WO2018023840A1 - 一种人造血管及其制备方法 - Google Patents

一种人造血管及其制备方法 Download PDF

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WO2018023840A1
WO2018023840A1 PCT/CN2016/096307 CN2016096307W WO2018023840A1 WO 2018023840 A1 WO2018023840 A1 WO 2018023840A1 CN 2016096307 W CN2016096307 W CN 2016096307W WO 2018023840 A1 WO2018023840 A1 WO 2018023840A1
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blood vessel
artificial blood
heparin
autologous
solution
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PCT/CN2016/096307
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French (fr)
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董念国
邱雪峰
王滔
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华中科技大学同济医学院附属协和医院
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Publication of WO2018023840A1 publication Critical patent/WO2018023840A1/zh

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    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3625—Vascular tissue, e.g. heart valves
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3679—Hollow organs, e.g. bladder, esophagus, urether, uterus, intestine
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
    • 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
    • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
    • A61L33/0005—Use of materials characterised by their function or physical properties
    • A61L33/0011—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
    • 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
    • A61L2300/23—Carbohydrates
    • A61L2300/236—Glycosaminoglycans, e.g. heparin, hyaluronic acid, chondroitin
    • 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
    • A61L2300/42—Anti-thrombotic agents, anticoagulants, anti-platelet 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
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

  • the invention belongs to the field of tissue engineering blood vessels, and more particularly to an artificial blood vessel and a preparation method thereof.
  • autologous blood vessels such as the saphenous vein and the internal mammary artery are the most important bridge vessels for coronary artery bypass surgery and other small-caliber vascular bypass surgery.
  • the two small-calibre vessels that are currently approved by the US Food and Drug Administration (FDA) for clinical trials are:
  • Nicolas L'Heureux et al. applied the acellular matrix-engineered blood vessels without synthetic materials to the clinic for the first time.
  • the 1-month clinical patency of the venous fistula was 78% (7/9), and the 6-month patency rate was 60% (5/8).
  • Nicolas L’Heureux et al. prepared the autologous acellular matrix tube, the preparation process was complicated. The preparation of a pipeline took 7-9 months, costing 15,000 US dollars, and the long-term patency rate was not ideal, which was difficult to be applied to the clinic.
  • the tubular structure of the fiber finally decellularized to form tissue engineered blood vessels with collagen fibers as the main component (containing a small amount of residual PGA), ie, allogeneic acellular matrix tubing, large animal arteriovenous fistula transplantation experiment 6 months patency rate 100 %(3/3), the FDA approved a 6-month patency rate of 63% in the second phase clinical trial, and 28%, 18%, and 15% in the 12, 18, and 24 months, respectively.
  • the surrounding cells can not enter the wall to participate in the remodeling.
  • the extracellular matrix component of the tube wall can not be effectively renewed, especially the elastic fiber that maintains the elasticity of the arterial wall, which seriously affects the mechanical properties. And patency rate.
  • the present invention provides an artificial blood vessel and a preparation method thereof, which are characterized in that an artificial blood vessel is obtained by subjecting an autologous tissue tube to decellularization and covalently binding heparin modification, thereby
  • the invention solves the technical problem that the prior art artificial blood vessel has poor mechanical properties, weak anticoagulant function and low vascular patency rate due to immune reaction.
  • an artificial blood vessel comprising a tube wall composed of an autologous extracellular matrix covalently bound to an anticoagulant molecule.
  • the anticoagulant molecule is heparin, and the heparin content is 5 to 10 micrograms per milligram of artificial blood vessel.
  • the artificial blood vessel wall has a thickness of 124.9 to 690.5 microns, preferably a thickness of 400 to 650 microns.
  • the wall burst pressure is 3157 ⁇ 216 mmHg
  • the stitching tension is 3.94 ⁇ 0.46N
  • the maximum tensile stress is 2.41 ⁇ 0.22 MPa
  • the maximum tensile strain is 30.63 ⁇ 2.74%.
  • a method of preparing an artificial blood vessel comprising the steps of:
  • Decellularization treatment the autologous tissue tube is removed by chemical decellularization of 3-[(3-cholamidopropyl)-diethylamine]-propanesulfonic acid reagent to obtain the autologous acellular matrix tube. ;
  • the anticoagulant molecule is covalently bound to the surface of the autologous acellular matrix tube obtained in the step (1) to obtain an artificial blood vessel.
  • the step (1) comprises the following steps:
  • (1-2) Decellularization treatment the autologous tissue tube is placed in the decellularization reagent equipped with the step (1-1), and treated at room temperature for 2 to 3 hours, and then replaced every 2 to 3 hours.
  • the cell reagent is replaced 4 to 6 times, preferably 5 times, to obtain an autologous acellular matrix tube.
  • the step (2) comprises the following steps:
  • the autologous acellular matrix tube obtained in the step (1) is placed in a container containing the heparin grafting reagent, and the artificial blood vessel is obtained by shaking for 3 to 5 hours.
  • the concentration of EDC in the step (2-1) heparin grafting reagent is preferably 40 mg/ml
  • the concentration of the Sulfo-NHS is preferably 20 mg/ml
  • the concentration of the heparin is preferably 60 mg/ml.
  • the pH of the MES buffer in the step (2-1) is 5.5.
  • the microfilter pore size of the step (c) in the step (2-1) is 0.2 ⁇ m.
  • the present invention utilizes the principle of foreign body reaction to prepare a self-organized tissue tube by placing a non-toxic, non-degradable Teflon tube under the skin, and using the same as a raw material to prepare the artificial blood vessel of the present invention, and the tissue structure and mechanical properties are close to normal arteries.
  • tissue structure and mechanical properties are close to normal arteries.
  • the CHAPS+EDTA decellularization method used in the present invention is relatively mild and has good preservation of the structure and mechanical properties of the extracellular matrix, and has good decellularization effect.
  • the decellularized treatment allows the autologous acellular matrix wall structure to allow surrounding cells to enter.
  • the wall of the tube participates in the reconstruction of the wall, effectively synthesizes elastic fibers and collagen fibers, improves the mechanical properties and patency rate after remodeling in vivo, and is close to the structure and mechanical properties of normal autologous arteries.
  • the present invention uses Sulfo-NHS/EDC-mediated heparin covalently bound to tissue engineered blood vessels without cytotoxicity in vivo, has good biocompatibility, good anticoagulant function, and high patency rate.
  • the artificial blood vessel of the invention has short preparation period, low cost, and can be prepared into different inner diameters, and is completely superior to the previous acellular matrix pipeline, and has good clinical application prospect.
  • Example 1 is a test result of bursting pressure of an autologous tissue pipe before and after decellularization according to Example 4;
  • Example 2 is a test result of tensile strength of a self-organized tissue suture before and after decellularization according to Example 4;
  • Example 3 is a test result of maximum tensile stress of an autologous tissue tube before and after decellularization according to Example 4;
  • Example 4 is a test result of maximum tensile strain of an autologous tissue tube before and after decellularization according to Example 4;
  • Figure 5 is a result of ultrasound examination 1 month after artificial blood vessel transplantation described in Example 5, A is a lateral vascular graft of the artificial blood vessel, and B is a contralateral normal carotid artery;
  • Figure 6 is a result of ultrasound examination 2 months after the artificial blood vessel transplantation described in Example 5, A is The artificial carotid artery was transplanted into the lateral carotid artery, and B was the contralateral normal carotid artery;
  • Example 7 is a CT angiographic examination result 1 month after the artificial blood vessel transplantation described in Example 5;
  • Example 8 is a result of DAPI staining of autologous tissue tubes before and after decellularization described in Example 6, A is before decellularization, and B is after decellularization;
  • Figure 10 is a qualitative and stable detection of heparin grafting of the acellular matrix of grafted heparin by toluidine blue as described in Example 7,
  • A is a complete autologous tissue conduit covalently bound to heparin
  • B is a covalently bound heparin The longitudinally sectioned autologous tissue duct.
  • the invention utilizes the principle of foreign body reaction to place a non-toxic, non-degradable Teflon tube in the subcutaneous tissue to form a tubular tissue. Then, after decellularization and heparinization treatment of the tubular tissue, an autologous extracellular matrix tube, that is, the artificial blood vessel according to the present invention is obtained, and the specific scheme is divided into the following steps:
  • the specific steps of the artificial blood vessel preparation method of the present invention are as follows:
  • the cell reagent is replaced 4 to 6 times, preferably 5 times, to obtain an autologous acellular matrix tube.
  • the heparin concentration is preferably 60 mg / ml;
  • the autologous acellular matrix tube obtained in the step (1) is placed in a container containing the heparin grafting reagent, and the artificial blood vessel is obtained by shaking for 3 to 5 hours.
  • CHAPS is a zwitterionic detergent with both nonionic and ionic detergent properties, including disruption of lipid-lipid, lipid-protein interactions or dissolution of the pulp and nucleus, which has a milder decellularization effect.
  • Extracellular matrix structure is less destructive.
  • EDTA is an agent that regulates permeability. By changing the concentration, it can be configured as a hypotonic or hypertonic solution, which mainly causes cell lysis and cannot remove cells or cellular components.
  • the decellularized material used in the experiment is an autologous tissue tube prepared by the principle of foreign body reaction. The tissue structure and mechanical properties are close to those of normal arterial vessels, and its purpose is to use for autologous vascular replacement, without considering immune rejection, CHAPS+EDTA decellularization. The method is relatively mild and preserves well the structure and mechanical properties of the extracellular matrix.
  • Sulfo-NHS/EDC activates the carboxyl group on heparin to a succinimide ester, which then covalently binds to the amino group on the surface of the collagen, ultimately anchoring the heparin to the collagen surface by covalent bonds.
  • Sulfo-NHS/EDC-mediated heparin covalent binding to tissue engineered blood vessels is not cytotoxic in vivo and has good biocompatibility.
  • the accurately weighed reagent is added into a clean wide-mouth glass bottle, shaken on a shaker for 2 to 3 hours, so that the reagent is fully dissolved. At this time, the solution becomes clear, and a decellularization reagent is prepared and stored at 4 ° C. .
  • the autologous tissue tube was placed in a sterile centrifuge tube, and 10 ml of the prepared 3-[(3-cholamidopropyl)-diethylamine]-propanesulfonic acid solution was added to each tube, and placed on a shaker at room temperature for 3 hours. The 3-[(3-cholamidopropyl)-diethylamine]-propanesulfonic acid solution was then replaced every 3 hours for 5 times.
  • a solution configuration Add the reagent in the a liquid formula to a clean 50 ml centrifuge tube, mix well by shaking, avoid light, and overnight at 4 °C.
  • B solution configuration first add 20ml MES-buffer to a 50ml clean centrifuge tube, then slowly add the weighed heparin to the centrifuge tube, shake it while adding it, and finally put it on the shaker overnight to fully dissolve the heparin.
  • Heparin grafting reagent configuration the next day, mix the a and b volumes in equal volume, incubate for 30 min at room temperature, adjust the pH to 7 with 1 M NaOH solution, and finally filter with 0.2 ⁇ m filter, place in a 50 ml sterile centrifuge tube, protected from light. Store at 4 ° C for later use.
  • Example 1 is a preferred embodiment.
  • the Teflon tube with the diameter matching the inner diameter of the blood vessel to be replaced is cut to the appropriate length, disinfected with 75% alcohol for 30 minutes, placed in the subcutaneous tissue, and after 2 weeks, the subcutaneous Teflon tube is taken out along with the wrapped new tissue.
  • the accurately weighed reagent is added into a clean wide-mouth glass bottle, shaken on a shaker for 2 to 3 hours, so that the reagent is fully dissolved. At this time, the solution becomes clear, and a decellularization reagent is prepared and stored at 4 ° C. .
  • the autologous tissue tube was placed in a sterile centrifuge tube, and 10 ml of the prepared 3-[(3-cholamidopropyl)-diethylamine]-propanesulfonic acid solution was added to each tube, and placed on a shaker at room temperature for 2 hours. The 3-[(3-cholamidopropyl)-diethylamine]-propanesulfonic acid solution was then replaced every 2 hours for 5 times.
  • a solution configuration Add the reagent in the a liquid formula to a clean 50 ml centrifuge tube, mix well by shaking, avoid light, and overnight at 4 °C.
  • B solution configuration first add 20ml MES-buffer to a 50ml clean centrifuge tube, then slowly add the weighed heparin to the centrifuge tube, shake it while adding it, and finally place it on the shaker. Night, so that heparin is fully dissolved.
  • Heparin grafting reagent configuration the next day, the a and b liquids were mixed in equal volume, incubated at room temperature for 50 min, adjusted to pH 7 with 1 M NaOH solution, finally filtered through a 0.2 ⁇ m filter, placed in a 50 ml sterile centrifuge tube, protected from light. Store at 4 ° C for later use.
  • the accurately weighed reagent is added into a clean wide-mouth glass bottle, shaken on a shaker for 2 to 3 hours, so that the reagent is fully dissolved. At this time, the solution becomes clear, and a decellularization reagent is prepared and stored at 4 ° C. .
  • the autologous tissue tube was placed in a sterile centrifuge tube, and 10 ml of the prepared 3-[(3-cholamidopropyl)-diethylamine]-propanesulfonic acid solution was added to each tube and placed on a shaker at room temperature for 2.5 h. Then, the 3-[(3-cholamidopropyl)-diethylamine]-propanesulfonic acid solution was changed every 5 hours for 5 times.
  • a solution configuration Add the reagent in the a liquid formula to a clean 50 ml centrifuge tube, mix well by shaking, avoid light, and overnight at 4 °C.
  • B solution configuration first add 20ml MES-buffer to a 50ml clean centrifuge tube, then slowly add the weighed heparin to the centrifuge tube, shake it while adding it, and finally put it on the shaker overnight to fully dissolve the heparin.
  • Heparin grafting reagent configuration the next day, the a and b liquids were mixed in equal volume, incubated at room temperature for 60 min, adjusted to pH 7 with 1 M NaOH solution, finally filtered through a 0.2 micron filter, placed in a 50 ml sterile centrifuge tube, protected from light. Store at 4 ° C for later use.
  • the decellularized autologous acellular matrix tube was placed in a numbered 6-well plate, and 5 ml of the prepared heparin grafting reagent was added to each well sample, shaken, protected from light, and 4 h, to obtain the person.
  • the autologous tissue tubing (autologous extracellular matrix tubing) after decellularization as described in Example 1 was subjected to burst pressure, suture tensile strength, maximum tensile stress and maximum tensile force, respectively, and the non-decellularized autologous tissue tubing.
  • the tensile strains were tested and compared separately. The results showed that the differences between the measured values of the two autologous tissue tubes were not statistically significant, indicating that the decellularization treatment of the autologous tissue tubes has no effect on the mechanical properties of the autologous tissue tubes. Large, specific test methods and results are as follows:
  • the burst pressure is accomplished by a self-made pressure measuring system with a precision pressure gauge.
  • the pressure measuring system is filled with PBS buffer, and the length of the tissue to be tested is connected to the pressure measuring system with a length of 5 cm.
  • the wire is fixed with a No. 7 wire, and the pressure in the pressure measuring system is slowly increased until the pressure drops suddenly.
  • the autologous extracellular matrix tube is used. A break occurs and the maximum pressure value is recorded. This value is the burst pressure.
  • Suture retention strength is the force at which the suture tears tissue. It is measured here according to ANSI/AAMI/ISO 7198. Prepare a 2cm long tissue tube to be tested, and sew the 6-0Prolene thread to one end of the pipe. The margin is 2mm, and another one is sewn at a 120-degree angle. A total of 3 stitches are stitched, and each stitch is separately knotted to form a ring. The seamless end of the pipe and one of the sutures are respectively fixed on the universal tensile testing machine, the tensile speed is adjusted to 50mm/min, the maximum tensile force is recorded, and the other two sutures are fixed in turn for testing, and three tests are performed. The results obtained by averaging the results are the tensile strength of the suture.
  • the tissue tube to be tested was cut into a 5 mm wide ring, and the thickness and diameter were measured and recorded. Hang the two ends on the paper clip, and then fix the two paper clips on the universal tensile tester.
  • the initial stretch length is about 10% of the pull length, and the tension speed is adjusted to 50mm/min.
  • the tensile stress and the maximum tensile strain are the maximum stresses and strains when the ring is broken.
  • Example 1 The artificial blood vessels prepared in Example 1 were subjected to autologous vascular grafting experiments on miniature pigs. A total of 8 small pigs were transplanted with autologous vascular grafts, 5 of which were followed up for 1 month, and 3 were followed up for 2 months. Ultrasound examination, 1 month follow-up end point before the death of another CTA examination, ultrasound examination of vascular patency, patency rate of 100% (5/5) at 1 month, patency rate of 67% (2/3) at 2 months .
  • CT angiography revealed that the vascular graft was unobstructed, without stenosis and tumor-like changes, and matched well with the common carotid artery of normal small pigs. ( Figure 7, the arrow points to the anastomosis)
  • the autologous tissue tubes prepared in Example 1 before and after decellularization were sectioned and subjected to DAPI staining (Fig. 8, A is before decellularization, B is after decellularization), blue fluorescence represents nuclei, and autologous tissue tubes can be observed before decellularization. In many nuclei, no blue fluorescence was detected in the autologous tissue tube after decellularization, and the nuclear staining was negative, which proved that the wall cells were completely removed.
  • heparin-grafted qualitative and stability test of the acellular matrix of grafted heparin in Example 1 was carried out using toluidine blue (Fig. 10, A is a complete autologous tissue tube covalently bound to heparin, and B is covalently bound to heparin).
  • the longitudinally dissected autologous tissue tube), the left side of the graphs A and B is the qualitative test results of heparin binding, and the autologous acellular matrix tube shows a specific blue color, indicating that heparin is successfully bound to the autologous acellular matrix tube, Figure 10A
  • the heparin binding stability test was performed, that is, the autologous tissue tube of the grafted heparin on the left side of FIGS. 10A and B was eluted for 12 hours on a shaker at 37 ° C in a water bath to measure the stability of heparin grafting.

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Abstract

一种人造血管及其制备方法，通过将自体组织管道采用3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸试剂化学脱细胞方法脱去管道的细胞成分，然后将肝素以共价结合的方式结合到自体脱细胞基质管道的表面，获得人造血管。这种方法制备的人造血管无免疫源性，制备周期短、费用低，并可制备成不同内径，具备良好抗凝血功能，无需复杂的体外制备过程，通畅率高，细胞能够有效进入管壁参与重构，完全优于以往同种异体或异种脱细胞基质管道，具有很好的临床应用前景。

Description

一种人造血管及其制备方法 【技术领域】
本发明属于组织工程血管领域，更具体地，涉及一种人造血管及其制备方法。
【背景技术】
目前，自体血管如大隐静脉、乳内动脉是临床上用于冠状动脉搭桥手术和其他小口径血管搭桥手术最为主要的桥血管。目前美国食品药品监督管理局(FDA)批准进行临床试验的两个脱细胞基质管道小口径血管分别是：
(1)Nicolas L’Heureux等首次将不含人工合成材料的脱细胞基质组织工程血管应用于临床，大动物恒河猴腹主动脉移植实验6周通畅率为100％(n＝3)，动静脉瘘管I期临床实验1个月通畅率为78％(7/9)，6个月通畅率为60％(5/8)。Nicolas L’Heureux等虽然制备出自体脱细胞基质管道，但制备过程复杂，制备一根管道时间7-9月，花费1.5万美金，远期通畅率不理想，很难推广应用于临床。
(2)Laura Niklason等将人尸体血管平滑肌细胞体外种植于可降解聚乳酸(PGA)管上，然后于生物反应器中体外动态培养8周，PGA降解后形成富含细胞和细胞外基质(胶原纤维)的管状组织，最后脱细胞形成以胶原纤维为主要成分的组织工程血管(含少量残余PGA)，即同种异体脱细胞基质管道，大动物狒狒动静脉瘘管移植实验6个月通畅率100％(3/3)，FDA批准二期临床试验6月通畅率63％，12、18、24月通畅率分别是28％、18％、15％。由于是同种异体移植物存在移植免疫反应，并且制备过程复杂，制备时间至少2个月，易导致血栓形成、内膜增生，通畅率不佳，而且使用尸体血管平滑肌细胞有传播如艾滋病、乙型肝炎病毒等疾病风险。
上述两种脱细胞基质管道由于管壁结构致密，周围细胞不能进入管壁参与重构，管壁细胞外基质成分不能有效更新，特别不能合成维持动脉管壁弹性的弹力纤维，严重影响其机械性能和通畅率。
Charles Sparks,Gordon Campbell,Yasuhide Nakayama及Rotmans Joris等不同研究小组制备未脱细胞自体血管移植物回植体内动脉，存在力学性能薄弱、血栓形成、管壁细胞增生导致血管通畅率低，同时制备过程中使用对机体有毒性的生物材料，很难实现临床转化。
【发明内容】
针对现有技术的以上缺陷或改进需求，本发明提供了一种人造血管及其制备方法，其目的在于通过将自体组织管道经过脱细胞以及共价结合肝素修饰，获得一种人造血管，由此解决现有技术的人造血管力学性能差、抗凝血功能薄弱以及免疫反应导致血管通畅率低的技术问题。
为实现上述目的，按照本发明的一个方面，提供了一种人造血管，包括由自体细胞外基质构成的管壁，所述管壁共价结合有抗凝分子。
优选地，所述抗凝分子为肝素，所述肝素含量为每毫克人造血管5～10微克。
优选地，所述人造血管管壁厚度为124.9～690.5微米，优选厚度为400～650微米。
优选地，所述管壁爆破压力为3157±216mmHg，缝合张力为3.94±0.46N，最大拉伸应力为2.41±0.22MPa，最大拉伸应变为30.63±2.74％。
按照本发明的另一个方面，提供了一种人造血管的制备方法，包括如下步骤：
(1)脱细胞处理：将自体组织管道采用3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸试剂化学脱细胞方法脱去管道的细胞成分，获取自体脱细胞基质管道；
(2)共价结合抗凝分子：将抗凝分子以共价结合的方式结合到步骤(1)获取的自体脱细胞基质管道的表面，获得人造血管。
优选地，所述步骤(1)包括如下步骤：
(1-1)脱细胞试剂的配置：将3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸、EDTA.Na2、NaCl、NaOH和无菌去离子水制备成500mL溶液，其摩尔浓度分别为6～10mmol/L、20～30mmol/L、0.10～0.15mmol/L和0.8～1.2mol/L，即为脱细胞试剂，其中3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸摩尔浓度优选为8mmol/L、EDTA.Na2摩尔浓度优选为25mmol/L、NaCl摩尔浓度优选为0.12mmol/L，NaOH摩尔浓度优选为1mol/L；
(1-2)脱细胞处理：将所述自体组织管道放入装有步骤(1-1)配置好的脱细胞试剂中，室温下处理2～3h，然后每2～3h更换一次所述脱细胞试剂，共更换4～6次，优选为5次，获取自体脱细胞基质管道。
优选地，所述步骤(2)包括如下步骤：
(2-1)肝素接枝试剂的配制
(a)采用0.5mol/L的MES缓冲液配制含30～50mg/ml EDC和10～30mg/ml的Sulfo-NHS溶液，得到A溶液；
(b)采用0.5mol/L的MES缓冲液配制40～80mg/ml的肝素溶液得到B溶液；
(c)将所述A溶液和B溶液等体积混合均匀，室温下孵育30～60分钟，然后用氢氧化钠溶液调节溶液pH值至7.0，最后用微过滤器过滤，得到清液为无菌肝素接枝试剂。
(2-2)将步骤(1)得到的自体脱细胞基质管道置于装有所述肝素接枝试剂的容器中，震荡处理3～5小时，即得到所述的人造血管。
优选地，所述步骤(2-1)肝素接枝试剂中EDC的浓度优选为40mg/ml，所述Sulfo-NHS的浓度优选为20mg/ml，所述肝素的浓度优选为60mg/ml。
优选地，所述步骤(2-1)中MES缓冲液的pH值为5.5。
优选地，所述步骤(2-1)中的(c)步骤的微过滤器孔径为0.2微米。
总体而言，通过本发明所构思的以上技术方案与现有技术相比，能够 取得下列有益效果。
(1)本发明利用异物反应原理，将无毒、体内不降解的Teflon管置于皮下制备得到自体组织管道，并以其为原料制备得到本发明的人造血管，组织结构及力学性能接近正常动脉，用于自体血管替代物时不用考虑免疫排斥反应，而且无传播如艾滋病、乙型肝炎病毒等疾病风险。
(2)本发明采用的CHAPS+EDTA脱细胞方法相对温和且对细胞外基质的结构和力学性能保存较好，脱细胞效果好，通过脱细胞处理使得自体脱细胞基质管壁结构允许周围细胞进入管壁参与管壁重构，有效合成弹力纤维和胶原纤维，提高体内重构后力学性能和通畅率，并接近正常自体动脉结构及力学性能。
(3)本发明采用Sulfo-NHS/EDC介导的肝素共价结合组织工程血管在体内没有细胞毒性，具有良好的生物相容性，良好的抗凝血功能，通畅率高。
(4)本发明的人造血管制备周期短、费用低，并可制备成不同内径，完全优于以往脱细胞基质管道，具有很好的临床应用前景。
【附图说明】
图1是实施例4所述的脱细胞前后自体组织管道爆破压力检测结果；
图2是实施例4所述的脱细胞前后自体组织管道缝线抗拉强度检测结果；
图3是实施例4所述的脱细胞前后自体组织管道最大拉伸应力检测结果；
图4是实施例4所述的脱细胞前后自体组织管道最大拉伸应变检测结果；
图5是实施例5所述的人造血管移植术后1个月超声检查结果，A为人造血管移植侧颈动脉，B为对侧正常颈动脉；
图6是实施例5所述的人造血管移植术后2个月超声检查结果，A为 人造血管移植侧颈动脉，B为对侧正常颈动脉；
图7是实施例5所述的人造血管移植术后1个月CT血管造影检查结果；
图8是实施例6所述的脱细胞前后自体组织管道切片后进行DAPI染色结果，A为脱细胞前，B为脱细胞后；
图9是实施例6所述的脱细胞前后的自体组织管道标本DNA定量检测结果；
图10是实施例7所述的甲苯胺蓝对接枝肝素的脱细胞基质管道进行肝素接枝的定性以及稳定性检测，A为共价结合肝素的完整自体组织管道，B为共价结合肝素的纵向剖开的自体组织管道。
【具体实施方式】
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
本发明利用异物反应原理，将无毒、体内不降解的Teflon管置于皮下组织，使之形成管状组织。然后对管状组织进行脱细胞和肝素化修饰处理后，获得自体细胞外基质管道，即本发明所述的人造血管，具体方案分为下面几步：
(A)将管状Teflon(外径1/16英寸，约1.59mm)，置于皮下组织2～5周后取出，对获得的自体组织管道进行力学性能检测和组织学检测，其中4、5周自体组织管道具有最佳力学性能和组织学结构组，为节省制备时间，采用4周自体管道进行脱细胞和共价结合肝素处理。
(B)采用化学脱细胞方法(CHAPS试剂)，脱去自体组织管道细胞成分，获取自体脱细胞基质管道。
(C)采用Sulfo-NHS/EDC将肝素共价结合到自体脱细胞基质管道， 获得具有抗凝血活性的自体细胞外基质管道，此即所述的人造血管。
本发明的人造血管制备方法具体步骤如下：
(1)自体组织管道的制备
将直径与需要替代的血管内径相匹配的特氟龙(Teflon)管裁剪至合适长度，75％酒精消毒30分钟，置于皮下组织，2～5周后取出，优选4周将皮下Teflon管连同包绕新生组织取出，抽除Teflon管，获得自体组织管道。4周对应的自体组织管道以及最终的人造血管产品厚度最合适，对应的人造血管产品性能最佳。
(2)脱细胞处理
(2-1)脱细胞试剂的配置：将3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸(简称CHAPS试剂)、EDTA.Na2、NaCl、NaOH和无菌去离子水制备成500mL溶液，其摩尔浓度分别为6～10mmol/L、20～30mmol/L、0.10～0.15mmol/L和0.8～1.2mol/L，即为脱细胞试剂，其中CHAPS试剂摩尔浓度优选为8mmol/L，EDTA.Na2的摩尔浓度优选为25mmol/L，NaCl的摩尔浓度优选为0.12mmol/L、NaOH的摩尔浓度优选为1mol/L；
(2-2)脱细胞处理：将所述自体组织管道放入装有步骤(1-1)配置好的脱细胞试剂中，室温下处理2～3h，然后每2～3h更换一次所述脱细胞试剂，共更换4～6次，优选5次，获取自体脱细胞基质管道。
(2-3)洗涤步骤：将步骤(1-2)获取的自体脱细胞基质管道采用无菌PBS溶液充分洗涤，以洗脱残余的脱细胞试剂。
(3)共价结合抗凝分子，本发明中抗凝分子优选肝素：
(3-1)肝素接枝试剂的配置
(a)采用0.5mol/L的MES缓冲液(pH值为5.5)配制含30～50mg/mlEDC(碳二亚胺)和10～30mg/ml的Sulfo-NHS(N-羟基琥珀酰亚胺)溶液，得到A溶液，其中EDC的浓度优选为40mg/ml，Sulfo-NHS的浓度优选为20mg/ml；
(b)采用0.5mol/L的MES缓冲液配制40～80mg/ml的肝素溶液得到B溶液，肝素浓度优选为60mg/ml；
(c)将所述A溶液和B溶液等体积混合均匀，室温下孵育30～60分钟，然后用氢氧化钠溶液调节溶液pH值至7.0，最后用0.2微米的微过滤器过滤，得到清液为无菌肝素接枝试剂。
(3-2)将步骤(1)得到的自体脱细胞基质管道置于装有所述肝素接枝试剂的容器中，震荡处理3～5小时，即得到所述的人造血管。
CHAPS是一种两性离子去垢剂，同时具有非离子型和离子型去垢剂的性能，包括破坏脂-脂、脂-蛋白相互作用或者溶解包浆及细胞核，其去细胞作用较为温和，对细胞外基质结构破坏性小，EDTA是一种调节渗透性的试剂，通过改变浓度，可以配置成低渗或者高渗性溶液，其作用主要是导致细胞裂解，并不能去除细胞或者细胞成分，本实验所用的脱细胞材料为通过异物反应原理制备的自体组织管道，组织结构及力学性能较正常动脉血管接近，且其目的是用于自体血管替代物，不用考虑免疫排斥反应，CHAPS+EDTA脱细胞方法相对温和且对细胞外基质的结构和力学性能保存较好。
Sulfo-NHS/EDC可以将肝素上的羧基激活成为琥珀酰亚胺酯，琥珀酰亚胺酯然后与胶原蛋白表面的氨基发生共价结合，最终将肝素依靠共价键锚定在胶原表面。Sulfo-NHS/EDC介导的肝素共价结合组织工程血管在体内是没有细胞毒性，且具有良好的生物相容性。
以下为实施例：
实施例1
(1)自体组织管道的制备
将直径与需要替代的血管内径相匹配的特氟龙(Teflon)管裁剪至合适长度，75％酒精消毒30分钟，置于皮下组织，4周后将皮下Teflon管连同 包绕新生组织取出，抽除Teflon管，获得自体组织管道，其厚度为324.1±57.4微米(n＝6，n为检测样本的个数，以下n的含义相同)。
(2)脱细胞处理
(2-1)脱细胞试剂的配置
Figure PCTCN2016096307-appb-000001
按照上表的配方，将精准称量的试剂加入干净的广口玻璃瓶中，摇床上振荡2～3h，使试剂充分溶解，此时溶液变澄清，制备成脱细胞试剂，于4℃保存备用。
(2-2)自体组织管道脱细胞
将自体组织管道放入无菌离心管中，每管加入10ml配置好的3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸溶液，置于摇床上，室温处理3h，然后每3h更换一次3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸溶液，共5次。脱细胞完成后，换用无菌PBS溶液10ml，放置于摇床上，洗涤30min，依法重复洗涤5遍，以洗脱残余3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸试剂，避免细胞毒性。最后将处理好的脱细胞管道置入生理盐水4℃保存备用。
(3)共价结合肝素
(3-1)共价结合肝素试剂
A溶液配方
Figure PCTCN2016096307-appb-000002
B溶液配方
Figure PCTCN2016096307-appb-000003
A溶液配置：将a液配方中的试剂按量加入干净50ml离心管中，振荡混匀，避光，4℃过夜。
B溶液配置：先将20ml MES-buffer加入50ml干净离心管中，然后缓慢将称量好的肝素加入离心管中，一边加入一边晃动，最后置于摇床上过夜，以使肝素充分溶解。
肝素接枝试剂配置：次日，将a、b液等体积混合，室温孵育30min，用1M NaOH溶液调节PH至7，最后用0.2微米过滤器过滤，置于50ml无菌离心管中，避光4℃保存备用。
(3-2)共价结合肝素
将脱细胞处理好的自体脱细胞基质管道放置于编号的6孔板中，每孔样本加入配置好的肝素接枝试剂5ml，摇床、避光、5h，即得到本发明所述的人造血管，其厚度为525±125微米(n＝6)，完成后置于生理盐水溶液中4℃保存备用。体外细胞毒性试验检测证实无细胞毒性。实施例1为优选实施例。
实施例2
(1)自体组织管道的制备
将直径与需要替代的血管内径相匹配的特氟龙(Teflon)管裁剪至合适长度，75％酒精消毒30分钟，置于皮下组织，2周后将皮下Teflon管连同包绕新生组织取出，抽除Teflon管，获得自体组织管道，其厚度为154.3±56.4微米(n＝6)。
(2)脱细胞处理
(2-1)脱细胞试剂的配置
Figure PCTCN2016096307-appb-000004
Figure PCTCN2016096307-appb-000005
按照上表的配方，将精准称量的试剂加入干净的广口玻璃瓶中，摇床上振荡2～3h，使试剂充分溶解，此时溶液变澄清，制备成脱细胞试剂，于4℃保存备用。
(2-2)自体组织管道脱细胞
将自体组织管道放入无菌离心管中，每管加入10ml配置好的3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸溶液，置于摇床上，室温处理2h，然后每2h更换一次3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸溶液，共5次。脱细胞完成后，换用无菌PBS溶液10ml，放置于摇床上，洗涤30min，依法重复洗涤5遍，以洗脱残余3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸试剂，避免细胞毒性。最后将处理好的脱细胞管道置入生理盐水4℃保存备用。
(3)共价结合肝素
(3-1)共价结合肝素试剂
A溶液配方
Figure PCTCN2016096307-appb-000006
B溶液配方
Figure PCTCN2016096307-appb-000007
A溶液配置：将a液配方中的试剂按量加入干净50ml离心管中，振荡混匀，避光，4℃过夜。
B溶液配置：先将20ml MES-buffer加入50ml干净离心管中，然后缓慢将称量好的肝素加入离心管中，一边加入一边晃动，最后置于摇床上过 夜，以使肝素充分溶解。
肝素接枝试剂配置：次日，将a、b液等体积混合，室温孵育50min，用1M NaOH溶液调节PH至7，最后用0.2微米过滤器过滤，置于50ml无菌离心管中，避光4℃保存备用。
(3-2)共价结合肝素
将脱细胞处理好的自体脱细胞基质管道放置于编号的6孔板中，每孔样本加入配置好的肝素接枝试剂5ml，摇床、避光、3h，即得到本发明所述的人造血管，其厚度为249.9±125微米(n＝6)，完成后置于生理盐水溶液中4℃保存备用。体外细胞毒性试验检测证实无细胞毒性。
实施例3
(1)自体组织管道的制备
将直径与需要替代的血管内径相匹配的特氟龙(Teflon)管裁剪至合适长度，75％酒精消毒30分钟，置于皮下组织，5周后将皮下Teflon管连同包绕新生组织取出，抽除Teflon管，获得自体组织管道，其厚度为352.1±43.2微米(n＝6)。
(2)脱细胞处理
(2-1)脱细胞试剂的配置
按照上表的配方，将精准称量的试剂加入干净的广口玻璃瓶中，摇床上振荡2～3h，使试剂充分溶解，此时溶液变澄清，制备成脱细胞试剂，于4℃保存备用。
(2-2)自体组织管道脱细胞
将自体组织管道放入无菌离心管中，每管加入10ml配置好的3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸溶液，置于摇床上，室温处理2.5h，然后每2.5h更换一次3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸溶液，共5次。脱细胞完成后，换用无菌PBS溶液10ml，放置于摇床上，洗涤30min，依法重复洗涤5遍，以洗脱残余3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸试剂，避免细胞毒性。最后将处理好的脱细胞管道置入生理盐水4℃保存备用。
(3)共价结合肝素
(3-1)共价结合肝素试剂
A溶液配方
Figure PCTCN2016096307-appb-000009
B溶液配方
Figure PCTCN2016096307-appb-000010
A溶液配置：将a液配方中的试剂按量加入干净50ml离心管中，振荡混匀，避光，4℃过夜。
B溶液配置：先将20ml MES-buffer加入50ml干净离心管中，然后缓慢将称量好的肝素加入离心管中，一边加入一边晃动，最后置于摇床上过夜，以使肝素充分溶解。
肝素接枝试剂配置：次日，将a、b液等体积混合，室温孵育60min，用1M NaOH溶液调节PH至7，最后用0.2微米过滤器过滤，置于50ml无菌离心管中，避光4℃保存备用。
(3-2)共价结合肝素
将脱细胞处理好的自体脱细胞基质管道放置于编号的6孔板中，每孔样本加入配置好的肝素接枝试剂5ml，摇床、避光、4h，即得到所述的人 造血管，其厚度为570.5±120微米(n＝6)，完成后置于生理盐水溶液中4℃保存备用。体外细胞毒性试验检测证实无细胞毒性。
实施例4
将实施例1所述的进行脱细胞处理后的自体组织管道(自体细胞外基质管道)与未脱细胞处理的自体组织管道分别进行爆破压力、缝线抗拉强度以及最大拉伸应力和最大拉伸应变分别进行检测和比较，检测结果表明两种自体组织管道各项测试测量值的差异均不具有统计学意义，说明本发明对自体组织管道的脱细胞处理对自体组织管道的力学性能影响不大，具体测试方法及结果如下：
(1)爆破压力(Burst pressure)检测
爆破压力由自制的、带有精密压力表的测压系统完成。测压系统中填满PBS缓冲液，将长度为5cm的待测组织管道连接于测压系统，用7号丝线固定，缓慢增加测压系统内的压力，直至压力骤降，自体细胞外基质管道出现破口，记录下最大压力值，此值即为爆破压力。
未脱细胞自体组织管道爆破压为3696±194mmHg(n＝6),脱细胞后爆破压为3157±216mmHg(n＝6)，P＞0.05，差异不具有统计学意义。(图1)
(2)缝线抗拉强度(Suture retention strength)检测
缝线抗拉强度(Suture retention strength)是指缝线将组织撕裂时的力，此处依照ANSI/AAMI/ISO 7198标准进行测定。准备2cm长的待测组织管道，将6-0Prolene线缝于管道的一端，边距为2mm，间隔120度角另缝一根，总共缝3针，将每根缝线单独打结形成一个环，将管道的无缝线端和其中1根缝线分别固定在万能拉力试验机上，调节拉伸速度为50mm/min，记录下最大拉力，依次固定另外2根缝线进行测试，将3次测试结果取平均值所得结果即为缝线抗拉强度。
未脱细胞自体组织管道缝合张力为4.97±0.55N(n＝6)，脱细胞后缝合张力为3.94±0.46N(n＝6)，P＞0.05，差异不具有统计学意义。(图2)
(3)最大拉伸应力(Ultimate tensile strength，UTS)和最大拉伸应变(ultimate strain)
将待测组织管道切成5mm宽的圆环，测定并记录其厚度和直径。将其两端挂在回型针上，然后将2根回型针固定在万能拉伸试验机上，初始拉伸长度为拉断长度的10％左右，调节拉伸速度为50mm/min，最大拉伸应力和最大拉伸应变分别为圆环被拉断时的最大应力和应变。
未脱细胞自体组织管道最大拉伸应力为3.16±0.30MPa(n＝6),脱细胞后最大拉伸应力为2.41±0.22MPa(n＝6)，P＞0.05，差异不具有统计学意义。(图3)
未脱细胞自体组织管道最大拉伸应变为24.33±2.15％(n＝6),脱细胞后最大拉伸应变为30.63±2.74％(n＝6)，P＞0.05，差异不具有统计学意义。(图4)
实施例5
将实施例1制备得到的人造血管对小型猪进行自体血管移植实验，实验总共完成8只小型猪自体血管移植物移植，其中5只随访1个月，另外3只随访2个月，处死前行超声检查，1个月随访终点处死前另外行CTA检查，超声检查血管通畅性，1个月时通畅率为100％(5/5)，2个月时通畅率为67％(2/3)。
(1)小型猪颈总动脉移植实验:移植术后1个月超声示血管移植物段无血栓形成，内膜光滑、无粥样斑块、无明显内膜增生。(图5，其中A为移植血管超声，B为对侧正常血管超声)
(2)小型猪颈总动脉移植实验：移植术后2个月超声示血管移植物段无血栓形成，内膜光滑，彩色多普勒示血管移植物血流通畅。(图6，其中A为移植血管超声，B为对侧正常血管超声)
CT血管造影发现血管移植物通畅，无狭窄及瘤样变，与正常小型猪颈总动脉匹配较好。(图7，箭头所指处为吻合口)
实施例6
将脱细胞前后的实施例1制备的自体组织管道切片后进行DAPI染色(图8，A为脱细胞前，B为脱细胞后)，蓝色荧光代表细胞核，脱细胞前自体组织管道可以观察到很多细胞核，脱细胞后自体组织管道没有检测到蓝色荧光存在，细胞核染色阴性，证明管壁细胞脱除完全。
对脱细胞前后的自体组织管道标本进行DNA定量检测，脱细胞前标本DNA含量为0.081±0.010μg/㎎(n＝6)，脱细胞后标本DNA含量为0.011±0.003μg/㎎(n＝6)，P＜0.05，其差异具有统计学意义。(图9)
实施例7
利用甲苯胺蓝对实施例1中接枝肝素的脱细胞基质管道进行肝素接枝的定性以及稳定性检测(图10，A为共价结合肝素的完整自体组织管道，B为共价结合肝素的纵向剖开的自体组织管道)，图示A、B左侧为肝素结合定性检测结果，自体脱细胞基质管道管呈现特异性蓝色，表示肝素成功地结合在自体脱细胞基质管道上，图10A、B右侧为肝素结合稳定性检测，即将图10A和B左侧的接枝肝素的自体组织管道在37摄氏度水浴的摇床洗脱12小时，以检测肝素接枝的稳定性。自体脱细胞基质管道管颜色比左侧稍浅但未消失，表示经过水浴洗脱后，少部分未稳定结合的肝素被洗脱掉，大部分肝素与自体脱细胞基质管道结合稳定，未被洗脱。肝素结合定量检测结果:单位质量自体脱细胞基质管道共价结合肝素质量为8.2±0.9μg/mg(n＝6)。
本领域的技术人员容易理解，以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种人造血管，其特征在于，包括由自体细胞外基质组成的管壁，所述管壁共价结合有抗凝分子。
  2. 如权利要求1所述的人造血管，其特征在于，所述抗凝分子为肝素，所述肝素含量为每毫克人造血管5～10微克。
  3. 如权利要求1所述的人造血管，其特征在于，所述人造血管管壁厚度为124.9～690.5微米，优选厚度为400～650微米。
  4. 如权利要求1所述的人造血管，其特征在于，所述管壁爆破压力为3157±216 mmHg，缝合张力为3.94±0.46 N，最大拉伸应力为2.41±0.22 MPa，最大拉伸应变为30.63±2.74％。
  5. 一种人造血管的制备方法，其特征在于，包括如下步骤：
    (1)脱细胞处理：将自体组织管道采用3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸试剂化学脱细胞方法脱去管道的细胞成分，获取自体脱细胞基质管道；
    (2)共价结合抗凝分子：将抗凝分子以共价结合的方式结合到步骤(1)获取的自体脱细胞基质管道的表面，获得人造血管。
  6. 如权利要求5所述的人造血管的制备方法，其特征在于，所述步骤(1)包括如下步骤：
    (1-1)脱细胞试剂的配置：将3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸、EDTA.Na2、NaCl、NaOH和无菌去离子水制备成500 mL溶液，其摩尔浓度分别为6～10 mmol/L、20～30 mmol/L、0.10～0.15 mmol/L和0.8～1.2mol/L，即为脱细胞试剂，其中3-[(3-胆酰胺丙基)-二乙胺]-丙磺酸摩尔浓度优选为8 mmol/L、EDTA.Na2摩尔浓度优选为25 mmol/L、NaCl摩尔浓度优选为0.12 mmol/L，NaOH摩尔浓度优选为1 mol/L；
    (1-2)脱细胞处理：将所述自体组织管道放入装有步骤(1-1)配置好的脱细胞试剂中，室温下处理2～3 h，然后每2～3 h更换一次所述脱细胞 试剂，共更换4～6次，优选为5次，获取自体脱细胞基质管道。
  7. 如权利要求5所述的人造血管的制备方法，其特征在于，所述步骤(2)包括如下步骤：
    (2-1)肝素接枝试剂的配制
    (a)采用0.5 mol/L的MES缓冲液配制含30～50mg/ml EDC和10～30mg/ml的Sulfo-NHS溶液，得到A溶液；
    (b)采用0.5 mol/L的MES缓冲液配制40～80mg/ml的肝素溶液得到B溶液；
    (c)将所述A溶液和B溶液等体积混合均匀，室温下孵育30～60分钟，然后用氢氧化钠溶液调节溶液pH值至7.0，最后用微过滤器过滤，得到清液为无菌肝素接枝试剂。
    (2-2)将步骤(1)得到的自体脱细胞基质管道置于装有所述肝素接枝试剂的容器中，震荡处理3～5小时，即得到所述的人造血管。
  8. 如权利要求7所述的人造血管的制备方法，其特征在于，所述步骤(2-1)肝素接枝试剂中EDC的浓度优选为40 mg/ml，所述Sulfo-NHS的浓度优选为20 mg/ml，所述肝素的浓度优选为60 mg/ml。
  9. 如权利要求3所述的人造血管的制备方法，其特征在于，所述步骤(2-1)中MES缓冲液的pH值为5.5。
  10. 如权利要求6所述的人造血管的制备方法，其特征在于，所述步骤(2-1)中的(c)步骤的微过滤器孔径为0.2微米。
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