following formula
  • Weight loss(%) (W 0 -Wt)/W 0 ⁇ 100%
  • W0 and Wt represent the dry weight of the samples before and after degradation, respectively.
  • the sample material was rinsed with distilled water in advance, wiped off to show excess water, and fresh human whole blood was used.
  • Experimental group take 15 mg of electrospinning sample and put it in EP tube, add 1 mL normal saline and 0.1 mL whole blood, negative control group: add 1 mL normal saline and 0.1 mL whole blood to EP tube, positive control group: add 1 mL to EP tube Ultrapure water and 0.1 mL of whole blood. All the above samples were incubated at 37°C for 2h, and the hemolysis reaction test was performed.
  • Data processing Take the mean value of OD of the three samples in the sample group and the control group, respectively. Calculate the hemolysis rate of the sample according to the formula.
  • H% is the hemolysis rate
  • the absorbance of the OD t sample the absorbance of the OD nc negative control sample
  • the OD pc absorbance of the positive control sample According to the GB/T1423.2-1993 standard, the in vitro hemolysis of the material was evaluated by measuring the lysis of erythrocytes and the degree of hemoglobin dissociation caused by the contact between the material and red blood cells in vitro, and the hemolysis reaction of more than 5% was positive.
  • Preparation of cell culture medium 500 mL of RPMI1640 medium + 50 mL of fetal bovine serum + 5 mL of penicillin/streptomycin double antibody.
  • the samples were sterilized with 75% alcohol in advance, and then irradiated with ultraviolet rays on the back and front of the ultra-clean bench for 30 minutes each.
  • the electrospinning samples were respectively cut into square films with a side length of 1.82 cm, 2 mL of complete medium was added, and they were bathed together at 37° C. for 24 hours to obtain a spinning sample extract.
  • L929 cells (adherent cells) were externally cultured with cell culture liquid, proliferated for more than three generations, and waited until the culture flask was full. Rinse three times with PBS (not against the cells to avoid washing out the cells). Then, it was digested with 50 ⁇ L of 0.25% trypsin for 30 s (37° C.) to make it into a cell suspension. Immediately add 3-5 mL of complete medium and transfer it to a centrifuge tube, centrifuge at 1000 rpm for 5 min, pour off the upper waste liquid, add 5 mL of PBS to the centrifuge tube, and rinse with a pipette to make the cells evenly dispersed. 1 ⁇ L was dropped onto a counting plate for cell counting, and the cell concentration was adjusted to 5 ⁇ 10 4 cells/mL.
  • Co-culture Seed the diluted L929 cells in a 96-well plate, 100 ⁇ L per well, 5000-8000 cells per well; culture at 37°C, 5% CO 2 for 24 hours, the cells are completely adherent and discarded Remove the culture medium; add 100 ⁇ L of the extract of the experimental group and the positive solution (10% DMSO (200 ⁇ L) in the positive control group) to the wells, 6 duplicate wells in each group, and incubate in a 37°C incubator for 24 h.
  • CCK-8 detection Take out the 96-well plate at preset time points (24h, 48h), aspirate the stock solution and add it around the sample wells, and add 10 ⁇ L of CCK-8 reagent and 100 ⁇ L of complete medium to each well (first Mix the two solutions), and after incubating in a 37°C incubator for 2 h, use a multifunctional microplate reader (absorbance 450 nm) to detect the absorbance (OD) value.
  • a s is the absorbance of the experimental well (with polymer extract, with cell culture medium, with CCK-8);
  • a c is the absorbance of the control well (without polymer extract, with cell culture medium, with CCK-8) 8);
  • Ab is the absorbance of the experimental wells (without polymer extract, without cell culture medium, with CCK-8).
  • the healing of the intestinal anastomosis mainly goes through the stages of inflammation, cell proliferation, and structural reorganization of the intestinal wall.
  • the functional repair is a very long process, which involves digestion and absorption, internal and external secretion, nerve repair and transitional compound movement. Therefore, the mechanics and histology must meet the indicators before we consider that the anastomotic stoma is healed.
  • burst pressure test an indicator of mechanical healing
  • abdominal adhesion score reflecting the local inflammation near the anastomosis
  • anastomotic tissue HE staining and Masson staining and immunohistochemistry Staining to assess the degree of inflammatory cell infiltration, collagen deposition.
  • the PTMC samples were soaked in 75% alcohol for 10 min, and sterilized by ultraviolet light for 30 min.
  • 180 male Sprague-Dawley rats of general grade, body weight 200 ⁇ 10g were anesthetized by intraperitoneal injection with 10% chloral hydrate according to body weight.
  • group A was PTMC group
  • group B was TCS/PTMC group
  • group C was blank control group.
  • Four time points were set for each group, 7 days, 14 days, 21 days, and 28 days.
  • the group consisted of 5 rats. The abdomen was shaved and the rat cecum was found by laparotomy.
  • the anastomotic tissue burst pressure is an important mechanical index to detect the anastomotic healing strength. It reflects the pressure that the intestine can withstand, and is generally used to detect the anastomotic healing strength.
  • the burst pressure test of the intestinal segment of the anastomosis was performed, and the in vitro pressure measurement method was used.
  • the anastomotic stoma and its surrounding about 5cm intestinal canal were cut out and the intestinal contents were washed out with normal saline.
  • Appropriately separate the abdominal adhesions expose the intestinal segments at each anastomotic site, connect one end of the intestinal tube to a pressure gauge (YB-150A precision pressure gauge), tie and fix it with two silk threads, and ligate the other end of the anastomosis with two silk threads to seal the intestine. cavity, and make the bowel and the manometer at the same level.
  • a pressure gauge YB-150A precision pressure gauge
  • a peristaltic pump to inject methylene blue diluent (0.16mg/mL) into the intestinal tube at a constant rate at a rate of 10mL/min, pay attention to observe the anastomosis, and record the pressure gauge when the anastomosis overflows with blue liquid (or the pressure suddenly drops). The reading is the anastomotic burst pressure.
  • H&E staining is also known as hematoxylin-eosin staining, hematoxylin Hematoxylin, eosin Eosin.
  • the basic principle is to use the basic dye hematoxylin and the acid dye eosin to interact with the nucleus and cytoplasm, respectively, so that the microstructure of the cell changes its refractive index through color, so that the cell image can be clearly presented under the light microscope, and Can provide a good nuclear cytoplasmic contrast staining.
  • Hematoxylin is a blue-violet basic dye that can color the nucleus.
  • the structure colored by hematoxylin itself is acidic and has basophili.
  • Eosin is a pink acid dye that can stain the cytoplasm red.
  • the structure colored by eosin itself is basic and acidophilic. Structures that are not easily stained by hematoxylin and eosin are neutrophilic.
  • the nucleus was blue-purple, the cytoplasm, muscle fibers, collagen fibers, red blood cells were red to varying degrees, and calcium salts and bacteria were blue or blue-purple.
  • Rats were sacrificed at designated time points, and the spinning scaffolds and intestinal wrapping tissues were taken out, fixed in 4% formaldehyde solution, dehydrated in ethanol solution, embedded in paraffin, sectioned (4 ⁇ m), and stained with H&E. The stained sections were observed by light microscopy.
  • the specific process of slice preparation is as follows:
  • Masson staining is mainly used for differential staining of collagen fibers and muscle fibers, and is used to observe the proliferation and distribution of fibrous connective tissue in diseased tissue.
  • the staining results showed that nuclei were black, muscle fibers were red, and collagen fibers were blue. step:
  • the tissue was fixed in 10% neutral formalin solution, rinsed with running water, and routinely dehydrated and embedded;
  • Sections are routinely dewaxed to water
  • PTMC homopolymers were synthesized by ring-opening polymerization catalyzed by Sn(Oct) 2 ( Figure 2). The copolymerization reaction is shown in Table 2.
  • Figure 4 is a 1 H-NMR spectrum of homopolymer PTMC. It clearly shows that the chemical shifts ⁇ at 4.171 ppm (a) belong to the methylene proton next to the oxygen in the PTMC block, ⁇ at 1.984 ppm (b) belongs to the other methylene protons in the PTMC block, and the ratio of the areas of the peaks consistent with the product.
  • the electrospun fibers were uniformly distributed, with diameters ranging from 5 ⁇ m to 10 ⁇ m ( Figure 5).
  • the mechanical properties of the anastomotic scaffolds prepared by electrospinning of PTMC are listed in Table 4.
  • the mechanical properties of scaffold materials under physiological conditions are an important indicator of implant materials. In order to observe the mechanical properties of materials under physiological conditions, the samples were immersed in PBS solution for 24 hours before the test, and it was found that the mechanical properties of the scaffolds did not differ much. , the tensile strength and elastic modulus decreased slightly, and the elongation at break increased to a certain extent, which was caused by the loose and porous structure of the scaffold absorbing water.
  • PTMC with high molecular weight has good mechanical properties and shape retention, and the molecular weight of PTMC 1 and PTMC 2 is too low to achieve a good support. When the reaction time exceeds 30h, the mechanical properties of PTMC are not very different with the increase of reaction time.
  • PTMC has stable mechanical properties in both dry and wet states, which ensures its reliability in practical applications.
  • the dried samples were dissolved in a mixed solution of chloroform/DMF (9/1, V/V), the concentration of the prepared solution was 5.5%, and the samples were placed on a shaker for 36 hours at 37°C to fully dissolve the samples to obtain uniform co-dissolving spinning.
  • Silk stock solution Fill the stock solution into a 2.5 mL syringe that includes a metal needle with an inner diameter of 0.5 mm.
  • the specific electrospinning conditions are shown in Table 1. The thickness of the sample after spinning is determined by the specific electrospinning time.
  • the resulting fibers were further dried in a vacuum oven at room temperature for 24 hours to remove residual organic solvent and moisture. Mechanical property tests as well as in vitro degradation tests were performed using the spun samples.
  • PTMC is mainly degraded by enzymatic hydrolysis in vitro and surface erosion in vivo. Feijen et al. all believed that the enzyme played an interfacial activation role, so the degradation rate in the enzyme solution in vitro was faster than that in vivo.
  • the degradation rates of PTMC materials with the same size and different thickness are very different.
  • the subjects of this animal experiment are male rats.
  • the intestinal healing period of rats is about 14 days. Therefore, the anastomotic stent implanted in the intestine must meet the requirements of mechanical strength for at least two weeks and degradation in about three weeks.
  • the effect of molecular weight on the weight loss rate was first studied (Fig. 6(a)), and PTMC 1-7 were cut into membranes of the same size (10.0 ⁇ 10.0 ⁇ 0.4 mm). The results showed that with the increase of the molecular weight of PTMC, the degradation accelerated , and no acidic substances were present during the degradation process (Fig. 6(b)).
  • PTMC 4 is selected, the length and width are 10.0mm, and the thicknesses are 0.10mm, 0.20mm, 0.30mm, 0.40mm, and 0.50mm, respectively. It can be found that the greater the thickness, the slower the degradation.
  • PTMC 4 when the thickness is 0.2 mm, the degradation rate in the enzyme solution in vitro satisfies our condition. Based on the combined factors of electrospinning, in vitro enzymatic solution degradation and mechanical properties, we believe that scaffolds with a molecular weight of 150,000 to 250,000 and a thickness of 0.2 mm are in line with our expectations. In this experiment, a PTMC 4 sample with a thickness of 0.2 mm was selected as an anastomotic scaffold and implanted into the cecum of mice to observe the subsequent in vivo degradation and healing promotion.
  • the microscopic morphology of the in vivo anastomotic stent was observed after being taken out at the corresponding time point (Fig. 6(c)).
  • the mass and length of the anastomotic stents decreased to varying degrees (Fig. 6(e), (f)).
  • the mass decreased by more than 40%, while the length decreased by 50%, achieving a mechanical support that lasted for two weeks.
  • After degraded in vivo, its morphology has begun to fragment, but the macroscopic whole remains well (Fig. 6(d)).
  • the wound healing process can be affected by bacterial infection, which will delay wound healing.
  • bacteria infection There are numerous microorganisms and bacteria in the gut, with the number of bacteria reaching 10 14 and more than 1000 species.
  • the pathogenic bacteria faced by intestinal healing have a higher density, and they will disrupt the normal physiological process of wound healing. Due to the particularity of the surgical site, it is difficult to maintain a relatively clean environment for the intestinal anastomosis. At this time, the antibacterial and bacterial isolation effects of the anastomotic stent play a very important role.
  • the antibacterial agent TCS was added to the sample in a certain proportion to make the material have antibacterial properties and ensure the relative cleanliness of the wound.
  • the samples without TCS did not have the ability to resist bacteria, and the samples with added TCS had obvious inhibition zones against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus.
  • the degradation of PTMC is a surface dissolution degradation, it is a process of gradually degrading from the surface to the interior, which allows TCS to be released slowly and has a sterilizing effect.
  • the anastomotic stent directly contacts the intestinal anastomosis site, if the material causes red blood cells to rupture, it often results in the release of adenosine diphosphate, which accelerates platelet aggregation and triggers thrombosis.
  • the hemolysis of the anastomotic stent was evaluated by direct contact of the in vitro material with blood. The experimental results are shown in Figure 8. The hemolysis rate is less than 0.1%, which is far lower than the upper limit of 5% for implantable medical devices.
  • L929 cells were used for cytotoxicity and cytocompatibility in vitro experiments to assess the cytocompatibility of pure PTMC with TCS-supplemented PTMC (Figure 9). L929 cells were incubated with the sample for 24h and 48h, and the cytotoxicity value of the sample added with TCS was not much different from that of the pure homopolymer, and the cell viability remained above 90%. It has a bactericidal effect without harming tissue cells.
  • the adhesion of the anastomotic stent-assisted healing group was significantly less than that of the blank control group, because the anastomotic stent effectively blocked the direct contact between the wound and the intestinal contents, reduced the occurrence of infection, and enabled the anastomotic stoma to have a better repair and healing speed.
  • the anastomotic burst pressure can effectively reflect the firmness of the anastomotic site healing after a period of postoperative intestinal anastomosis. This mechanical index can quantitatively reveal the amount of tension that the anastomotic stoma can withstand.
  • the balance between deposition of submucosa collagen synthesis and the rate of remodeling is a critical factor in the healing process of the gut. Both insufficient and excessive tissue repair can affect normal bowel function, with insufficient repair leading to ulcers and fistulas, and over repair leading to fibrosis and strictures.
  • the remodeling rate of collagen was much higher than the deposition rate in the first 4 days after surgery, starting on day 5 postoperatively, collagen deposition predominated, and finally reached a peak in the proliferative phase on day 7. Delay or damage to the peak of the proliferative phase can lead to anastomotic dehiscence. Excessive deposition of collagen and inflammation can lead to narrowing of the anastomosis. Therefore, on the 7th day after the operation, the adhesions were separated without affecting the local anastomosis, and then the cecum of the surgical operation segment was obtained, and the burst pressure test of the cecal segment of the anastomosis was performed (Fig. 11).
  • the burst pressure of the 20 surviving rats in the control group was 183 mmHg, which was lower than that of the 20 surviving rats in the anastomotic stent group by 190 mmHg (PTMC group) and 205 mmHg (TCS/PTMC group).
  • the anastomotic stent obviously promotes wound healing, and the addition of TCS isolates the wound from bacteria, which is beneficial to wound healing. There were statistical differences among the three groups (P ⁇ 0.001).
  • tissue damage During acute and chronic intestinal inflammation, macrophages and neutrophils induce local tissue damage by secreting reactive oxygen species and tissue-degrading enzymes. If tissue damage is severe, myofibroblasts migrate to the defect site. Inflammation is associated with the infiltration of immune cells, such as T cells, macrophages, and neutrophils, and it also frequently causes severe damage to the tissues where inflammation occurs. This ongoing inflammation and tissue degradation may thus lead to fibrosis and stenosis formation.
  • immune cells such as T cells, macrophages, and neutrophils
  • the healing process of tissue can be divided into inflammatory phase, proliferative phase and remodeling phase, and the three processes are not strictly defined [54] .
  • the seventh day is the node of the inflammatory and proliferative phases
  • the 14th day is the node of the proliferative and remodeling phases.
  • the inflammatory phase is characterized by the aggregation and infiltration of inflammatory cells dominated by neutrophils
  • the proliferative phase is characterized by an increase in the number of fibroblasts and the production of a large number of disordered weak collagen fibers.
  • TGF-beta transforming growth factor-beta
  • ⁇ -SMA ⁇ -smooth muscle actin
  • TGF- ⁇ is increased in myofibroblasts from fibrotic sites in patients with experimental enterocolitis and Crohn's disease.
  • Transforming growth factor can induce the expression of type I collagen, and can effectively stimulate the expression of ⁇ -SMA. Detecting the level of TGF- ⁇ in the wound can intuitively understand the speed and quality of the recovery of the wound.
  • tumor necrosis factor- ⁇ (TNF- ⁇ ) was selected as a monitoring indicator, and the efficacy of anastomotic stents in preventing infection was tested by immunohistochemical analysis.
  • intestinal anastomosis includes three processes: physical healing, histological healing and physiological healing.
  • Physical healing means that the intestinal cavity can be closed after anastomosis, the intestinal contents cannot enter the abdominal cavity, and the intestinal wall can withstand a certain pressure.
  • Histological healing refers to the complete integration of the anastomotic mucosal layers in histology.
  • the intestinal tract at both ends of the anastomosis restores its original innervation and realizes integrated and orderly intestinal relaxation and peristalsis. This process is called physiological healing.
  • anastomotic stents can effectively isolate unfavorable factors such as bacteria and viruses, and create a relatively clean environment for the wound, which is crucial for the healing of the anastomotic stoma.
  • Tension at the anastomosis is a major cause of poor healing. This tension can come from the tissue, or it can be caused by insufficient blood supply to persistently tight blood vessels.
  • the intestinal anastomosis stent prepared in this experiment has tissue flexibility, which can relieve this tension to a large extent, so the anastomotic stoma will have a better healing effect.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Surgery (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Vascular Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Pulmonology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Textile Engineering (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Cardiology (AREA)
  • Mechanical Engineering (AREA)
  • Transplantation (AREA)
  • Physiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medical Informatics (AREA)
  • Materials For Medical Uses (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

一种基于PTMC的生物可吸收柔性弹性体的肠吻合支架及其制备方法，采用静电纺丝方法，制备了PTMC为基材的肠道吻合支架，并根据降解速率与机械性能筛选出适合植入体内的合适范围。支架的PTMC均聚物材料负载具有杀菌作用的三氯生TCS，使支架具备组织修复调控功能，使得伤口在恶劣的多细菌肠道环境中实现更快的愈合，用于调控术后组织愈合与功能修复。进行体内动物肠道吻合实验，验证了实际效果和方法的可行性。

Description

基于PTMC的生物可吸收柔性弹性体的肠吻合支架及其制备方法 技术领域
本发明具体涉及高分子材料技术领域，具体涉及一种基于PTMC的生物可吸收柔性弹性体的肠吻合支架及其制备方法。
背景技术
胃肠道重建吻合术是腹部外科中最常见的手术操作之一，在消化道外科发展的近一个世纪来，吻合口瘘的发生率并未明显下降，一直成为困扰胃肠外科手术成功率的世界性难题之一。消化道良恶性肿瘤、消化道穿孔、消化道梗阻、出血、缺血等肠道病变，往往需要切除部分病变肠道后再进行吻合，传统方法多以手工缝合吻合，近几十年来多以管状吻合器进行端端或端侧吻合、或直线切割闭合器进行侧侧吻合。无论何种吻合方式，均无法防治吻合口瘘这一致死性并发症。
目前，国内外结直肠外科医师普遍接受和实践的，是暂时性改道转流手术，诸如暂时性回肠造口或结肠造口，此类附加手术可确切地避免因为吻合口瘘所导致的并发症，但尚无文献支持是否可以减少吻合口瘘的发生概率。然而，改道手术需要计划性二次手术行回纳，再次回纳同时也意味着再次的消化道重建与吻合，同样存在着吻合口瘘、吻合口狭窄等相关并发症的发生概率，但发生相比首次手术概率较低。在吻合口两端血供良好、对合无张力的情况下，实现肠道内容物，尤其是粪性内容物的在吻合口区域的隔离，实现相对隔绝、清洁的局部环境，是预防吻合口瘘以及诸如腹膜炎，腹腔脓肿等并发症的有效策略。其策略实现的关键技术瓶颈是理想辅助吻合材料的突破。
肠道吻合的目的是为了恢复吻合口两端肠道的物理学、组织学和生理学功能。目前，传统的吻合器存在的主要问题包括：(1)金属吻合器不可生物降解，导致体内永久性滞留；(2)可降解高分子材料吻合器，缺乏与创面组织之间的力学匹配性；(3)吻合器不具有组织修复调控功能，无法对肠道正常功能的恢复进行合理调控。如发明专利CN 111449707A提出一种肛肠吻合器，包括手柄座、传动组件、击发组件和吻切组件；传动组件包括设置于手柄座内部的丝杆以及设置于手柄座尾端并与所述丝杆尾端相连的调节机构；丝杆前端固定安装有抵钉座；击发组件包括设置于手柄座上的活动手柄以及套设于丝杆上的直推杆；吻切组件包括推钉片、钉仓套、钉仓和环形刀。该发明中推钉片、钉仓套和钉仓均采用金属材质制成，零件无法在体内降解，只能选择永久性滞留体内或者二次手术取出。专利CN109480943A由可降解材料制成，采用钉体穿孔固定的方式，并在钉体后端设计了支撑架，但是吻合环硬度 大、无弹性，不能很好地适应肠道蠕动，异物感明显。类似的还有发明专利CN103230265A，其选用了可降解材料聚乙交酯、聚丙交酯为原料，应用于胃肠道吻合。该吻合器具有易碎解的功能，但是同样缺乏与肠道组织的力学匹配性。理想的吻合器应具备以下特点：(1)有效隔离肠道内容物；(2)吻合器植入操作对吻合口肠壁破坏小；(3)操作简便易行。目前市场上的吻合装置均无法同时满足上述要求。
从生产的角度来看，支架必须易于制造成各种不同的长度和直径以适应不同的个体，并且不需要任何复杂的储存过程。所有这些都必须符合要求，同时保持支架的经济性和可承受性。
发明内容
为了解决现有技术存在的技术缺陷，本发明提供了一种基于PTMC的生物可吸收柔性弹性体的肠吻合支架及其制备方法，具有与肠道弹性相匹配的、具有组织修复调控功能，可以明显降低肠道吻合口瘘及其它并发症的发生概率。
本发明采用的技术解决方案是：一种基于PTMC的生物可吸收柔性弹性体的肠吻合支架,所述的肠吻合支架整体采用PTMC均聚物材料制成，所述的PTMC均聚物为高分子医用材料PTMC单体采用开环聚合的方法合成聚合物材料，所述的肠吻合支架的厚度为0.05-0.3mm。
所述的生物柔性弹性体肠吻合支架的PTMC均聚物材料负载三氯生(triclosan，TCS)。
所述的肠吻合支架内还设有植物纤维素管套，所述的肠吻合支架是无间隙套嵌式结构，内部为植物纤维素材料的管，外部为PTMC均聚物材料。
一种生物可吸收柔性弹性体的肠吻合支架的制备方法，其特征在于，通过以下步骤制备：
(1)PTMC的开环聚合：将TMC单体转移到反应容器中，在N2氛围下将催化剂Sn(Oct)2溶解在无水甲苯溶液中，用移液器取100ppm加入到反应容器中共聚反应，保证整个过程无水无氧，24h后将产物溶解，待完全溶解，将聚合物溶液进行提纯，重复多次，纯化后的共聚物真空干燥箱中干燥48h，然后储存在干燥柜中；
(2)静电纺丝制备吻合支架：将干燥后的样品溶解于CHCl 3/DMF混合溶液中，配置的溶液浓度为5-10.0％，加入混合溶液0.1～1.0wt％的抗菌剂，混合后37℃置于摇床待样品充分溶解，以获得均匀的共溶解纺丝原液，将原液装入2.5毫升的注射器，该注射器包括一根内径为0.5毫米的金属针，纺丝后样品厚度为0.2±0.01mm，所得纤维在真空干燥箱中室温进一步干燥，以除去残留的有机溶剂和水分。
所述的步骤(1)中产物溶解条件为用CHCl 3或DMF或THF进行溶解，置于摇床，摇 床温度设定为37℃。
所述的步骤(1)中提纯条件用正己烷或乙醇进行提纯，并且用玻璃棒不断搅拌。
所述的步骤(2)CHCl 3/DMF混合溶液中的CHCl 3/DMF＝1:1。
所述的步骤(2)的纺丝步骤具体为：将一定尺寸的植物纤维素管套在静电纺丝接收器上进行纺丝，控制参数可得到相应尺寸的管，所述的针头推速V＝1.0～5.0ml/h，辊轮转速V＝100～500RMP，温度T＝25～35℃；湿度WET＝20～40％。
本发明的有益效果是：本发明提供了一种基于PTMC的生物可吸收柔性弹性体的肠吻合支架及其制备方法，采用静电纺丝方法，制备了PTMC为基材的肠道吻合支架，并根据降解速率与机械性能筛选出适合植入体内的合适范围。负载具有杀菌作用的TCS，使其具备组织修复调控功能，使得伤口在恶劣的多细菌肠道环境中实现更快的愈合，用于调控术后组织愈合与功能修复。进行体内动物肠道吻合实验，以验证实际效果和方法的可行性。
附图说明
图1为体内实验过程；(1)盲肠切口；(2)吻合支架的植入；(3)间断法全层缝合。
图2为本发明PTMC合成工艺示意图。
图3为PTMC红外光谱图。
图4为是均聚物PTMC的 1H-NMR谱。
图5为静电纺丝样品的SEM显微图像。
图6为(A)不同分子量的PTMC膜在酶溶液中的降解时间，(B)不同分子量的PTMC膜经酶降解后脂肪酶溶液的pH曲线，(C)不同厚度的PTMC膜的降解时间，(D)吻合支架植入后的SEM，(E)吻合支架降解的物理图，(F)吻合支架在相应时间从大鼠身上的重量损失，(G)吻合支架在相应时间从大鼠身上的重量损失长度损失。
图7为不含三氯生的样品(上图)和含三氯生的样品(下图)的抗菌效果，A为金黄色葡萄球菌，B为大肠杆菌。
图8为不同样品的溶血率。
图9为样品的细胞毒性作用。
图10为肠吻合术后不同时间的腹部粘连评分。
图11为术后7天不同样品组的吻合口破裂压力。
图12为H&E染色和Masson染色结果。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整的描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发 明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获的的所有其他实施例，都属于本发明保护的范围。
材料
Poly(ethylene glycol)(PEG,Mn＝5000),辛酸亚锡((Sn(Oct) 2)，四氢呋喃(THF),N,-二甲基甲酰胺(DMF),三氯甲烷(CHCl 3),三氯生(TCS)，甲苯，正己烷，脂肪酶(Lipase from Aspergillus oryzae；solution,≥100,000U/g)were purchased from Sigma-Aldrich Co.LLC.Polymer grade 1,3-trimethylene carbonate(TMC,Daigang Biology,China)。所有试剂和化学品均为分析级，无需进一步纯化即可使用。
小鼠成纤维细胞系L929由中国科学院典型培养物保藏中心(中国上海)提供。培养皿购自康宁公司(纽约，美国)。在Dulbecco’s改良Eagle培养基(DMEM，Gibco)中添加10％胎牛血清(FBS，Gibco)，100IU/ml青霉素和100mg/ml硫酸链霉素进行培养。所有细胞均培养在37℃、5％CO 2、完全湿化条件下的培养箱中。
温州医科大学(中国温州)实验动物中心提供的雄性Sprague-Dawley大鼠(200±20g)，在25℃和湿度55％的条件养殖。所有动物实验均按照伦理学委员会评估并批准的指南进行。
PTMC为基材的肠道吻合支架的制备步骤
PTMC-b-PEG-b-PTMC的开环聚合
采用开环聚合的方法合成了PTMC均聚物。简而言之，将计量好的TMC单体转移到带有磁力搅拌棒的完全干燥的玻璃反应器中。在N 2氛围下将Sn(Oct) 2溶解在无水甲苯溶液中，用移液器取100ppm加入到反应容器中。共聚反应在130±2℃下反应24h，保证整个过程无水无氧。24h后将产物溶解于三氯甲烷中，待完全溶解，将聚合物溶液用过量的正己烷来提纯产品，重复3次。纯化后的均聚物在40℃的真空干燥箱中干燥48h，然后储存在干燥柜中。
静电纺丝制备吻合支架
将干燥后的样品溶解于CHCl 3/DMF(9/1,V/V)混合溶液中，制备的溶液浓度为5.5％，37℃置于摇床36h待样品充分溶解，以获得均匀的共溶解纺丝原液。将原液装入2.5毫升的注射器，该注射器包括一根内径为0.5毫米的金属针。具体的静电纺丝条件详见表1 [30]。纺丝后样品厚度为0.2±0.01mm。所得纤维在真空干燥箱中室温进一步干燥24小时，以除去残留的有机溶剂和水分。使用纺丝后的样品进行力学性能测试以及体外降解测试。
Table 1.静电纺丝条件
Figure PCTCN2022088873-appb-000001
表征
理化表征
用配备ATR附件的Nicolet Magna-560光谱仪测定了聚合物PTMC的FTIR-ATR光谱。用Bruker谱仪测定了均聚物PTMC的 1H-NMR光谱，所有的 1H-NMR均以四甲基硅烷(TMS)为内参比，以氘代氯仿(CDCl 3)为溶剂，以ppm为单位记录了样品的化学位移(D)。使用日立冷场发射电镜SU8010场发射扫描电子显微镜拍摄了样品静电纺丝之后的微观形貌、以及植入动物体内降解之后的微观形貌。使用DSC8000(美国PerkinElmer)进行DSC分析，以10℃/min的升温速率记录了聚合物PTMC的热性能。使用乌氏粘度计在25℃恒温水浴中测定PTMC的特性粘数，测试结果为三次实验的平均值应力应变在电子万能材料试验机(Instron 5944)上进行，静电纺丝后的样品处理为尺寸为45.0mm×25.0mm片状材料，SD大鼠盲肠尺寸为45.0mm×25.0mm×0.3mm，用生理盐水冲洗干净，擦拭掉表面多余水分。
体外酶降解
取尺寸为10.0×10.0mm PTMC膜，置于1mL脂肪酶溶液，放置于37℃空气浴中，每天振荡8h，振幅为65次/分。酶溶液每3天更换一次以保持酶的活性，分别在第1、5、10、15、20、30、40、50天后取出样品，随机抽取3个平行样条。用蒸馏水充分洗涤后，滤纸吸干表面水分，37℃真空干燥12h至恒质量。记录干燥样品的质量以及含有降解产物的介质的酸碱度。
体内生物降解行为，通过记录吻合支架植入前后的质量以及尺寸得到。吻合支架取出后使用蒸馏水清洗干净，滤纸吸干表面水分。失重率通过以下公式计算
Weight loss(％)＝(W 0－Wt)/W 0×100％
其中W 0和Wt分别表示样品降解前和降解后的干重。
生物学表征
均聚物的溶血性研究
样品材料事先用蒸馏水冲洗干净，擦拭净表明多余水分，使用新鲜人类全血。实验组：取静电纺丝样品15mg置于EP管，加入1mL生理盐水和0.1mL全血，阴性对照组：EP管中加入1mL生理盐水和0.1mL全血，阳性对照组：EP管中加入1mL超纯水和0.1mL全血。以上所有样品37℃孵育2h，进行溶血反应试验。
所有试样3000rpm离心10分钟，若上清液尚未清亮则再重复离心一次。拍照后吸取上清液移入孔板中，每个样品的上清液制三个平行样，每个样移取200μL，用721分光光度计在540nm波长处测定吸光度(OD)并记录结果。
数据处理：分别取样品组和对照组3个试样OD的均值。依公式计算样品的溶血率。
Figure PCTCN2022088873-appb-000002
其中H％是溶血率，OD t样品的吸光度，OD nc阴性对照样品的吸光度，OD pc阳性对照样品的吸光度。按照GB/T1423.2-1993标准，通过对材料与血红细胞在体外接触过程中，所致红细胞溶解和血红蛋白游离程度的测定，评价材料的体外溶血性，大于5％溶血反应为阳性。
细胞毒性研究
采用CCK-8法进行实验样品的毒理性研究，实验步骤如下：
细胞培养液配制：RPMI1640培养基500mL+胎牛血清50mL+青霉素/链霉素双抗5mL。
实验组浸提液的制备：样品事先使用75％酒精杀菌消毒，然后超净台紫外线反正面各照30min。静电纺丝样品分别裁成边长为1.82cm的正方形薄膜，加入2mL完全培养基，在37℃的环境下共浴24小时，获得纺丝样品浸提液。
细胞准备：用细胞培养液体外培养L929细胞(贴壁细胞)，增殖三代以上，待到长满培养瓶。PBS冲洗三遍(不要对着细胞，以防冲掉细胞)。之后用0.25％胰蛋白酶50μL将其消化30s(37℃)，使其变成细胞悬液。马上加入3-5mL完全培养基并转移到离心管，1000rpm离心5min，倒掉上层废液，向离心管中加入5mLPBS，移液管吹洗使细胞混散均匀。取1μL滴加到计数板上进行细胞计数，将细胞浓度调整至5×10 4个/mL。
共培养：将稀释后的L929细胞接种于96孔板中，每孔100μL，每孔需要5000-8000个细胞；在37℃、5％CO 2的环境中培养24小时，细胞完全贴壁后弃去培养液；分别将实验组浸提液以及阳性溶液(阳性对照组为10％DMSO(200μL))各100μL加入孔中，每组6个复孔，在37℃培养箱中孵育24h。
CCK-8检测：分别在预设时间点(24h、48h)取出96孔板，将原液吸出加到样品孔周围，于各孔中分别加入10μL的CCK-8试剂和100μL的完全培养基(先将两种溶液混合)，在37℃培养箱中孵育2h后，用多功能酶标仪(吸光度450nm)检测吸光度(OD)值。
细胞相对增殖率计算：取每组6孔OD值的平均值，按照如下公式计算各组的细胞相对增殖率(RGR)：
Figure PCTCN2022088873-appb-000003
式中：A s为实验孔的吸光度(有聚合物提取液、有细胞培养基、有CCK-8)；A c为对照孔的吸光度(无聚合物提取液、有细胞培养基、有CCK-8)；
A b为实验孔的吸光度(无聚合物提取液、无细胞培养基、有CCK-8)。
抗菌性研究
分别将冻存的大肠杆菌和金黄色葡萄球菌的细菌原液取50μL加到装有5mL细菌培养液的离心管中，细菌培养箱孵育24h待用。材料裁剪为直径是1.0cm的圆形片状材料，75％酒精冲洗干净后擦拭净表面多余水分，紫外消毒30min。分别取稀释后的细菌100μL均匀涂在培养基上，将圆形片状材料放在涂有细菌的培养基上，37℃细菌培养箱孵育24h。
体内生物相容性研究
大鼠体内实验
肠道吻合口处的愈合主要经过炎症反应、细胞增殖、肠壁结构重组等阶段，实现力学、组织学和功能学三方面的修复才能达到最终愈合。其中功能学方面的修复是一个相当漫长的过程，其中涉及消化吸收、内外分泌、神经修复及移行复合运动方面，因而力学和组织学必须达到指标，我们才认为吻合口完成愈合。结合手术的操作，在动物实验阶段需要达到以下指标：爆破压实验(力学愈合的指标)：腹腔黏连评分(体现吻合口附近局部炎症情况)；吻合口组织HE染色和Masson染色以及免疫组化染色(评估炎症细胞浸润程度，胶原沉积情况)。
实验前，将PTMC样品用75％酒精浸泡10min，紫外线杀菌消毒30min。通级别Sprague-Dawley雄性大鼠180只，体重200±10g，根据体重使用10％的水合氯醛进行腹腔注射麻醉。实验共设置三组，A组为PTMC组，B为TCS/PTMC组，C组为空白对照组，每组设置四个时间点，分别为7天、14天、21天、28天，设置平行组为5只大鼠。腹部剃毛剖腹找到大鼠盲肠，盲肠中上处进行切口，尺寸10±1mm，将盲肠内容物清理干净，A、B组放入实验样品随后缝合，C组直接进行缝合。吻合口处的缝合均采用4针单纯间断法全层缝合，以盲肠横截面为对象，分别在对应时钟位置的3、6、9、12各个位置全层间断缝合，针距约为0.4cm，间距约为0.5cm。术后两组大鼠麻醉清醒后即刻予以自由进食、饮水。
术后一鼠一笼独立饲养，密切观察老鼠的进食、排便以及行为活动情况。对比两组吻合方式所需时间，术后一般情况及死亡情况。各组大鼠分别在到达相应时间点后麻醉剖腹，观察并记录腹腔黏连情况，是否有腹腔感染以及有无吻合口漏现象。
腹腔黏连评分(Adhesion score)
对术后SD大鼠腹腔黏连进行分级评定 [31]，得到量化结果 [32]。评分标准为(0-3分)：
0分：无黏连；
1分：轻度黏连，仅仅在吻合口附近有上存在组织覆盖，易分离；
2分：中度黏连，吻合部位与腹腔内组织发生黏连，难以分离，但尚能分离；
3分：重度黏连，吻合口被腹腔内组织或其它脏器组织黏连包裹黏连。
吻合口爆破压
吻合口组织爆破压是检测吻合口愈合强度的重要的力学指标，它反映了肠道所能承受压力的大小，普遍被用来检测吻合口的愈合强度。
术后第7天进行吻合口肠段爆破压试验，采用体外测压法。切取吻合口及其周围约5cm肠管用生理盐水冲洗出肠内容物。适当分离腹腔粘连，暴露各个吻合口部位肠段，将肠管一端连接压力计(YB-150A精密压力表)，用两道丝线捆扎固定，跨过吻合口的另一端同样用两道丝线结扎封闭肠腔，并使肠管与压力计处于同一水平面。用蠕动泵以10mL/min的速度向肠管内匀速注入亚甲蓝稀释液(0.16mg/mL)，注意观察吻合口，并记录吻合口有蓝色液体溢出(或压力突然下降)时压力计上的读数，为吻合口爆破压。
H&E染色
H&E染色又称苏木素-伊红染色法，苏木素Hematoxylin，伊红Eosin。其基本原理是用碱性染料苏木素和酸性染料伊红分别与细胞核和细胞质发生作用，使细胞的微细结构通过颜色而改变它的折射率，从而在光镜下能清晰地呈现出细胞图像，并能提供良好的核浆对比染色。
检验原理：苏木素是蓝紫色的碱性染料，可以使细胞核着色，被苏木素着色的结构本身为酸性，具有嗜碱性(Basphili)。伊红是粉红色的酸性染料，可以将细胞质染成红色，被伊红着色的结构本身为碱性，具有嗜酸性(Acidophilic)。不易被苏木素和伊红着色的结构具有嗜中性(Neutrophilic)。
结果：细胞核呈蓝紫色，细胞质、肌纤维、胶原纤维、红细胞呈不同程度的红色，钙盐和细菌呈蓝色或蓝紫色。
分别于指定时间点处死大鼠，将纺丝支架及肠包裹组织取出，经4％甲醛溶液固定，乙醇溶液脱水后，石蜡包埋，切片(4μm)，进行H&E染色。染色后的切片通过光学显微镜观察。切片制备具体过程如下：
(1)取材，经固定后，常规石蜡包埋，4μm切片；
(2)切片常规用二甲苯脱蜡，经各级乙醇至水洗：二甲苯(I)5min→二甲苯(II)5min→100％乙醇2min95％的乙醇1min→80％乙醇1min→75％乙醇1min→蒸馏水洗2min；
(3)苏木素染色5min，自来水冲洗；
(4)盐酸乙醇分化30s；
(5)自来水浸泡15min或温水(约50℃)5min；
(6)置伊红液2min。
Masson染色
Masson染色主要用于胶原纤维和肌纤维的鉴别染色，用于观察病变组织中纤维结缔组织的增生和分布。染色结果为细胞核呈黑色，肌纤维呈红色，胶原纤维呈蓝色。步骤：
组织固定于10％中性福尔马林溶液，流水冲洗，常规脱水包埋；
(1)切片常规脱蜡至水；
(2)取适量的Weigert铁苏木素A液和Weigert铁苏木素B液等量混合，即为Weigert铁苏木素染色液。用配制好的Weigert铁苏木素染色液染色5min-10min；
(3)酸性乙醇分化液分化5-15s，水洗；
(4)Masson蓝化液返蓝3-5min，水洗；
(5)蒸馏水洗1min；
(6)丽春红品红染色液染色5-10min；
(7)按照蒸馏水：弱酸溶液＝2:1的比例配置弱酸工作液，用弱酸工作液洗1min；
(8)磷钼酸溶液洗1-2min；
(9)用配置好的弱酸工作液洗1min；
(10)95％乙醇快速脱水；
(11)无水乙醇脱水三次，每次5-10s；
(12)二甲苯透明3次，每次1-2min。
免疫组化染色
石蜡切片脱蜡至水,随后组织切片置于盛有柠檬酸抗原修复缓冲液(PH6.0)的高压锅内进行抗原修复。用3％过氧化氢溶液(双氧水：纯水＝1:9)阻断内源性过氧化物酶，加入加用3％BSA进行封闭，然后在4℃与一抗一起孵育过夜。加入与一抗相应种属的二抗室温孵育50min，细胞核用苏木素染色。每次孵育后，将细胞用PBS洗涤两次。用荧光显微镜(NIKON ECLIPSE TI-SR)对染色的细胞照相。
结果和讨论
PTMC的合成及其表征结果
在Sn(Oct) 2催化下，通过开环聚合合成了PTMC均聚物(图2)。共聚反应如表2所示。
不同分子量的均聚物其降解速率和力学性能差异很大，高分子量的PTMC具有更好的降解速率以及形状保持度，且形状的不同对其降解速率影响很大 [35]。植入肠道中要求产品具有合适的降解速度和优异的力学性能，因而本实验探讨了反应时间对PTMC的分子量的影响。控制反应时间以得到不同分子量的PTMC。PTMC分子量与玻璃化转变温度结果见表1。数据表明，随着分子量增大PTMC的玻璃化转变温度降低，这是因为分子量低结晶度增高从而Tg增大。
PTMC的红外光谱如图3所示。在PTMC观察到-CH 2-(2970和2909cm -1)和C＝O(1735cm -1)以及-O-(1218cm -1)的伸缩振动 [36]。图4是均聚物PTMC的 1H-NMR谱。它清楚地显示出化学位移δ在4.171ppm(a)属于PTMC块中氧旁边的亚甲基质子，δ在1.984ppm(b)属于PTMC块中的其他亚甲基质子，且峰的面积之比与产物一致。
表2 PTMC聚合物的合成反应时间和物理数据
Figure PCTCN2022088873-appb-000004
a.Determined by[η]＝KM α，K＝1.986×10 -4，α＝0.789 [37]
静电纺丝样品及其力学性能评价
静电纺丝纤维分布均匀，直径在5μm-10μm之间(图5)。
用PTMC静电纺丝制备的吻合支架的机械性能列于表4。支架材料在生理条件下力学性能是作为植入材料的一个重要指标，为了观测材料在生理条件下的力学性能，在测试前，将样品浸泡在PBS溶液中24h，发现支架的机械性能差别不大，拉伸强度和弹性模量稍有降低，断裂伸长率都一定程度的增加，这是因为支架疏松多孔的结构吸水所导致。分子量高的PTMC具有好的机械性能和形状保持度，PTMC 1与PTMC 2的分子量过低，无法达到一个好的支撑的作用。当反应时间超过30h后，随反应时间的增加，PTMC的力学性能差异不是很大。PTMC作为吻合支架在干湿态下机械性能都很稳定，这保证它在实际应用中的可靠性。
表4.PTMC聚合物的机械性能
Figure PCTCN2022088873-appb-000005
a.弹性模量(已制造)，b.弹性模量(预湿24小时)，c.抗张强度(制造时)，d.抗张强度(预湿24小时)，e.断裂伸长率(制造)，f.断裂伸长率(预湿24h)
将干燥后的样品溶解于chloroform/DMF(9/1,V/V)混合溶液中，制备的溶液浓度为5.5％，37℃置于摇床36h待样品充分溶解，以获得均匀的共溶解纺丝原液。将原液装入2.5毫升的注射器，该注射器包括一根内径为0.5毫米的金属针。具体的静电纺丝条件详见表1。纺丝后样品厚度由具体电纺时间决定。所得纤维在真空干燥箱中室温进一步干燥24小时，以除去残留的有机溶剂和水分。使用纺丝后的样品进行力学性能测试以及体外降解测试。
静电纺丝条件
Figure PCTCN2022088873-appb-000006
Figure PCTCN2022088873-appb-000007
体外生物降解评价
PTMC主要通过体外酶解和体内表面侵蚀降解。Feijen等均认为酶起到了界面活化作用，因而体外酶溶液中的降解速度快于体内降解。相同尺寸而不同厚度的PTMC材料其降解速率差异十分大。作为肠道支架，我们研究了静电纺丝后不同分子量以及不同厚度的膜状材料的降解差异。
本次动物实验的对象是雄性大鼠，大鼠肠道愈合周期为14天左右，因而植入肠道的吻合支架需满足至少持续两周的力学性能强度，以及三周左右降解的要求。首先研究了分子量对失重率的影响(图6(a))，将PTMC 1-7裁剪为同同尺寸(10.0×10.0×0.4mm)的膜片，结果显示随着PTMC分子量的增加，降解加快，且降解过程无酸性物质(图6(b))。当PTMC分子量过低时，如PTMC 1和PTMC 2所显示的那样，在50天时仅失重20％，降解太慢；分子 量在15万以上时，PTMC在50天的的失重可达到60％，符合植入大鼠肠道的条件。其次我们研究了不同厚度的材料对失重率的影响(图6(C))。选择PTMC 4，长度与宽度为10.0mm，厚度分别为0.10mm、0.20mm、0.30mm、0.40mm、0.50mm。可以发现厚度越大，降解越慢。对于PTMC 4，当厚度为0.2mm，体外酶溶液中的降解速率是满足我们的条件的。在基于静电纺丝与体外酶溶液降解以及机械性能综合因素，我们认为分子量在15万～25万内、厚度为0.2mm的支架是符合我们的预期的。本实验选取厚度为0.2mm的PTMC 4样品作为吻合支架植入老鼠盲肠，来观测后续体内降解与促愈合的作用。
体内吻合支架于相应时间点取出后观测微观形貌(图6(c))。吻合支架的质量和长度均有不同程度的减小(图6(e),(f))，28天之后质量减少40％以上，而长度则减少50％，达到了持续两周的力学性能支持。体内降解后其形貌已经开始碎裂，但是宏观整体保持较好(图6(d))。
体外生物评价
抗菌
众所周知，伤口愈合过程可能会受到细菌感染，这将延迟伤口愈合。肠道中微生物与细菌数不胜数，细菌数量级别达到10 14，种类超过1000种，与其他上皮相比，肠道愈合面临的致病菌具有更高的密度，它们会扰乱正常的伤口愈合生理过程。由于手术部位的特殊性，肠道吻合口保持一个相对干净的环境很难，这时吻合支架的抗菌与隔菌效果便起到了十分重要的作用。
为了解决这个问题，将抗菌剂TCS以一定比例添加到样品中，使材料具备抗菌性，保证伤口处的相对清洁。如图7所示，不含TCS的样品没有抵抗细菌的能力，添加了TCS的样品对铜绿假单胞菌、大肠杆菌和金黄色葡萄球菌有明显的抑制区。由于PTMC降解是表面溶蚀降解，是一个由表面渐渐深入内部降解的过程，这使得TCS可以缓慢释放出来并一直起到杀菌的作用。
溶血率
由于吻合支架直接接触肠吻合口部位，如果材料导致红细胞破裂，往往会导致二磷酸腺苷被释放，加速血小板聚集，从而引发血栓。通过体外材料直接接触血液，对吻合支架的溶血性进行评估，实验结果如图8所示，其溶血率为不到0.1％，远低于植入型医疗器械5％的上限值。
细胞毒性
L929细胞用于细胞毒性和细胞相容性的体外实验，以评估纯PTMC与添加TCS的PTMC的细胞相容性(图9)。L929细胞与样品一起孵育24h、48h，且添加TCS的样品细胞毒性数值与单纯均聚物相差不大，细胞存活率均保持在90％以上，这表明TCS的添加量是有效可 行的，能够在起到杀菌作用的同时不会对组织细胞产生伤害。
伤口恢复情况评价
腹腔黏连评分以及吻合口爆破压
分别于术后第7、14、21、28天剖腹，对于腹腔黏连情况做评分，结果详见图10。当腹腔由于损伤刺激或者感染时，局部会产生一种纤维蛋白原的胶状液，它很快会转变成纤维蛋白的凝结物并覆盖在创伤的黏膜表面，起到修复保护作用。纤维蛋白具有较大的粘附性，会使得相互贴近的腹腔粘膜连在一起。创伤愈合后，如果机体能很好地吸收掉这些纤维蛋白，就不会有痕迹。如果吸收不全，黏连则会持续存在，严重的情况会成为粘连性肠梗阻，影响肠道正常生理活动。吻合支架辅助愈合组黏连情况明显少于空白对照组，这是因为吻合支架有效阻隔了伤口与肠道内容物的直接接触，减少了感染的发生，使得吻合口具有更好的修复愈合速度。
吻合口爆破压能够有效反映肠道吻合口术后愈合一段时间后，吻合口部位愈合的牢固度，通过该力学指标，能够定量地揭示吻合口所能承受的张力大小。在肠道愈合的过程中，粘膜下层胶原合成的沉积和重塑速率之间的平衡是关键的因素。组织修复不足和过度都会影响正常的肠道功能，修复不足会造成溃疡和瘘，过度修复则会导致纤维化和狭窄。术后前4天，胶原的重塑速率远高于沉积速率，术后第5天开始，胶原沉积占主导地位，最终在第7天达到增殖期峰值。达到增殖期峰值的延迟或损伤可导致吻合口裂开。胶原蛋白的过渡沉积和炎症会导致吻合口的狭窄。因而选择术后第7天，在不影响吻合口局部的情况下，分离黏连，然后取得手术操作段盲肠，行吻合口盲肠段爆破压试验(图11)。对照组存活20只的大鼠的爆破压为183mmHg，低于吻合支架组存活的20只大鼠190mmHg(PTMC组)，205mmHg(TCS/PTMC组)。吻合支架明显对伤口愈合起到了促进作用，且TCS的加入使伤口处与细菌隔绝，有利于伤口愈合。三组存在统计学差异(P<0.001)。
组织学分析
在急性和慢性肠道炎症期间，巨噬细胞和中性粒细胞通过分泌活性氧自由基和组织降解酶诱导局部组织损伤。如果组织损伤严重，肌成纤维细胞会迁移到缺损部位。炎症与免疫细胞的浸润有关，如T细胞、巨噬细胞和中性粒细胞，它也经常对发生炎症的组织造成严重损害。这种持续的炎症和组织降解可能因此导致纤维化和狭窄的形成。
术后相应时间点，我们将吻合口附近肠壁组织进行H&E和Masson染色(图12)。按照时间顺序，组织的愈合过程可以分为炎症期、增生期和重塑期，三个过程没有严格的界定 [54]。一般而言，第七天是炎症期和增生期的节点，14天是增生期和重塑期的节点。炎症期表现为以中性粒细胞为主的炎性细胞的聚集和浸润；增生期表现为成纤维细胞数量增加、产生大量 无序排列的弱胶原纤维，重塑期急性炎症明显减少，取而代之的是慢性炎症的标志——多核巨细胞的产生，胶原纤维明显增多。HE染色可以放映炎性细胞浸润程度。在术后对应时间点，对照组炎症细胞浸润明显高于吻合支架辅助组，添加TCS的吻合支架组的炎症细胞明显比没有添加TCS的吻合支架组少。Masson染色也验证了上述规律，支架组因为隔绝细菌减少了炎症反应，有利于纤维的再生，而TCS的添加进一步促进了伤口愈合。
免疫组化
伤口的修复由细胞分泌的生长因子完成，如转化生长因子-β(TGF-β)。TGF-β是α-平滑肌肌动蛋白(α-SMA)最有效和最重要的诱导因子。在实验性小肠结肠炎和克罗恩病患者的纤维化部位的肌成纤维细胞中，TGF-β增加。转化生长因子都能诱导i型胶原的表达，并且能有效地刺激α-SMA的表达。对伤口处TGF-β水平做检测，可以直观了解伤口恢复程度的快慢与好坏。
由于伤口感染是受伤患者死亡的主要原因之一，因此选择肿瘤坏死因子-α(TNF-α)作为监测指标，通过免疫组织化学分析来测试吻合支架在预防感染方面的功效。
结论
吻合后肠道的愈合是一个复杂而漫长的生理学过程。实际上，肠道吻合包括物理愈合、组织学愈合及生理学愈合三个过程。物理学愈合是指吻合后的肠道能够肠腔的封闭，肠道内容物不能进入腹腔，肠壁可以承受一定的压力。组织学愈合是指吻合口粘膜层在组织学上完成结合。吻合口两端的肠道恢复原有的神经支配，实现一体化有序地肠道舒缩和蠕动，这个过程称为生理学愈合。
吻合支架的植入，可以有效隔绝细菌病毒等不利因素，为伤口创造一个相对清洁干净的环境，这对吻合口的愈合至关重要。
吻合处的张力是导致愈合不良的主要原因。这种张力可能来自于组织，也可能是因为持续紧绷的血管供血不足所导致。本实验制备的肠道吻合支架具备组织柔顺性，在很大程度上可以缓解这种张力，因而吻合口会有一个更好的愈合效果。
各位技术人员须知：虽然本发明已按照上述具体实施方式做了描述，但是本发明的发明思想并不仅限于此发明，任何运用本发明思想的改装，都将纳入本专利专利权保护范围内。
以上所述仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (8)

  1. 一种基于PTMC的生物可吸收柔性弹性体的肠吻合支架,其特征在于,所述的肠吻合支架整体采用PTMC均聚物材料制成，所述的PTMC均聚物为高分子医用材料PTMC单体采用开环聚合的方法合成聚合物材料，所述的肠吻合支架的厚度为0.05-0.3mm。
  2. 根据权利要求1所述的生物可吸收柔性弹性体的肠吻合支架，其特征在于，所述的生物柔性弹性体肠吻合支架的PTMC均聚物材料负载三氯生(triclosan，TCS)。
  3. 根据权利要求1所述的生物可吸收柔性弹性体的肠吻合支架，其特征在于，所述的肠吻合支架内还设有植物纤维素管套，所述的肠吻合支架是无间隙套嵌式结构，内部为植物纤维素材料的管，外部为PTMC均聚物材料。
  4. 一种权利要求1所述的生物可吸收柔性弹性体的肠吻合支架的制备方法，其特征在于，通过以下步骤制备：
    (1)PTMC的开环聚合：将TMC单体转移到反应容器中，在N2氛围下将催化剂Sn(Oct)2溶解在无水甲苯溶液中，用移液器取100ppm加入到反应容器中共聚反应，保证整个过程无水无氧，24h后将产物溶解，待完全溶解，将聚合物溶液进行提纯，重复多次，纯化后的共聚物真空干燥箱中干燥48h，然后储存在干燥柜中；
    (2)静电纺丝制备吻合支架：将干燥后的样品溶解于CHCl 3/DMF混合溶液中，配置的溶液浓度为5-10.0％，加入混合溶液0.1～1.0wt％的抗菌剂，混合后37℃置于摇床待样品充分溶解，以获得均匀的共溶解纺丝原液，将原液装入2.5毫升的注射器，该注射器包括一根内径为0.5毫米的金属针，纺丝后样品厚度为0.2±0.01mm，所得纤维在真空干燥箱中室温进一步干燥，以除去残留的有机溶剂和水分。
  5. 根据权利要求1所述的制备方法，其特征在于，所述的步骤(1)中产物溶解条件为用CHCl 3或DMF或THF进行溶解，置于摇床，摇床温度设定为37℃。
  6. 根据权利要求1所述的制备方法，其特征在于，所述的步骤(1)中提纯条件用正己烷或乙醇进行提纯，并且用玻璃棒不断搅拌。
  7. 根据权利要求1所述的制备方法，其特征在于，所述的步骤(2)CHCl 3/DMF混合溶液中的CHCl 3/DMF＝1:1。
  8. 根据权利要求1所述的制备方法，其特征在于，所述的步骤(2)的纺丝步骤具体为：将一定尺寸的植物纤维素管套在静电纺丝接收器上进行纺丝，控制参数可得到相应尺寸的管，所述的针头推速V＝1.0～5.0ml/h，辊轮转速V＝100～500RMP，温度T＝25～35℃；湿度WET＝20～40％。
PCT/CN2022/088873 2021-04-30 2022-04-25 基于ptmc的生物可吸收柔性弹性体的肠吻合支架及其制备方法 WO2022228356A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2023565596A JP2024515141A (ja) 2021-04-30 2022-04-25 Ｐｔｍｃに基づく生体吸収性柔軟性エラストマー腸吻合ステント及びその製造方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110487743.6A CN113288505B (zh) 2021-04-30 2021-04-30 基于ptmc的生物可吸收柔性弹性体的肠吻合支架及其制备方法
CN202110487743.6 2021-04-30

Publications (1)

Publication Number Publication Date
WO2022228356A1 true WO2022228356A1 (zh) 2022-11-03

Family

ID=77320690

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/088873 WO2022228356A1 (zh) 2021-04-30 2022-04-25 基于ptmc的生物可吸收柔性弹性体的肠吻合支架及其制备方法

Country Status (3)

Country Link
JP (1) JP2024515141A (zh)
CN (1) CN113288505B (zh)
WO (1) WO2022228356A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113288505B (zh) * 2021-04-30 2023-02-03 中国科学院大学温州研究院(温州生物材料与工程研究所) 基于ptmc的生物可吸收柔性弹性体的肠吻合支架及其制备方法
CN113893390B (zh) * 2021-10-12 2023-12-01 国科温州研究院(温州生物材料与工程研究所) 一种新型肠道柔性吻合支架

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19926008A1 (de) * 1999-06-08 2000-12-14 Peter Sterk Biofragmentabler Stent
CN101133973A (zh) * 2007-10-09 2008-03-05 浙江铭众科技有限公司 胃肠吻合支架及其制备方法
US20100298928A1 (en) * 2007-10-19 2010-11-25 Micell Technologies, Inc. Drug Coated Stents
CN102989044A (zh) * 2012-12-10 2013-03-27 辽宁省计划生育科学研究院 一种可生物降解医用高分子管材及其制备方法
CN105944153A (zh) * 2016-05-24 2016-09-21 德州海利安生物科技股份有限公司 显影型可降解修复胆道支架
CN108577916A (zh) * 2018-03-08 2018-09-28 戴庆涛 一种胃肠外科的手术用吻合器的制备方法
CN109966567A (zh) * 2017-12-27 2019-07-05 天津大学 一种可控降解支架材料及其制备方法
CN113288505A (zh) * 2021-04-30 2021-08-24 中国科学院大学温州研究院(温州生物材料与工程研究所) 基于ptmc的生物可吸收柔性弹性体的肠吻合支架及其制备方法
CN113413491A (zh) * 2021-04-30 2021-09-21 中国科学院大学温州研究院(温州生物材料与工程研究所) 基于PTMC-b-PEG-b-PTMC共聚物的生物柔性弹性体肠吻合支架及制备方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8299316B2 (en) * 2007-12-18 2012-10-30 Ethicon, Inc. Hemostatic device
US20090163936A1 (en) * 2007-12-21 2009-06-25 Chunlin Yang Coated Tissue Engineering Scaffold

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19926008A1 (de) * 1999-06-08 2000-12-14 Peter Sterk Biofragmentabler Stent
CN101133973A (zh) * 2007-10-09 2008-03-05 浙江铭众科技有限公司 胃肠吻合支架及其制备方法
US20100298928A1 (en) * 2007-10-19 2010-11-25 Micell Technologies, Inc. Drug Coated Stents
CN102989044A (zh) * 2012-12-10 2013-03-27 辽宁省计划生育科学研究院 一种可生物降解医用高分子管材及其制备方法
CN105944153A (zh) * 2016-05-24 2016-09-21 德州海利安生物科技股份有限公司 显影型可降解修复胆道支架
CN109966567A (zh) * 2017-12-27 2019-07-05 天津大学 一种可控降解支架材料及其制备方法
CN108577916A (zh) * 2018-03-08 2018-09-28 戴庆涛 一种胃肠外科的手术用吻合器的制备方法
CN113288505A (zh) * 2021-04-30 2021-08-24 中国科学院大学温州研究院(温州生物材料与工程研究所) 基于ptmc的生物可吸收柔性弹性体的肠吻合支架及其制备方法
CN113413491A (zh) * 2021-04-30 2021-09-21 中国科学院大学温州研究院(温州生物材料与工程研究所) 基于PTMC-b-PEG-b-PTMC共聚物的生物柔性弹性体肠吻合支架及制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AJIRO HIROHARU; HARAMIISHI YOSHIAKI; CHANTHASET NALINTHIP; AKASHI MITSURU: "Polymer design using trimethylene carbonate with ethylene glycol units for biomedical applications", POLYMER JOURNAL, NATURE PUBLISHING GROUP UK, LONDON, vol. 48, no. 7, 1 January 1900 (1900-01-01), London , pages 751 - 760, XP037325147, ISSN: 0032-3896, DOI: 10.1038/pj.2016.35 *

Also Published As

Publication number Publication date
CN113288505B (zh) 2023-02-03
CN113288505A (zh) 2021-08-24
JP2024515141A (ja) 2024-04-04

Similar Documents

Publication Publication Date Title
WO2022228357A1 (zh) 基于PTMC-b-PEG-b-PTMC共聚物的生物柔性弹性体肠吻合支架及制备方法
WO2022228356A1 (zh) 基于ptmc的生物可吸收柔性弹性体的肠吻合支架及其制备方法
Lee et al. Tissue anti-adhesion potential of ibuprofen-loaded PLLA–PEG diblock copolymer films
JP2003517352A (ja) 吸収性のポリオキサエステルによる接着を防止する方法
Yamaoka et al. Novel adhesion prevention membrane based on a bioresorbable copoly (ester‐ether) comprised of poly‐L‐lactide and Pluronic®: In vitro and in vivo evaluations
CN105194735A (zh) 一种脱细胞生物羊膜及种以及京尼平交联脱细胞生物羊膜的制备方法
WO2011150328A1 (en) Wet-electrospun biodegradable scaffold and uses therefor
JPH03502651A (ja) 反復カーボネート単位を含むホモポリマーおよびコポリマーから製造された医療用具
Li et al. Reconstruction of abdominal wall with scaffolds of electrospun poly (L-lactide-co caprolactone) and porcine fibrinogen: An experimental study in the canine
Shamirzaei Jeshvaghani et al. Fabrication, characterization, and biocompatibility assessment of a novel elastomeric nanofibrous scaffold: A potential scaffold for soft tissue engineering
Wu et al. Tannic acid-loaded hydrogel coating endues polypropylene mesh with hemostatic and anti-inflammatory capacity for facilitating pelvic floor repair
KR102048418B1 (ko) 3d 프린팅으로 형성되며 장막-배양으로 보강된 인공 식도 및 그 제조방법
Wu et al. Bioabsorbable flexible elastomer of PTMC‐b‐PEG‐b‐PTMC copolymer as intestinal anastomosis scaffold
Liu et al. Sutureless transplantation using a semi-interpenetrating polymer network bioadhesive for ocular surface reconstruction
Wei et al. A novel tetra-PEG based hydrogel for prevention of esophageal stricture after ESD in a porcine model
CN111714696A (zh) 一种复合肌腱防粘连膜及制备方法
CN107007620B (zh) 对氧环己酮与l-苯丙氨酸共聚物及其应用
CN103495211B (zh) 用于心脏手术的可吸收防粘连膜及其制备方法
Ren et al. Poly (trimethylene carbonate) flexible intestinal anastomosis scaffolds to reduce the probability of intestinal fistula and obstruction
CN114053486B (zh) 一种可吸收生物活性膜及其制备方法和用途
CN113599576A (zh) 一种可降解气管外支架及其制备方法
CN112755255A (zh) 一种生物可降解的多孔医用支架材料及其制备方法
Xu et al. Biodegradation of poly (l-lactide-co-glycolide) tube stents in bile
Horii et al. Regeneration of functional bladder using cell-seeded amnion and P (LA/CL) scaffolds
Wang et al. Anti‐Adhesive, Platelet Gathering Effects of c‐RGD Modified Poly (p‐dioxanone‐co‐l‐Phe) Electrospun Membrane and Its Comprehensive Application in Intestinal Anastomosis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22794826

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023565596

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22794826

Country of ref document: EP

Kind code of ref document: A1