WO2022143701A1 - 促内皮化生物材料、心脏瓣膜及其制备和应用 - Google Patents

促内皮化生物材料、心脏瓣膜及其制备和应用 Download PDF

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WO2022143701A1
WO2022143701A1 PCT/CN2021/142207 CN2021142207W WO2022143701A1 WO 2022143701 A1 WO2022143701 A1 WO 2022143701A1 CN 2021142207 W CN2021142207 W CN 2021142207W WO 2022143701 A1 WO2022143701 A1 WO 2022143701A1
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preparation
promoting
biological material
growth factor
biomaterial
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PCT/CN2021/142207
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English (en)
French (fr)
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刘兆刚
朱雨婷
邝大军
林浩昇
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杭州启明医疗器械股份有限公司
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Priority to EP21914435.9A priority Critical patent/EP4272772A1/en
Priority to CN202180075228.5A priority patent/CN116490226A/zh
Publication of WO2022143701A1 publication Critical patent/WO2022143701A1/zh
Priority to US18/211,416 priority patent/US20230330306A1/en

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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
    • 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
    • A61L27/3683—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
    • 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
    • A61L2300/414—Growth factors
    • 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
    • A61L2430/20—Materials or treatment for tissue regeneration for reconstruction of the heart, 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
    • 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 present application relates to the technical field of implanted biomaterials, in particular to a biomaterial for promoting endothelialization, a heart valve, and the preparation and application thereof.
  • the surface treatment of the interventional valve device can capture endothelial progenitor cells after the interventional valve is placed in the body, promote the endothelialization of the valve device, enhance the biocompatibility of the valve device and prolong the service life of the valve device.
  • the heart valve device includes a stent and a valve installed in the stent. In existing studies, the capture factor for capturing endothelial progenitor cells is loaded on the stent, and there is a problem that the load is not firm. To solve this problem, a cross-linking agent must be used.
  • the present application provides a pro-endothelializing heart valve and its preparation and application by directly chemically or physically loading the pro-endothelializing growth factor on the valve surface without the use of additional cross-linking agents.
  • An endothelial-promoting biomaterial the surface of the biomaterial is loaded with endothelial-promoting growth factors.
  • Endothelialization-promoting growth factor refers to a class of substances that can capture endothelial progenitor cells to make endothelial progenitor cells adhere, grow and differentiate on the surface of biomaterials, so as to induce the formation of endothelial functional cell layers on the surface of biomaterials.
  • the loading amount of the endothelial-promoting growth factor is as follows: the surface of the biological material with a unit surface area of 1 cm 2 contains 0.1-20 ug of the endothelial-promoting growth factor.
  • the biological material is a glutaraldehyde cross-linked biological material.
  • the glutaraldehyde cross-linked biological material is a biological tissue treated by glutaraldehyde cross-linking; the biological source of the biological tissue is pig or bovine, and the source part is pericardium, heart valve, blood vessel, ligament, muscle, intestine or skin.
  • These biological tissues are rich in collagen fibers, collagen fibers are rich in amino and carboxyl groups, and glutaraldehyde forms a cross-linked network within and between collagens through the reaction of its aldehyde groups with amino or carboxyl groups.
  • the endothelial growth-promoting growth factor is CD34 antibody, CD133 antibody or vascular endothelial growth factor VEGF.
  • the CD34 antibody is loaded on the surface of the biomaterial, and through specific binding with the CD34 antigen on the surface of the endothelial progenitor cells, the endothelial progenitor cells are captured, and the endothelial progenitor cells can adhere, grow and differentiate on the surface of the biomaterial, and form a functional endothelial cell layer.
  • the CD34 antibody of the present application is a mouse anti-human CD34 antibody, KG-1a type.
  • the antibody itself can be obtained by cloning methods well known in the art, and can also be obtained commercially.
  • the CD133 antibody and the VEGF growth factor may act by substantially similar principles.
  • the endothelial growth-promoting growth factor is loaded on the surface of the biological material by chemical grafting or physical coating.
  • a preparation method of a biomaterial for promoting endothelialization comprising:
  • the glutaraldehyde-crosslinked biomaterial after scavenging free glutaraldehyde is exposed to a solution containing endothelial growth-promoting growth factor, the pH of which is neutral or weakly alkaline.
  • the two ends of a part of glutaraldehyde are respectively connected with the amino group or carboxyl group of collagen fibers, and cross-linked in the network structure, and one end of a part of glutaraldehyde is connected with the amino group of collagen fibers.
  • carboxyl group is connected, the other end is in the free state, that is the residual aldehyde group, and some free glutaraldehyde that does not participate in the reaction, this part of the free glutaraldehyde needs to be removed before the endothelin-promoting factor coating, to Eliminate its possible disruption of endothelial growth factor activity.
  • the treatment method is a chemical grafting method.
  • the chemical grafting mechanism CD34 antibody or CD133 antibody or VEGF growth factor contains amino functional groups, and biological materials contain residual aldehyde groups.
  • the aldehyde group on the valve undergoes a chemical reaction, and the antibody is grafted to the surface of the valve material.
  • the concentration of the endothelialization-promoting growth factor is 1-50 ug/ml.
  • the solution containing the endothelial growth-promoting growth factor further contains a polyamino substance.
  • the polyamino substance is at least one of polyethylene glycol ammonia, aminopropylene glycol, amino acid and polypeptide.
  • the concentration of the polyamino substance is 1-50ug/ml.
  • the solvent of the solution containing the endothelial growth-promoting growth factor is PBS buffer, phosphate buffer or Tris-hydrochloric acid buffer; the pH value is 7-8.
  • the reaction is terminated when the content of the endothelial-promoting growth factor on the surface of the endothelial-promoting biomaterial reaches 0.1-20 ug/cm 2 .
  • the reaction time is 1-6 h.
  • concentration of promoting endothelial growth factor is 1 ⁇ 50ug/ml
  • the content of promoting endothelial growth factor on the surface of the biomaterial can reach 0.1 ⁇ 20ug/cm 2 after reaction for 1 ⁇ 6h.
  • the chemical grafting reaction is carried out at normal room temperature, and it is not necessary to control the reaction temperature deliberately.
  • the temperature of the reaction is 0-37°C.
  • the relative movement between the glutaraldehyde-crosslinked biological material and the solution containing the endothelial growth-promoting growth factor can be achieved by cyclic spraying or stirring and shaking the reaction system during the reaction, for example, glutaraldehyde.
  • the aldehyde cross-linked biological material is soaked in the reaction solution and then placed in a shaker to react.
  • the glutaraldehyde cross-linked biological material and the solution containing the endothelial growth-promoting growth factor are relatively static, for example, the glutaraldehyde cross-linked membrane can be directly immersed in the reaction solution for a static reaction.
  • the dynamic contact and static contact mentioned in this application can be understood in the same way unless otherwise specified.
  • the preparation method further includes: exposing the glutaraldehyde cross-linked biological material after the reaction is completed to a softener solution.
  • the softener solution is selected from pure glycerin or a mixed solution of glycerol and polyethylene glycol; the volume percentage of glycerin is 20-100%; when the volume percentage of glycerin is 100%, it is pure glycerin, and when When the volume percentage of glycerol is less than 100%, it is a mixed solution of glycerol and polyethylene glycol.
  • glycerin solution is glycerol (20%-100%) + PEG.
  • the glycerol solution is soaked for 0.5-4 hours and taken out, free glycerol is removed, dehydrated or dried for dry storage; it can also be directly soaked in glycerol solution for preservation.
  • the softener can also be a mixed solution of glycerin and water; a mixed solution of glycerol and alcohols; a mixed solution of glycerol, water and alcohols; polyethylene glycol; a mixed solution of polyethylene glycol and water; a mixed solution of polyethylene glycol and alcohols ; Polyethylene glycol, water and alcohol mixed solution.
  • the volume percentage of glycerol is 20% to 100%; in the solution containing polyethylene glycol, the volume percentage of polyethylene glycol is 20% to 100%.
  • the preparation method further includes: dehydration or drying after removing free glycerol from the glutaraldehyde cross-linked biological material treated with the softener.
  • Dehydration treatment is an optional and preferred step, and preservation after dehydration is more conducive to the preservation of antibody activity; of course, it can also be directly soaked in the corresponding solution for preservation after softening treatment. For the choice of soaking method, it is necessary to not affect CD34 antibody activity.
  • stored in an inert environment For example, pure glycerin solution, glycerol and water mixed solution, glycerol and alcohol mixed solution, pure polyethylene glycol solution, polyethylene glycol and water mixed solution, polyethylene glycol and alcohol mixed solution, etc.
  • the storage temperature can be stored at low temperature or normal temperature, preferably at -10 to 25°C.
  • a method for preparing an endothelium-promoting biological material comprising: exposing the glutaraldehyde cross-linked biological material after removing free glutaraldehyde to a solution containing an end-capping substance and a solution containing an endothelial growth-promoting factor in sequence.
  • the end-capping substance is a substance that blocks residual aldehyde groups in the glutaraldehyde cross-linked biological material, and is used to block the residual aldehyde groups of the glutaraldehyde cross-linked biological material, so as to eliminate the influence of the residual aldehyde groups on the activity of endothelial growth-promoting factors.
  • the treatment method is a physical grafting method, and the physical grafting mechanism is as follows: firstly, the residual aldehyde groups on the surface of the biological material are blocked, and then the matrix solution in which the endothelial growth-promoting growth factor is dissolved is coated on the surface of the biological material.
  • the capping species comprises at least one group reactive with residual aldehyde groups of the glutaraldehyde-crosslinked biomaterial.
  • the group that reacts with the residual aldehyde group is an amino group; the pH value of the solution containing the end-capping substance is neutral or weakly alkaline.
  • the end-capping substance is a polyamino substance.
  • the end-capping substance is one or more combinations of polyethylene glycol ammonia, aminopropylene glycol, amino acids and polypeptides.
  • the mass percentage concentration of the capping substance is 0.1% to 0.5%.
  • the solvent in the end-capping substance-containing solution is PBS buffer, phosphate buffer or Tris-hydrochloric acid buffer; the pH is 7-8.
  • the exposure time of the glutaraldehyde-crosslinked biological material in the solution containing the capping substance is terminated when the residual aldehyde group amount of the glutaraldehyde-crosslinked biological material is lower than a detectable value.
  • detectable value means that the residual aldehyde groups cannot be detected by conventional detection methods, and at this time, it can also be considered that all the residual aldehyde groups are completely blocked.
  • the exposure time is 1-6 hours.
  • the contact reaction for 1-6h can completely block the residual aldehyde groups.
  • a reducing agent can also be used to reduce the residual aldehyde group to a hydroxyl group to eliminate the effect of the residual aldehyde group on the activity of endoglin-stimulating factor.
  • the reducing agent can be selected from sodium borohydride or potassium borohydride.
  • the biomaterial After the removal of residual aldehyde groups as described above, the biomaterial is exposed to the endothelial growth factor solution, and the endothelial growth factor is grafted to the surface of the biomaterial by a physical coating method.
  • the endothelial growth-promoting growth factor is CD34 antibody, CD133 antibody or vascular endothelial growth factor VEGF.
  • the concentration of the endothelial growth-promoting growth factor in the endothelial growth-promoting growth factor and the exposure time in the endothelial growth-promoting factor solution are such that the content of the endothelial growth-promoting factor on the surface of the glutaraldehyde cross-linked biomaterial after the preparation reaches 0.1 ⁇ 20ug/ cm2 meter.
  • the solvent of the endothelial growth-promoting factor solution is PBS buffer, phosphate buffer or Tris-hydrochloric acid buffer; the endothelial growth-promoting factor concentration is 1-50 ug/ml.
  • the exposure time in the endothelial growth-promoting factor solution is 1-6 hours.
  • reaction temperature in the endothelial growth-promoting factor-containing solution is 0-37°C.
  • the pH value of the solution containing the endothelial growth-promoting growth factor is neutral.
  • the exposure modes of the glutaraldehyde-crosslinked biological material in the solution containing the capping substance and in the solution containing the endothelial growth-promoting growth factor are both dynamic contact or static contact.
  • the method further includes: exposing the glutaraldehyde cross-linked biological material after the reaction is completed to the softener solution.
  • the requirements of the softening treatment step, the drying and dehydration step and the storage conditions are the same as those of the chemical grafting method.
  • a biomaterial for promoting endothelialization prepared by the preparation method.
  • the biomaterials prepared in the present application can be used for interventional biological valves, such as through minimally invasive intervention, and can also be used in surgical biological valves, such as surgical implantation.
  • An endothelium-promoting heart valve comprises a cylindrical stent and a valve installed in the stent, wherein the valve is the endothelium-promoting biomaterial of the present application.
  • a preparation method of a heart valve for promoting endothelialization the heart valve comprises a stent and a valve sewn on the stent, the valve is a glutaraldehyde cross-linked biological material, and the heart valve is carried out according to the aforementioned method. (chemical grafting or physical coating) for endothelial growth factor grafting treatment.
  • a preparation method of a pro-endothelialization heart valve the heart valve comprises a stent and a valve, the valve is a glutaraldehyde cross-linked biological material, and the valve is subjected to the aforementioned endothelinization-promoting factor loading treatment (chemical grafting or physical coating) and then sewn onto the stent.
  • a pro-endothelialized heart valve prepared by the preparation method.
  • the pro-endothelializing heart valve of the present application can be a valve through minimally invasive intervention, or a valve implanted through surgery.
  • An interventional system includes a heart valve and a delivery tube, the heart valve is delivered through the delivery tube, and the heart valve is the pro-endothelialization heart valve as described above.
  • the present application has at least one of the following beneficial effects:
  • the antibody selected in this application has high activity to capture endothelial progenitor cells, high specificity and capture ability, and faster endothelialization.
  • the residual aldehyde group on the surface of the biological material is directly used as the linking group to react with the amino group of the endothelial growth factor, which not only eliminates the possible influence of the residual aldehyde group on the activity of the endothelial growth factor, Again, the treatment step for residual aldehyde groups was omitted.
  • the endothelial growth-promoting factor is directly combined on the surface of the biological material, and the endothelial cells are directly deposited on the surface of the biological material. After implanting in the human body, it can be maintained for a long time Endothelialization of the membrane surface avoids the risk of shedding attached to surfaces such as substrates.
  • FIG. 1 is a process flow diagram of the application.
  • FIG. 2 is a graph showing the results of characterization of the grafting uniformity of the membrane material of the present application.
  • FIG. 3 is a graph showing the results of the characterization of the adhesion of the membrane material of the present application to platelets.
  • FIG. 4 is a graph showing the results of characterization of the in vitro cell adhesion of the membrane material of the application.
  • FIG. 5 is a graph showing the results of characterization of the in vitro cell proliferation performance of the membrane material of the application.
  • FIG. 6 is a graph showing the characterization results of the anti-scour resistance of the antibody coating of the membrane material of the present application.
  • Figure 7 is a graph of the characterization results of the long-term stability of the antibody coating of the membrane material of the present application.
  • the surface of the glutaraldehyde cross-linked biological material is grafted with endothelial growth-promoting factor. Sterilize after handling and store by appropriate means. Taking the grafted CD34 antibody as an example, the process flow is generally shown in FIG. 1 .
  • the washing of free antibody, sterilization and antibody storage shown in the figure are all routine operations, and are not limited to the method of the present application.
  • the surface contact method selects chemical grafting method and physical coating method.
  • Chemical grafting The cross-linked biological material after removal of free glutaraldehyde is immersed in a solution containing endothelial growth-promoting growth factors and reacted under neutral or weak alkaline conditions.
  • Amino reagents for eliminating aldehyde groups include: polyethylene glycol ammonia, amino propylene glycol, amino acids, and polypeptides.
  • a reducing agent can also be used.
  • the reducing agent can be selected from sodium borohydride or potassium borohydride.
  • the concentration of the reducing agent solution is 0.1% to 2%, and the solvent is water, ethanol, methanol or a mixed solution.
  • the alcohol content is 0-100%, and the contact reaction is carried out at room temperature for 0.5-4 hours.
  • the chemical cross-linking method was also compared (through the cross-linking agent SANPAH, under ultraviolet irradiation, one end containing an N-hydroxysuccinimide group (NHS) was reacted with the amino group of the CD34 antibody, and the CD antibody was combined.
  • SANPAH cross-linking agent
  • layer-by-layer self-assembly immobilization of the negatively charged antibody molecule CD34 to the surface of the polyelectrolyte with the polycation as the outermost layer.
  • Softening treatment The biological material after surface contact treatment is soaked in a softener solution for softening treatment.
  • Dehydration freeze-drying or ethanol dehydration.
  • Sterilization preservation The dry biological material after dehydration treatment is sterilized and preserved.
  • Sterilization method ethylene oxide or irradiation sterilization.
  • the antibodies used in the following examples are mouse anti-human CD34 antibodies, KG-1a type, animal-derived cloned antibodies, which can be obtained by conventional cloning methods or obtained through commercial channels.
  • the antibodies used in the following examples are all obtained from commercial channels. .
  • the solution is a PBS solution containing 20ug/ml CD34 antibody and pH 7.4 .
  • the antibody solution containing the valve was placed on a shaker, and the temperature was controlled to be 20°C for 2h shaking reaction.
  • the valve was taken out and soaked in pure glycerol solution for 1 h at room temperature. The excess glycerol on the surface was removed, and centrifuged rapidly at 1000 r/min for 5 min, the preparation process was completed, and a heart valve with CD34 antibody immobilized on the surface was obtained.
  • the valve is finally packaged and sterilized with ethylene oxide.
  • the sewn heart valve was washed with normal saline to remove free glutaraldehyde, and then the heart valve containing residual aldehyde groups was immersed in PBS solution containing 0.1 wt% polyethylene glycol ammonia and pH 7.4.
  • the solution containing the valve was placed on a shaker, and the temperature was controlled to 37 °C for 4 h of shaking reaction. Place the valve and soak it in a solution containing 10ug/ml CD34 antibody for 0.5h at room temperature.
  • the valve was then soaked in pure glycerol solution for 2 hours, the valve was taken out, and centrifuged rapidly at 1000 r/min for 2 minutes, the preparation process was completed, and a heart valve with CD34 antibody immobilized on the surface was obtained.
  • the valve is finally packaged and sterilized with ethylene oxide.
  • Embodiment 6 First reduction and then physical coating
  • the concentration of sodium borohydride is 0.2% (concentration by mass)
  • the solvent is a mixed solution of water and ethanol, and the content of ethanol is 50% (concentration by volume)
  • the sewn heart valve is washed with normal saline to remove free amyloid.
  • Dialdehyde was put into the above-prepared solution and soaked for 2h at room temperature. Wash with normal saline. Then place the valve and soak it in a solution containing 10ug/ml CD34 antibody for 2h at room temperature.
  • valve was then soaked in pure glycerol solution for 2 hours, the valve was taken out, and centrifuged rapidly at 1000 r/min for 1 minute, the preparation process was completed, and a heart valve with CD34 antibody immobilized on the surface was obtained. The valve is finally packaged and sterilized with ethylene oxide.
  • the cross-linked membrane was placed in a 20ug/ml anti-CD34 antibody solution (there are also cationic compounds such as chitosan in the solution), and the membrane was treated at 4°C for 12 hours to obtain an anti-CD34 antibody-modified membrane.
  • the uniformity of fluorescence intensity distribution on the membrane surface was observed by immunofluorescence method to characterize the grafting uniformity on the membrane surface.
  • the specific process includes: (1) The membrane sheet immobilized with CD34 antibody (prepared in Examples 1 to 6, respectively) Rinse three times with PBS at room temperature, 5 min each time; (2) add fluorescent secondary antibody (PE-labeled rabbit anti-mouse IgG) to the membrane after rinsing at a molar ratio of 1:400, and incubate at room temperature for 1 hour in the dark; (3) Remove the unreacted fluorescent secondary antibody, and rinse three times with PBS at room temperature for 10 min each time; (4) Fix the treated membrane on a glass slide and send it for immunofluorescence detection.
  • the detection results are shown in Figure 2, and the results show that CD34 antibody can be detected in all the examples; the grafting uniformity of the method of the present application is good.
  • Enzyme-linked immunosorbent assay was used to detect the amount of CD34 antibody immobilized on the surface of the membrane by ELISA.
  • the test results are shown in Table 1.
  • the results show that the amount of CD34 antibody on the membrane surface prepared in Examples 1 to 6 ranges from 9.86 to 18.94 ⁇ g CD34 antibody on the surface of the biomaterial with a unit surface area of 1 cm 2 .
  • Example serial number Antibody content per unit area/( ⁇ g/cm 2 ) Example 1 12.85 ⁇ 1.01
  • Example 2 10.77 ⁇ 1.62
  • Example 3 9.86 ⁇ 0.82
  • Example 4 18.72 ⁇ 1.98
  • Example 5 15.88 ⁇ 1.18
  • Example 6 18.94 ⁇ 1.79
  • the LDH (lactate dehydrogenase) content released by the platelets adsorbed on the membrane material was measured by using the LDH content detection kit to detect the adhesion of the membrane material to the platelets.
  • the specific experimental process (1) The sample membrane (Example 1 , 2, 4 and 5, the control group is the glutaraldehyde cross-linked membrane not modified with CD34) and fresh platelet-rich plasma at 37 ° C for 30 min, carefully wash the membrane with PBS, and use 1% glutaraldehyde solution Fixed for 30min; (2) After washing with three distilled water, successively dehydrated with ethanol water gradient solution (30, 40, 50, 60, 70, 80, 90, 95, 100% v/v); (3) in air After natural drying, add 150 ⁇ L of LDH release reagent diluted 10 times with PBS to the well plate, shake the 96-well plate to make it evenly mixed, and then put it into a 37°C incubator to incubate for 1 hour; (4) Take 120 ⁇ L of the supernatant from
  • the solution was added to a new 96-well plate, and then three solutions (lactic acid solution, enzyme solution and 1X INT solution) were added according to the volume 1:1:1 to prepare the detection solution.
  • three solutions lactic acid solution, enzyme solution and 1X INT solution
  • the test results are shown in Figure 3, and the results show that the CD34-modified cross-linked membrane has better anti-platelet adhesion.
  • the blank group was in a 24-well cell culture plate without CD34-modified glutaraldehyde cross-linked membrane), after culturing for 10 min, 30 min, and 60 min, respectively, the original culture solution was discarded, and washed with PBS for 3 times; after FDA staining for 15 min, PBS Washed 5 times, photographed and observed with a fluorescence microscope, and counted and counted the cells on the photo with ImageJ software; the membrane without modified CD34 antibody was used as a control.
  • cells were cultured for a period of time, trypsinized and manually counted with a hemocytometer under a phase contrast microscope. Or use flow cytometry to measure the number of cells on the membrane surface.
  • the test results are shown in Figure 4. The results show that the CD34-modified cross-linked membrane has better adhesion to endothelial cells, which is significantly better than that of the unmodified cross-linked membrane.
  • the culture medium was discarded and washed 3 times with PBS; after 15 min of FDA staining, washed 5 times with PBS, and photographed with a fluorescence microscope for observation.
  • Cells on the surface of the photographs were counted either in software (IPP or ImageJ) or manually, using membranes with unmodified CD34 antibody as a control. Or cells were cultured for a period of time, trypsinized and manually counted with a hemocytometer under a phase contrast microscope.
  • the detection results are shown in Figure 5. The results show that the CD34-modified cross-linked membrane endothelial cells have better proliferation, high specificity and capture ability, and are very suitable for cell endothelialization.
  • the cytotoxicity of the prepared antibody membranes was observed by MTT method.
  • the specific process was as follows: (1) Cell culture: cultured and sorted endothelial cells, respectively inoculated into 48-well plates with 5000 cells per well, and the membrane materials were placed in the wells.
  • Fluorescence method or immunofluorescence method was used to observe the change of the fluorescence intensity on the surface of the membrane.
  • the specific test method put the antibody-modified dry membrane (prepared in Example 1 and Example 4) into a test tube containing PBS buffer solution, place it in a shaker, and keep it at a constant temperature. 37 °C, the speed is 100r/min. After 1 day, it was taken out, and the distribution of residual antibodies on the membrane surface was detected.
  • the antibody-modified dry film was placed in a sterilized container, sealed, and refrigerated at 4°C for 6 months. According to the above description, the washout resistance of the antibody film after storage for 6 months was tested.
  • the test results are shown in Figure 6. The results show that the cross-linked film grafted with CD34 has strong erosion resistance and can remain on the film surface for a long time.
  • the fluorescence intensity changes on the membrane surface were observed by fluorescence immunoassay.
  • Test method Take several pieces of the prepared antibody-modified dry film (prepared in Example 1 and Example 4) and put them into sterilization containers, and number them in sequence. (1) Put 6 pieces of dry film in a desiccator and store at room temperature for one month; (2) Put 6 pieces of dry film in a refrigerator at 4°C for one month. Fluorescence immunoassays were performed as described in fluoroimmunoassay, and fluorescence photographs were taken to examine the activity of the antibody coating. The results are shown in Fig. 7, and the results show that the membrane prepared in the present application has long-term antibody stability.

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Abstract

一种促内皮化生物材料及其制备方法，促内皮化的心脏瓣膜及其制备方法，介入系统。促内皮化生物材料包括生物材料和负载在生物材料表面的促内皮化生长因子，能够捕获内皮祖细胞，使内皮祖细胞在生物材料表面附着、生长和分化。制备促内皮化生物材料的方法包括化学接枝法和物理涂覆法。心脏瓣膜可通过先缝制再接枝或先接枝再缝制两种方式制备。直接将促内皮化生长因子负载在生物材料膜的表面，促进内皮细胞层在生物材料表面的形成。

Description

促内皮化生物材料、心脏瓣膜及其制备和应用 技术领域
本申请涉及植入生物材料技术领域，具体涉及一种促内皮化生物材料、心脏瓣膜及其制备和应用。
背景技术
近年来，介入生物材料例如介入瓣膜置换手术由于其操作方便、大大降低了对病人的二次损伤，从而取得了越来越广泛的临床应用。对介入瓣膜装置进行表面处理，介入瓣膜置于体内后可捕获内皮祖细胞，促进瓣膜装置的内皮化，增强瓣膜装置的生物相容性以及延长瓣膜装置的使用寿命。心脏瓣膜装置包括支架和安装于支架内的瓣膜，已有的研究中，捕获内皮祖细胞的捕获因子负载在支架上，存在负载不牢固的问题，为解决该问题必然过渡使用交联剂。
发明内容
本申请提供促内皮化心脏瓣膜及其制备和应用，通过将促内皮化生长因子直接通过化学或物理负载在瓣膜表面，无需额外交联剂的使用。
一种促内皮化生物材料，.生物材料的表面负载有促内皮化生长因子。
促内皮化生长因子是指一类可以捕获内皮祖细胞以使内皮祖细胞在生物材料的表面粘附、生长和分化的一类物质，以诱导内皮化功能细胞层在生物材料的表面生成。
可选的，所述促内皮化生长因子的负载量为：单位表面积1cm 2的生物材料表面含有0.1～20ug所述促内皮化生长因子。
可选的，所述生物材料为戊二醛交联生物材料。
所述戊二醛交联生物材料为戊二醛交联处理的生物组织；所述生物组织的生物来源为猪或牛、来源部位为心包膜、心脏瓣膜、血管、韧带、肌肉、肠或皮肤。这些生物组织中富含胶原蛋白纤维，胶原蛋白纤维富含氨基和羧基，戊二醛通过其醛基与氨基或羧基的反应将胶原蛋白内、胶原蛋白间形成交联网络。
可选的，所述促内皮化生长因子为CD34抗体、CD133抗体或血管内皮生长因子VEGF。
CD34抗体负载在生物材料表面，通过与内皮祖细胞表面CD34抗原的特异性结合，捕获内皮祖细胞，使内皮祖细胞在生物材料表面粘附、生长和分化，并形成功能性内皮细胞层。本申请的CD34抗体选择鼠抗人CD34抗体，KG-1a类型。该抗体本身可通过本领域熟知的克隆式获得，也可通过市购方式获得。CD133抗体和VEGF生长因子可通过基本类似的原理发生作用。
可选的，所述促内皮化生长因子通过化学接枝或物理涂覆负载于生物材料表面。
一种促内皮化生物材料的制备方法，包括：
将清除游离戊二醛后的戊二醛交联生物材料暴露于含促内皮化生长因子的溶液中进行反应，所述溶液的pH值为中性或弱碱性。
戊二醛交联处理后的生物材料中，一部分戊二醛的两端分别与胶原蛋白纤维的氨基或羧基连接，交联于网络结构中，一部分戊二醛的其中一端与胶原蛋白纤维的氨基或羧基连接，另一端处于游离状态即为所述残余醛基，还有部分未参与反应的游离戊二醛，该部分游离戊二醛在进行促内皮化因子涂覆前需将其清除，以消除其可能对促内皮生长因子活性的破坏。
该处理方法为化学接枝方法，化学接枝机理：CD34抗体或CD133抗体或VEGF生长因子含有氨基官能团，生物材料上含有残留醛基，当在中性或弱碱性条件下，抗体氨基官能团与瓣膜上醛基发生化学反应，抗体接枝到瓣膜材料表面。
可选的，所述含促内皮化生长因子的溶液中，促内皮化生长因子的浓度为1～50ug/ml。
进一步可选的，所述含促内皮化生长因子的溶液中还含有多氨基物质。
可选的，所述多氨基物质为聚乙二醇氨、氨基丙二醇、氨基酸和多肽中的至少一种。
可选的，所述多氨基物质的浓度为1～50ug/ml。
当促内皮化生长因子的浓度较小，促内皮化生长因子的接枝量不足以反应掉所有残余醛基时，加入多氨基物质竞争材料表面的残余醛基，消除残余醛基对促内皮化生长因子活性的影响。
可选的，所述含促内皮化生长因子的溶液的溶剂为PBS缓冲液、磷酸缓冲液或Tris-盐酸缓冲液；pH值为7～8。
可选的，所述反应以所述促内皮化生物材料表面的促内皮化生长因子含量达到0.1～20ug/cm 2时终止。
可选的，所述反应的时间为1～6h。在促内皮化生长因子的浓度为1～50ug/ml时，反应1～6h可使促内皮化生物材料表面的促内皮化生长因子含量达到0.1～20ug/cm 2。
化学接枝反应在常规室温下进行，可无需刻意控制反应温度。可选的，所述反应的温度为0～37℃。
可选的，反应过程中戊二醛交联生物材料与含促内皮化生长因子的溶液之间动态接触或静态接触
所述的动态接触过程中，戊二醛交联生物材料与含促内皮化生长因子的溶液之间相对运动，可以通过循环喷淋或反应过程中搅拌、晃动反应体系等实现，例如将戊二醛交联生物材料浸泡于反应溶液然后置于摇床内反应。所述静态接触过程中，戊二醛交联生物材料与含促内皮化生长因子的溶液之间相对静止，例如可直接将戊二醛交联膜浸泡于反应溶液中，静置反应。本申请中提到的动态接触和静态接触如无特殊说明均可同此理解。
可选的，所述制备方法还包括：将反应完成后的戊二醛交联生物材料暴露于柔顺剂溶液中。
抗体接枝后的生物材料，柔顺处理过程中对柔顺剂的要求为柔顺处理过程中不破坏抗体的活性。可选的，所述柔顺剂溶液选自纯甘油或甘油与聚乙二醇的混合溶液；所述甘油的体积百分比为20～100％；当甘油体积百分比为100％时即为纯甘油，当甘油体积百分比小于100％时即为甘油与聚乙二醇的混合溶液。
甘油溶液配方为甘油(20％～100％)+PEG，甘油溶液浸泡时间0.5～4h后取出，去除游离甘油，脱水或干燥后进行干态保存；也可以直接浸泡于甘油溶液中保存。
所述柔顺剂还可以是甘油和水混合液；甘油和醇类混合溶液；甘油、水和醇类混合溶液；聚乙二醇；聚乙二醇和水混合液；聚乙二醇和醇类混合溶液；聚乙二醇、水和醇类混合溶液。
在含甘油的方案中，甘油的体积百分比为20％～100％；在含聚乙二醇的方案中，聚乙二醇的体积百分比为20％～100％。
可选的，所述制备方法还包括：将柔顺剂处理后的戊二醛交联生物材料去除游离甘油后进行脱水或干燥处理。
脱水处理为可选择的优选步骤，脱水后保藏更有利于抗体活性的保持；当然也可在柔顺处理后直接浸泡于相应的溶液中进行保存，对于浸泡方式的选择，需在不影响CD34抗体活性的惰性环境中保存。例如纯甘油溶液、甘油和水混合溶液、甘油和醇类混合溶液、纯聚乙二醇溶液、聚乙二醇和水混合溶液、聚乙二醇和醇类混合溶液等中。保存温度可采用低温或常温保存，优选在-10～25℃条件下保存。
一种促内皮化生物材料的制备方法，包括：将清除游离戊二醛后的戊二醛交联生物材料依次暴露于含封端物质溶液和含促内皮生长因子溶液中。
所述封端物质为封闭戊二醛交联生物材料中残余醛基的物质，用于封闭戊二醛交联生物材料的残余醛基，消除残余醛基对促内皮生长因子活性的影响。该处理方法为物理接枝方法，物理接枝机理：先将生物材料表面残余醛基封闭，再将溶解有所述促内皮化生长因子的基质溶液涂覆于生物材料表面。
可选的，所述封端物质包含至少一个与所述戊二醛交联生物材料的残余醛基反应的基团。
可选的，与所述残余醛基反应的基团为氨基；所述含封端物质溶液的pH值为中性或弱碱性。
可选的，所述封端物质为多氨基物质。
可选的，所述封端物质为聚乙二醇氨、氨基丙二醇、氨基酸和多肽的一种或多种组合。
可选的，所述含封端物质溶液中，封端物质的质量百分比浓度为0.1％～0.5％。
可选的，所述含封端物质溶液中溶剂为PBS缓冲液、磷酸缓冲液或Tris-盐酸缓冲液；pH值为7～8。
可选的，所述戊二醛交联生物材料在含封端物质溶液中的暴露时间以所述戊二醛交联生物材料的残余醛基量低于可检测值终止。
低于可检测值即是指采用常规的检测手段无法检测到残余醛基，此时也可认为是所有的残余醛基被全部封闭。
可选的，所述暴露时间为1～6h。在封端物质采用如前所述质量百分比浓度为0.1％～0.5％条件下，接触反应1～6h可使残余醛基全部封闭。
在处理残余醛基的步骤中除采用含氨基的物质直接通过其氨基与残余醛基反应外，也可采用还原剂将残余醛基还原为羟基，消除残余醛基对促内皮因子活性的影响。还原剂可选择硼氢化钠或硼氢化钾。
在进行如上所述的残余醛基消除后，将生物材料暴露于促内皮化生长因子溶液中，通过物理涂覆法将促内皮化生长因子接枝再生物材料表面。
可选的，所述促内皮化生长因子为CD34抗体、CD133抗体或血管内皮生长因子VEGF。
可选的，所述促内皮化生长因子中促内皮生长因子的浓度以及在促内皮生长因子溶液中的暴露时间以制备完成后的戊二醛交联生物材料表面促内皮生长因子含量达到0.1～20ug/cm 2计。
可选的，所述促内皮生长因子溶液的溶剂为PBS缓冲液、磷酸缓冲液或Tris-盐酸缓冲液；促内皮生长因子浓度为1～50ug/ml。
可选的，在上述浓度范围内，促内皮生长因子溶液中的暴露时间为1～6h。
反应在常规室温下进行，可无需刻意控制反应温度。可选的，在含促内皮生长因子溶液中的反应温度为0～37℃。
可选的，所述含促内皮化生长因子溶液的pH值为中性。
可选的，所述戊二醛交联生物材料在含封端物质溶液中和含促内皮化生长因子溶液中的暴露方式均为动态接触或静态接触。
可选的，还包括：将反应完成后的戊二醛交联生物材料暴露于柔顺剂溶液中。
物理涂覆方法中，柔顺处理步骤、干燥脱水步骤以及保存条件要求均同化学接枝方法。
一种由所述制备方法制备得到的促内皮化生物材料。
本申请制备的生物材料可以用于介入生物瓣膜，例如通过微创介入，也可用于外科生物瓣膜，例如通过外科手术植入。
一种促内皮化的心脏瓣膜，包括圆筒状的支架和安装于支架内的瓣膜，所述瓣膜为本申请的促内皮化生物材料。
一种促内皮化心脏瓣膜的制备方法，所述心脏瓣膜包括支架和缝制在支架上的瓣膜，所述瓣膜为戊二醛交联生物材料，对所述心脏瓣膜按如前所述的方法(化学接枝或物理涂覆)进行促内皮生长因子接枝处理。
一种促内皮化心脏瓣膜的制备方法，所述心脏瓣膜包括支架和瓣膜，所述瓣膜为戊二醛交联生物材料，将所述瓣膜经如前所述的促内皮化因子负载处理(化学接枝或物理涂覆)后缝制在所述支架上。
一种由所述制备方法制备得到的促内皮化心脏瓣膜。
本申请的促内皮化心脏瓣膜可以是通过微创介入的瓣膜，也可以是通过外科手术植入的瓣膜。
一种介入系统，包括心脏瓣膜和输送管，所述心脏瓣膜经输送管输送，所述心脏瓣膜为如前所述的促内皮化心脏瓣膜。
与现有技术相比，本申请至少具有如下有益效果之一：
(1)本申请所选择的抗体选择捕获内皮祖细胞活性高，具有高特异性和捕获能力，内皮化速度更快。
(2)本申请的化学接枝方法中，无需另外增加交联剂，仅使用交联膜表面残余醛基接枝上大量抗体；降低成本，减少废液带来的污染。
(3)本申请的化学接枝方法中，直接以生物材料表面的残余醛基作为连接基团与促内皮生长因子的氨基反应，既消除了残余醛基可能对促内皮生长因子的活性影响，又省略了残余醛基的处理步骤。
(4)本申请的物理涂覆方法中，仅需通过封端物质或还原剂将残余醛基封闭后一步涂覆完成。
(5)本申请中不管是采用化学接枝法还是物理涂覆法都是将促内皮生长因子直接结合在生物材料表面，内皮细胞直接敷在生物材料表面，在植入人体后，能够长期维持膜表面内皮化，可避免附着在基质等表面的脱落风险。
附图说明
图1为本申请的一种工艺流程图。
图2为本申请膜材料接枝均匀性的表征结果图。
图3为本申请膜材料对血小板粘附性的表征结果图。
图4为本申请膜材料体外细胞粘附性的表征结果图。
图5为本申请膜材料体外细胞增殖性能的表征结果图。
图6为本申请膜材料抗体涂层耐冲刷性的表征结果图。
图7位本申请膜材料抗体涂层长期稳定性的表征结果图。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
除非另有定义，本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同。本文中在本申请的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本申请。
将戊二醛交联生物材料清洗掉表面游离戊二醛后，对戊二醛交联生物材料表面进行促内皮生长因子的表面接枝，接枝反应完成后处理掉游离促内皮生长因子，柔顺处理后灭菌并通过适当的方式进行保存。以接枝CD34抗体为例，其工艺流程大体如图1所示，图中所出现的清洗游离抗体、灭菌及抗体保存均为常规操作，不作为对本申请的方法的限定。
表面接枝：表面接触方法选择化学接枝法和物理涂覆法。
化学接枝：将清除游离戊二醛后的交联生物材料浸泡在含有促内皮化生长因子的溶液中，在中性或弱碱性条件下反应。
物理涂覆：将清除游离戊二醛后的交联生物材料在中性或弱碱性条件下用含氨基试剂消除醛基，处理好的瓣膜浸泡在含有促内皮化生长因子的中性溶液中涂覆。
消除醛基的氨基试剂包括：聚乙二醇氨、氨基丙二醇、氨基酸、多肽。
消除醛基除采用氨基试剂外，还可采用还原剂，还原剂可选择硼氢化钠或硼氢化钾，还原剂溶液浓度为0.1％～2％，溶剂为水、乙醇、甲醇或其混合溶液，醇含量为0～100％，室温条件下接触反应0.5～4h。
接枝步骤中，还对比了化学交联法(通过交联剂SANPAH在紫外线照射下，将其一端含有N-羟琥珀酰亚胺基团(NHS)与CD34抗体的氨基反应，将CD抗体结合到SANPAH上)和层层自组装法(将带负电荷的抗体分子CD34固定到聚阳离子为最外层的聚电解质表面)。
柔顺处理：表面接触处理后的生物材料浸泡于柔顺剂溶液中进行柔顺处理。
脱水处理：采用冷冻干燥或乙醇脱水。
灭菌保存：脱水处理后的干态生物材料进行灭菌保存。
灭菌方式：环氧乙烷或辐照灭菌。
以下以具体实施例进行说明：
以下实施例中所用抗体选择鼠抗人CD34抗体，KG-1a类型，动物源克隆抗体，可通过常规克隆手段获得，也可通过商购途径获得，以下实施例中所用抗体均为商购途径获得。
实施例1化学接枝
将缝制好的心脏瓣膜用生理盐水清洗，清除游离戊二醛，然后将含有残留醛基的心脏瓣膜浸泡在CD34抗体溶液中，溶液为含有20ug/ml CD34抗体、pH值为7.4的PBS溶液。然后将含有瓣膜的抗体溶液放置在摇床上，控制温度为20℃震荡反应2h。取出瓣膜，放置在纯甘油溶液中室温浸泡1h。清除表面多余甘油，1000r/min快速离心5min，制备过程完成，得到表面固定CD34抗体的心脏瓣膜。最后包装瓣膜，环氧乙烷灭菌。
实施例2化学接枝
将缝制好的心脏瓣膜用生理盐水清洗，清除游离戊二醛，然后将含有残留醛基的心脏瓣膜浸泡在CD34抗体溶液中，溶液为含有5ug/ml CD34抗体、50ug/ml聚乙二醇氨、pH值为7.4的PBS溶液。然后将含有瓣膜的抗体溶液放置在摇床上，控制温度为20℃震荡反应2h。取出瓣膜，放置在纯甘油溶液中室温浸泡1h。清除表面多余甘油，2000r/min快速离心5min。包装瓣膜，辐照灭菌。
实施例3化学接枝
将缝制好的心脏瓣膜用生理盐水清洗，清除游离戊二醛，然后将含有残留醛基的心脏瓣膜浸泡在CD34抗体溶液中，溶液为含有10ug/ml CD34抗体、pH值为7.4的硼酸盐溶液。然后将含有瓣膜的抗体溶液放置在摇床上，控制温度为20℃震荡反应1h。取出瓣膜，放置在甘油和乙醇混合溶液中室温浸泡1h。清除表面多余甘油，1000r/min快速离心10min，制备过程完成，得到表面固定CD34抗体的心脏瓣膜。最后包装瓣膜，环氧乙烷灭菌。
实施例4物理涂覆
将缝制好的心脏瓣膜用生理盐水清洗，清除游离戊二醛，然后将含有残留醛基的心脏瓣膜浸泡在含有0.1wt％聚乙二醇氨、pH值为7.4的PBS溶液中。将含有瓣膜的溶液放置在摇 床上，控制温度为37℃震荡反应4h。放置瓣膜在含有10ug/ml CD34抗体的溶液中室温浸泡0.5h。再用纯甘油溶液浸泡瓣膜2h，取出瓣膜，1000r/min快速离心2min，制备过程完成，得到表面固定CD34抗体的心脏瓣膜。最后包装瓣膜，环氧乙烷灭菌。
实施例5先接枝再缝合
将戊二醛交联膜用生理盐水清洗，清除游离戊二醛，然后将含有残留醛基的交联膜浸泡在CD34抗体溶液中，溶液为含有5ug/ml CD34抗体、50ug/ml聚乙二醇氨pH值为7.4的PBS溶液。然后将含交联膜的抗体溶液放置在摇床上，控制温度为20℃震荡反应2h。取出交联膜，进行切割、缝制在金属支架上。将缝制好的心脏瓣膜放置在纯甘油溶液中室温浸泡1h。清除表面多余甘油，1000r/min快速离心5min。包装瓣膜，环氧乙烷灭菌。
实施例6先还原再物理涂覆
硼氢化钠浓度为0.2％(质量百分浓度)，溶剂为水、乙醇混合溶液，乙醇含量为50％(体积百分浓度)，然后将缝制好的心脏瓣膜用生理盐水清洗，清除游离戊二醛，放入上述配制的溶液中，室温条件下浸泡2h。用生理盐水清洗干净。然后放置瓣膜在含有10ug/ml CD34抗体的溶液中室温浸泡2h。再用纯甘油溶液浸泡瓣膜2h，取出瓣膜，1000r/min快速离心1min，制备过程完成，得到表面固定CD34抗体的心脏瓣膜。最后包装瓣膜，环氧乙烷灭菌。
对比例1
CD34抗体和SANPAH(6-[(4-叠氮-2-硝基苯基)氨基]己酸琥珀酰亚胺酯)反应液制备：以化学交联剂SANPAH为介导，用4％NaOH(质量百分浓度)滴定无水乙醇，调整pH值到7.9，在避光下配制0.133mmol/L SANPAH溶液。按CD34抗体和SANPAH反应摩尔比为1:20加入抗体溶液中，室温避光混合反应2h，10000xg离心10min去除未反应的抗体和SANPAH。
对比例2
将交联膜置于20ug/ml的抗CD34抗体溶液(溶液中还有阳离子化合物，例如壳聚糖)中，4℃下作用12小时，得到抗CD34抗体修饰的膜。
接枝均匀性
通过免疫荧光法观察荧光在膜表面强度分布均匀性，以表征膜表面接枝均匀性，具体过程包括：(1)将固定有CD34抗体的膜片(分别为实施例1～实施例6制备)在室温下用PBS漂洗三次，每次5min；(2)向漂洗后的膜上按摩尔比1：400加入荧光二抗(PE标记的兔抗鼠IgG)，在室温下避光孵育1小时；(3)去除未反应的荧光二抗，用PBS在室温漂洗三次，每次10min；(4)将处理后的膜片固定于载玻片上，送检免疫荧光检测。检测结果如图2所示，结果表明，所有的实施例均能检测到CD34抗体；本申请方法的接枝均匀性较好。
接枝量和抗体活性
酶联接免疫吸附剂测定ELISA法检测固定于膜表面的CD34抗体量，具体过程包括：(1)固定了抗体的膜片(实施例1～实施例6制备)置于24孔板，用10％的BSA在37℃下阻断1小时，洗涤液(生理盐水)浸洗3×5min；(以湿膜表面直接固定BSA和干态膜表面直接固定BSA为阴性对照)；(2)加入HRP-IgG，100ul/孔，37℃作用1小时，洗涤液(生理盐水)浸洗3×5min；(3)加入TMB显色液，100ul/孔，37℃作用30min；(4)加入TMB终止液50ul/孔；(5)用酶标仪测定所得溶液的OD(450nm)值。
检测结果如表1所示，结果表明，实施例1～实施例6所制备得到是膜表面的CD34抗体量范围为：单位表面积1cm 2的生物材料表面含有9.86～18.94μg CD34抗体。
表1
实施例序号 单位面积抗体含量/(μg/cm 2)
实施例1 12.85±1.01
实施例2 10.77±1.62
实施例3 9.86±0.82
实施例4 18.72±1.98
实施例5 15.88±1.18
实施例6 18.94±1.79
对血小板粘附性
采用LDH含量检测试剂盒测定吸附在膜材料上的血小板释放的LDH(乳酸脱氢酶)含量，以检测膜材料对血小板的黏附量，具体实验过程：(1)将样品膜片(实施例1、2、4和5制备，对照组为未用CD34修饰的戊二醛交联膜)与新鲜富血小板血浆在37℃作用30min后，用PBS小心清洗膜片，并用1％的戊二醛溶液固定30min；(2)三蒸水清洗后，依次用乙醇水梯度溶液(30、40、50、60、70、80、90、95、100％v/v)逐级脱水；(3)空气中自然干燥后向孔板中加入150μL用PBS稀释10倍的LDH释放试剂并摇晃96孔板使其混合均匀，随即放入37℃恒温箱中孵育1小时；(4)取各孔的120μL上清液，加入到一个新的96孔板中，然后按照体积1:1:1加入三种溶液(乳酸溶液、酶溶液和1X INT溶液)配制成检测液。向每孔上清液中加入60μL检测液，混匀后，避光常温条件下在水平摇床上孵育25分钟，随后用酶标仪检测溶液在490nm处的吸光度。检测结果如图3所示，结果表明CD34修饰的交联膜抗血小板粘附性较好。
体外细胞粘附性
荧光显微镜法或流式细胞仪法观察记录膜表面细胞数量，具体实验过程：以1.5×10 4个/孔的密度将内皮细胞接种到底部置有样品膜片(实施例1和实施例4制备，空白组为未用CD34修饰的戊二醛交联膜)的24孔细胞培养板中，分别培养10min、30min、60min后，吸弃原培养液，PBS洗涤3次；FDA染色15min后，PBS清洗5次，并用荧光显微镜拍照观察，用ImageJ软件对照片上的细胞进行计数统计；以未修饰CD34抗体的膜片作为对照。或者细胞在培养一段时间后，用胰酶消化并用血球计数板在相差显微镜下进行人工计数。或采用流式细胞仪测定膜表面细胞的数量。检测结果如图4所示，结果表明CD34修饰的交联膜对内皮细胞粘附性较好，显著优于未修饰交联膜。
体外细胞增殖
荧光显微镜法或流式细胞仪法观察记录膜表面细胞数量，具体实验过程：以1.5×10 4个/孔的密度将内皮细胞接种到底部置有干膜(实施例1～实施例6制备，对照组为未用CD34修饰的戊二醛交联膜)的24孔细胞培养板中；培养1h后，吸弃未粘附的细胞，加入细胞培养液(DMEM培养基)继续培养24h后，吸弃培养液，PBS洗涤3次；FDA染色15min后，PBS清洗5次，并用荧光显微镜拍照观察。以未修饰CD34抗体的膜作为对照，通过在软件(IPP或ImageJ)或人工对照片表面的细胞进行计数。或细胞在培养一段时间后，用胰酶消化并用血球计数板在相差显微镜下进行人工计数。检测结果如图5所示，结果显示CD34修饰的交联膜内皮细胞增殖性较好，具有高特异性和捕获能力，非常适合细胞内皮化。
细胞毒性
MTT法观察制备得到的抗体膜的细胞毒性，具体过程：(1)细胞培养：培养分选的内皮细胞，分别以每孔5000个细胞分别接种于48孔板内，孔板内放有膜材料(实施例1～实施例6制备)，每孔加入细胞培养液(DMEM培养基)培养0小时，24小时，48小时，72小时，每组选择三个复孔；(2)呈色：拿出培养箱后，每孔加入500μl细胞培养液和50μl MTT溶液(5mg/ml)；37℃培养箱内孵育4小时；小心吸出细胞培养液，加入500μl DMSO，充分震荡10min，使结晶物充分溶解；(3)比色：每孔分别取出100μl，加入96孔板中进行检测；选择490nm波长，以对照孔调零，在酶标仪上测定各孔的吸光值；以时间为横坐标，吸光值为纵坐标绘制细胞生长曲线图。检测结果如表2所示，结果表明CD34修饰的交联膜无细胞毒性，生物相容性较好。
表2
Figure PCTCN2021142207-appb-000001
Figure PCTCN2021142207-appb-000002
抗体涂层耐冲刷性
荧光法或免疫荧光法观察膜表面荧光强度变化，具体试验方法：将抗体修饰干膜(实施例1和实施例4制备)放入装有PBS缓冲溶液的试管中，置于摇床中，恒温37℃，转速为100r/min。1天后取出，检测膜表面残留抗体分布情况。将抗体修饰干膜置于灭菌容器中，密封，置于4℃冷藏保存6个月，按照上述描述，检测保存6个月后的抗体膜的耐冲刷性。检测结果如图6所示，结果表明CD34接枝的交联膜耐冲刷性强，能够长期保留在膜表面。
抗体涂层长期稳定性
荧光免疫法观察膜表面荧光强度变化。试验方法：取制备好的抗体修饰干膜(实施例1和实施例4制备)若干片分别放入灭菌容器中，依次编号。(1)将6片干膜放入干燥器中，室温保存一个月；(2)将6片干膜放入冰箱4℃保存一个月。用荧光免疫法所描述进行荧光免疫检查，拍摄荧光照片，考察抗体涂层的活性。结果如图7所示，结果表明本申请制备得到的膜具有长期抗体稳定性。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对申请专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准。

Claims (41)

  1. 一种促内皮化生物材料，其特征在于，包括生物材料和负载于所述生物材料上的促内皮化生长因子。
  2. 根据权利要求1所述的生物材料，其特征在于，所述促内皮化生长因子的负载量为：单位表面积1cm 2的生物材料表面含有0.1～20ug所述促内皮化生长因子。
  3. 根据权利要求1所述的生物材料，其特征在于，所述生物材料为戊二醛交联生物材料。
  4. 根据权利要求1所述的生物材料，其特征在于，所述促内皮化生长因子为CD34抗体、CD133抗体或血管内皮生长因子VEGF。
  5. 根据权利要求1所述的生物材料，其特征在于，所述促内皮化生长因子通过化学接枝或物理涂覆负载于生物材料表面。
  6. 一种促内皮化生物材料的制备方法，其特征在于，包括：将清除游离戊二醛后的戊二醛交联生物材料暴露于含促内皮化生长因子的溶液中进行反应，所述溶液的pH值为中性或弱碱性。
  7. 根据权利要求6所述的制备方法，其特征在于，所述含促内皮化生长因子的溶液中，促内皮化生长因子的浓度为1～50ug/ml
  8. 根据权利要求7所述的制备方法，其特征在于，所述含促内皮化生长因子的溶液中还含有多氨基物质；所述多氨基物质为聚乙二醇氨、氨基丙二醇、氨基酸和多肽中的至少一种；所述多氨基物质的浓度为1～50ug/ml。
  9. 根据权利要求6所述的制备方法，其特征在于，所述含促内皮化生长因子的溶液中溶剂为PBS缓冲液、磷酸缓冲液或Tris-盐酸缓冲液；pH值为7～8。
  10. 根据权利要求6所述的制备方法，其特征在于，所述反应以所述促内皮化生物材料表面的促内皮化生长因子含量达到0.1～20ug/cm 2时终止。
  11. 根据权利要求6所述的制备方法，其特征在于，所述反应的时间为1～6h；所述反应的温度为0～37℃。
  12. 根据权利要求6所述的制备方法，其特征在于，反应过程中戊二醛交联生物材料与含促内皮化生长因子的溶液之间动态接触或静态接触。
  13. 根据权利要求6所述的制备方法，其特征在于，还包括：将反应完成后的戊二醛交联生物材料暴露于柔顺剂溶液中。
  14. 根据权利要求13所述的制备方法，其特征在于，所述柔顺剂溶液为甘油与聚乙二醇的混合溶液或纯甘油；所述甘油的体积百分比为20～100％。
  15. 根据权利要求13所述的制备方法，其特征在于，还包括：将柔顺剂处理后的戊二醛交联生物材料去除游离甘油后进行脱水或干燥处理。
  16. 一种促内皮化生物材料的制备方法，其特征在于，包括：将清除游离戊二醛后的戊二醛交联生物材料依次暴露于含封端物质溶液和含促内皮生长因子溶液中，所述封端物质为封闭戊二醛交联生物材料中残余醛基的物质。
  17. 根据权利要求16所述的制备方法，其特征在于，所述封端物质包含至少一个与所述戊二醛交联生物材料中残余醛基反应的基团。
  18. 根据权利要求17所述的制备方法，其特征在于，与所述残余醛基反应的基团为氨基； 所述含封端物质溶液的pH值为中性或弱碱性。
  19. 根据权利要求18所述的制备方法，其特征在于，所述封端物质为多氨基物质。
  20. 根据权利要求19所述的制备方法，其特征在于，所述封端物质为聚乙二醇氨、氨基丙二醇、氨基酸和多肽中的至少一种。
  21. 根据权利要求16所述的制备方法，其特征在于，所述含封端物质溶液中，封端物质的质量百分比浓度为0.1％～5％。
  22. 根据权利要求16所述的制备方法，其特征在于，所述含封端物质溶液中溶剂为PBS缓冲液、磷酸缓冲液或Tris-盐酸缓冲液；pH值为7～8。
  23. 根据权利要求16所述的制备方法，其特征在于，所述戊二醛交联生物材料在含封端物质溶液中的暴露时间以所述戊二醛交联生物材料的残余醛基量低于可检测值时终止。
  24. 根据权利要求23所述的制备方法，其特征在于，所述暴露时间为1～6h。
  25. 根据权利要求16所述的制备方法，其特征在于，所述封端物质为用于还原所述戊二醛交联生物材料中残余醛基的还原剂。
  26. 根据权利要求25所述的制备方法，其特征在于，所述还原剂为硼氢化钠或硼氢化钾。
  27. 根据权利要求16所述的制备方法，其特征在于，所述促内皮化生长因子为CD34抗体、CD133抗体或血管内皮生长因子VEGF。
  28. 根据权利要求16所述的制备方法，其特征在于，所述促内皮化生长因子中促内皮生长因子的浓度以及在促内皮生长因子溶液中的暴露时间以制备完成后的戊二醛交联生物材料表面促内皮生长因子含量达到0.1～20ug/cm 2计。
  29. 根据权利要求16所述的制备方法，其特征在于，所述促内皮生长因子溶液的溶剂为PBS缓冲液、磷酸缓冲液或Tris-盐酸缓冲液；促内皮生长因子浓度为1～50ug/ml。
  30. 根据权利要求29所述的制备方法，其特征在于，促内皮生长因子溶液中的暴露时间为1～6h；反应温度为0～37℃。
  31. 根据权利要求16所述的制备方法，其特征在于，所述含促内皮化生长因子溶液的pH值为中性。
  32. 根据权利要求16所述的制备方法，其特征在于，所述戊二醛交联生物材料在含封端物质溶液中和含促内皮化生长因子溶液中的暴露方式均为动态接触或静态接触。
  33. 根据权利要求16所述的制备方法，其特征在于，还包括：将反应完成后的戊二醛交联生物材料暴露于柔顺剂溶液中。
  34. 根据权利要求33所述的制备方法，其特征在于，所述柔顺剂溶液为甘油与聚乙二醇的混合溶液或纯甘油；所述甘油的体积百分比为20～100％。
  35. 根据权利要求33所述的制备方法，其特征在于，还包括：将柔顺剂处理后的戊二醛交联生物材料去除游离甘油后进行脱水或干燥处理。
  36. 一种由权利要求6～35任一所述制备方法制备得到的促内皮化生物材料。
  37. 一种促内皮化的心脏瓣膜，包括圆筒状的支架和安装于支架内的瓣膜，其特征在于，所述瓣膜为如权利要求1～5及36中任一所述的促内皮化生物材料。
  38. 一种促内皮化心脏瓣膜的制备方法，所述心脏瓣膜包括支架和缝制在支架上的瓣膜， 所述瓣膜为戊二醛交联生物材料，其特征在于，对所述心脏瓣膜按权利要求6～35任一所述的方法进行处理。
  39. 一种促内皮化心脏瓣膜的制备方法，所述心脏瓣膜包括支架和瓣膜，所述瓣膜为戊二醛交联生物材料，其特征在于，包括：将所述瓣膜经权利要求6～35任一所述方法处理后缝制在所述支架上。
  40. 一种由权利要求38或39所述制备方法制备得到的促内皮化心脏瓣膜。
  41. 一种介入系统，包括心脏瓣膜和输送管，所述心脏瓣膜经输送管输送，其特征在于，所述心脏瓣膜为权利要求37或40任一所述的促内皮化心脏瓣膜。
PCT/CN2021/142207 2020-12-31 2021-12-28 促内皮化生物材料、心脏瓣膜及其制备和应用 WO2022143701A1 (zh)

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