WO2022143700A1 - 抗折痕的脱水交联生物材料及其制备方法和应用 - Google Patents

抗折痕的脱水交联生物材料及其制备方法和应用 Download PDF

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WO2022143700A1
WO2022143700A1 PCT/CN2021/142206 CN2021142206W WO2022143700A1 WO 2022143700 A1 WO2022143700 A1 WO 2022143700A1 CN 2021142206 W CN2021142206 W CN 2021142206W WO 2022143700 A1 WO2022143700 A1 WO 2022143700A1
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cross
biological material
linking
linking agent
solution
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PCT/CN2021/142206
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English (en)
French (fr)
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卫星
林浩昇
邝大军
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杭州启明医疗器械股份有限公司
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Priority to EP21914434.2A priority Critical patent/EP4272771A1/en
Publication of WO2022143700A1 publication Critical patent/WO2022143700A1/zh
Priority to US18/211,381 priority patent/US20230330300A1/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
    • A61L27/36—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
    • A61L27/3604—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
    • 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
    • A61L27/362—Skin, e.g. dermal papillae
    • 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
    • A61L27/3629—Intestinal tissue, e.g. small intestinal submucosa
    • 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
    • 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 prosthesis biomaterials, in particular to a crease-resistant dehydrated cross-linked biomaterial and a preparation method and application thereof.
  • interventional biomaterials such as interventional valve replacement surgery have achieved more and more extensive clinical applications due to their convenience in operation and greatly reduced secondary damage to patients.
  • the interventional valve is stored in glutaraldehyde solution. When it is used at the operation site, it needs to be cleaned and loaded repeatedly, which increases the operation time and additional risks; toxicity.
  • Bioprosthetic valves can perfectly solve the above problems through dehydration and preloading.
  • the dehydration and preloading methods require the biological valve to have better elasticity and toughness due to the rehydration after the upper time crimping, so as to solve the crease problem after prolonged crimping.
  • the biological valve still cannot meet the crease problem after long-term crimping.
  • the existence of crease not only affects the hydromechanical properties of the valve, but also is more prone to calcification and fatigue tearing at the crease.
  • the present application provides a crease-resistant dehydrated and cross-linked biological material and a preparation method and application thereof, which solve the problem that the dry valve valve cannot be recovered after being rehydrated and held in a conveyor for a long time.
  • a method for preparing a crease-resistant dehydrated biological material by composite crosslinking characterized in that the biological material is subjected to (a) oxidation treatment, (b) first crosslinking treatment, (c) second crosslinking treatment and (d) ) obtained by dehydration treatment; wherein, (b) is carried out after (a) and (a) (b) (c) are all carried out before (d);
  • an oxidizing agent capable of converting hydroxyl groups into aldehyde groups is used, and the hydroxyl groups are derived from the mucopolysaccharides in the biological material
  • a first crosslinking agent capable of causing mutual cross-linking between the aldehyde groups of the mucopolysaccharides is used
  • a second cross-linking agent capable of causing the collagen fibers in the biomaterial to be cross-linked to each other is used in the second cross-linking treatment.
  • the collagen content of the biological material is 60%-90%.
  • the biological source of the biological material is pig, cow, horse or sheep; the source site of the biological material is pericardium, heart valve, blood vessel, ligament, muscle, intestine or skin.
  • the biological material includes porcine pericardium or bovine pericardium.
  • the oxidative treatment includes exposing the biological material to a solution containing an oxidant.
  • the hydroxyl group is the adjacent hydroxyl group of the mucopolysaccharide.
  • the oxidant is sodium periodate.
  • the mass percentage concentration of the oxidant in the oxidant-containing solution is 0.1% to 1%;
  • the solvent for configuring the oxidant solution is a conventional solvent for processing biological materials, which will not damage the structure of biological materials, such as water, PBS or physiological materials. salt water etc.
  • the exposure time of the biological material in the oxidant-containing solution is 1-12 hours.
  • the exposure mode of the biological material in the oxidant-containing solution is static contact or dynamic contact.
  • Static contact can be understood as the relative static between the biological material and the oxidant-containing solution during the oxidative treatment, such as soaking.
  • Dynamic contact can be understood as the relative movement between the biological material and the oxidant-containing solution during the oxidation treatment, for example, by circulating the oxidant-containing solution to the biological material, or stirring or shaking the reaction system when the biological material is immersed in the oxidant-containing solution.
  • the static contact or dynamic contact mentioned below can also be understood in the same way unless otherwise specified.
  • the biological material is exposed to a solution containing an oxidizing agent, and the oxidizing agent is used to oxidize the adjacent hydroxyl groups of the mucopolysaccharide in the biological material to an aldehyde group, and the mucopolysaccharide is connected to the collagen fiber in the biological material through its oxidized aldehyde group; after the oxidation treatment
  • the biological material is exposed to the first cross-linking agent for cross-linking treatment, and the mucopolysaccharides in the biological material are cross-linked through the first cross-linking agent; the biological material is exposed to the second cross-linking agent for cross-linking treatment, the biological material
  • the collagen fibers are cross-linked and connected by a second cross-linking agent; the cross-linked biological material is subjected to dehydration or drying treatment.
  • the biological material is exposed to a solution containing an oxidizing agent.
  • the oxidizing agent oxidizes the adjacent hydroxyl groups of the mucopolysaccharide molecules in the biological material to an aldehyde group, and on the other hand, the oxidized aldehyde group interacts with the collagen fibers in the biological material.
  • the amino groups are cross-linked.
  • the corresponding cross-linking agent is used to cross-link between the glycosaminoglycan molecules and the glycosaminoglycan molecules, and between the collagen fibers and the collagen fibers to form a network structure.
  • the sugar groups in the biomaterial are opened in situ and form a part of the cross-linked network.
  • the resulting cross-linked biomaterial has significantly improved elasticity and water absorption. After long-term compression in the conveyor, there is basically no crease after rehydration. At the same time, since the mucopolysaccharide molecule is a part of the biomaterial, the in situ opening and forming part of the cross-linked network not only solves the problem of crease recovery, but also solves the problem of immunogenicity.
  • An optional treatment sequence sequentially subjecting the biological material to (a) oxidation treatment, (b) first cross-linking treatment and (c) second cross-linking treatment: the oxidized biological material is sequentially exposed to the first cross-linking treatment. in the linker solution and the second crosslinker solution.
  • the first cross-linking agent has at least two active groups that react with aldehyde groups, the second cross-linking agent and the first cross-linking agent are different substances, and the second cross-linking agent can interact with the first cross-linking agent.
  • the reactive groups of the crosslinker react.
  • the cross-linking of the mucopolysaccharide is performed first, and then the cross-linking of the collagen fibers is performed.
  • the adjacent hydroxyl groups of the mucopolysaccharides in the biomaterial are oxidized to aldehyde groups, and a part of the aldehyde groups react with the amino groups of the collagen fibers in the biomaterials, resulting in the formation of cross-links between the mucopolysaccharides and the collagen fibers; the activity of the first cross-linking agent The group reacts with the remaining part of the aldehyde groups of the mucopolysaccharide, resulting in cross-linking between the mucopolysaccharides; the first cross-linking agent cross-links the mucopolysaccharide aldehyde groups and also introduces some free active groups, and the second cross-linking agent is mainly associated with The amino or carboxyl groups of the collagen fibers react with each other, resulting in
  • the first cross-linking agent is a small molecular substance with at least two amino groups.
  • Small molecular substances with at least two amino groups include amino acids, diamines, etc.; where amino acids can be selected from lysine; diamines are amino compounds containing two amino groups, such as ethylenediamine, propylenediamine, hexamethylenediamine , p-phenylenediamine, etc.
  • the first cross-linking agent is lysine, ethylenediamine or hexamethylenediamine.
  • the mass percentage concentration of the first cross-linking agent in the first cross-linking agent solution is 0.05% to 5%;
  • the solvent for configuring the first cross-linking agent solution is a conventional solvent for processing biological materials, which will not damage biological materials.
  • the structure of the material such as water, PBS or saline, etc.
  • the time for exposing the biological material to the first crosslinking agent solution is 0.5-12 hours.
  • the second cross-linking agent is glutaraldehyde; the mass percentage concentration of the second cross-linking agent in the second cross-linking agent solution is 0.05% to 25%, and the solvent of the second cross-linking agent solution is configured
  • the conventional solvent for processing biological materials will not destroy the structure of biological materials, such as water, PBS or physiological saline, etc.; the cross-linking time is 6h-3 weeks; the cross-linking temperature is 0-37°C.
  • the exposure modes of the biological material in the first cross-linking agent solution and the second cross-linking agent solution are both static contact or dynamic contact.
  • the first cross-linking agent is diamine and the second cross-linking agent is glutaraldehyde as an example.
  • the reaction principle is shown in Figure 2. Cross-linked collagen fibers.
  • first cross-linking agent as diamine and the second cross-linking agent as glutaraldehyde as an example
  • the entire reaction principle of oxidation and two-step cross-linking is shown in Figure 3: in the oxidation step, on the one hand, the oxidant will On the other hand, the oxidized aldehyde group is cross-linked with the amino group of collagen fibers in the biomaterial.
  • a first cross-linking agent such as a diamine links the mucopolysaccharide molecules in the biomaterial through the reaction of amino groups with aldehyde groups.
  • the second cross-linking agent such as glutaraldehyde connects the collagen fibers of the biomaterial through the reaction of aldehyde groups with amino groups or carboxyl groups, and finally forms a cross-linked network.
  • the cross-linked network MPS molecules are connected with collagen fibers, MPS molecules are connected with each other, and collagen fibers are connected with each other.
  • the mucopolysaccharide molecules of the biomaterial itself form part of the cross-linked network, and the polysaccharide group has good hydrophilic properties, which improves the hydrophilicity of the biomaterial and quickly flattens after the biomaterial absorbs water.
  • the residual amino groups from the first cross-linking agent are also cross-linked by the second cross-linking agent during the second cross-linking agent treatment process, so the treatment of the first cross-linking agent can be omitted. Residual amino step.
  • the method further includes an end-capping treatment step of exposing the cross-linked biological material to a solution containing an end-capping substance to block residual active groups; the residual active groups are derived from the second cross-linking agent.
  • the cross-linking agent active group can be understood as the active group derived from the corresponding cross-linking agent. In this manner, the cross-linking active group is the active group of the second cross-linking agent.
  • the end-capping substance is a polyamino substance.
  • the polyamino substance is polyethyleneimine, polylysine, ⁇ -polylysine, lysine or hexamethylenediamine.
  • the solvent for configuring the solution containing the end-capping substance is a conventional solvent for processing biological materials, which will not damage the structure of the biological material, such as water, PBS or physiological saline, etc.; the mass percentage concentration of the end-capping substance is 0.5% ⁇ 10%.
  • the exposure time of the biological material in the solution containing the capping substance is 0.5 h to 12 h.
  • the exposure mode of the biological material in the solution containing the capping substance is static contact or dynamic contact.
  • it also includes a post-crosslinking treatment step of exposing the end-capped biological material to a third cross-linking agent solution to cross-link residual active groups; the residual active groups are derived from the end-capping substance.
  • the end-capping substance is a polyamino substance
  • the polyamino substance includes at least two amino groups, a part of the amino group reacts with the residual aldehyde group, and the remaining part forms a residual amino group, and the content of the amino group in the polyamino substance increases, and after the end-capping treatment
  • the residual amino group content is high, and the cross-linking sites with the third cross-linking agent are many in the post-cross-linking treatment step, and the formed cross-linked network can further enhance the elasticity of the biomaterial.
  • the third crosslinking agent in the third crosslinking agent solution has at least one group reactive with amino group and one hydrophilic group.
  • the group that reacts with the amino group includes an ester bond or an epoxy group; the hydrophilic group includes an alcoholic hydroxyl group or an ether bond.
  • the third crosslinking agent is polyethylene glycol glycidyl ether.
  • the solvent for configuring the third cross-linking agent solution is a conventional solvent for processing biological materials, which will not destroy the structure of the biological material, such as water, PBS or physiological saline, etc.; the mass percentage concentration of the third cross-linking agent is 0.01% ⁇ 2%.
  • the exposure time of the biological material in the third cross-linking agent solution is 2-48 hours.
  • the exposure mode of the biological material in the third crosslinking agent solution is static contact or dynamic contact.
  • Another optional treatment sequence sequential (c) second cross-linking treatment, (a) oxidation treatment and (b) first cross-linking treatment on the biological material: sequential exposure of the biological material to a second cross-linking agent solution , the oxidant-containing solution and the first cross-linking agent solution.
  • Another optional treatment sequence the biological material is sequentially subjected to (a) oxidation treatment, (c) second cross-linking treatment and (b) first cross-linking treatment: the biological material is sequentially exposed to the oxidant-containing solution, the second in the crosslinker solution and the first crosslinker solution.
  • the second cross-linking agent has at least two reactive groups that react with amino groups or carboxyl groups
  • the first cross-linking agent and the second cross-linking agent are different substances
  • the first cross-linking agent can interact with the first cross-linking agent.
  • the reactive groups of the two crosslinkers react.
  • the collagen fibers are cross-linked first and then the mucopolysaccharide is cross-linked.
  • the adjacent hydroxyl groups of the mucopolysaccharides in the biomaterial are oxidized to aldehyde groups, and a part of the aldehyde groups are cross-linked with the amino groups of the collagen fibers in the biomaterials;
  • the second cross-linking agent reacts with the amino groups or carboxyl groups of the collagen fibers to connect the collagen fibers.
  • the second cross-linking agent cross-links the collagen fibers and also introduces some free active groups.
  • the first cross-linking agent mainly reacts with the aldehyde groups of the mucopolysaccharides to cross-link between the mucopolysaccharides.
  • the free reactive groups introduced in the second crosslinker crosslinking step are also crosslinked by the first crosslinker.
  • the step of blocking the residual reactive groups of the first crosslinker can be omitted.
  • the first cross-linking agent is a small molecular substance with at least two amino groups.
  • Small molecular substances with at least two amino groups include amino acids, polyamines, etc.; among them, amino acids can be selected from lysine, polylysine or ⁇ -polylysine, etc.; polyamines can be diamines, and diamines contain Amino compounds with two amino groups, for example, ethylenediamine, propylenediamine, hexamethylenediamine, p-phenylenediamine, etc.; polyamines can also be polyethyleneimine.
  • the first cross-linking agent is lysine, ethylenediamine, hexamethylenediamine, polyethyleneimine, polylysine or ⁇ -polylysine.
  • the mass percentage concentration of the first cross-linking agent in the first cross-linking agent solution is 0.5% to 10%;
  • the solvent of the first cross-linking agent solution is configured as a conventional solvent for processing biological materials, which will not destroy the structure of the biological material. , such as water, PBS or normal saline, etc.
  • the time for exposing the biological material to the first crosslinking agent solution is 0.5-12 hours.
  • the second cross-linking agent is glutaraldehyde; the mass percentage concentration of the second cross-linking agent in the second cross-linking agent solution is 0.05% to 25%;
  • the solvent is a conventional solvent for processing biological materials, which will not destroy the structure of biological materials, such as water, PBS or physiological saline, etc.; the cross-linking time is 6h-3 weeks; the cross-linking temperature is 0-37°C.
  • the exposure modes of the biological material in the first cross-linking agent solution and the second cross-linking agent solution are both static contact or dynamic contact.
  • it also includes a post-crosslinking treatment step of exposing the biological material treated with the first crosslinking agent to the third crosslinking agent solution to crosslink residual active groups; the residual active groups come from the first crosslinking agent. joint agent.
  • the first crosslinking agent is a bisamino small molecule substance
  • the residual active group after the first crosslinking agent is an amino group
  • the post-crosslinking treatment step of polyethylene glycol can be directly performed.
  • the third crosslinking agent in the third crosslinking agent solution has at least one group reactive with amino group and one hydrophilic group.
  • the group that reacts with the amino group includes an ester bond or an epoxy group; the hydrophilic group includes an alcoholic hydroxyl group or an ether bond.
  • the third crosslinking agent is polyethylene glycol glycidyl ether.
  • a catalyst can also be added to accelerate the progress of the reaction, and the catalyst can be selected from methylimidazole, triethylamine and tetramethylethylenediamine.
  • Polyethylene glycol glycidyl ether acts as a third cross-linking agent to react with residual amino groups to form a cross-linked network through polyethylene glycol.
  • the ability of the material to deform elastically; on the other hand, water is a benign solvent for polyethylene glycol, which shows high elasticity after immersion in water, which further improves the anti-crease performance of biomaterials.
  • the solvent for configuring the third cross-linking agent solution is a conventional solvent for processing biological materials, which will not destroy the structure of the biological material, such as water, PBS or physiological saline, etc.; the mass percentage concentration of the third cross-linking agent is 0.01% ⁇ 2%.
  • the exposure time of the biological material in the third cross-linking agent solution is 2-48 hours.
  • the exposure mode of the biological material in the third crosslinking agent solution is static contact or dynamic contact.
  • all of the above solutions with different treatment sequences further include a reduction step of exposing the post-crosslinking treated biological material to a reducing agent solution.
  • the amino group reacts with the aldehyde to form a schiff-base chemical bond, but the schiff-base is reversible, and is converted into a stable carbon-nitrogen single bond through a reduction reaction, avoiding the chemical bond exchange reaction during the pressing process.
  • the reducing agent in the reducing agent solution is sodium cyanoborohydride or sodium borohydride
  • the solvent for configuring the reducing agent solution is a conventional solvent for processing biological materials, which will not damage the structure of biological materials, such as water, PBS Or physiological saline, etc.
  • the mass percentage concentration of the reducing agent is 0.01%-2%
  • the exposure time of the biological material in the reducing agent solution is 1-12 h
  • the exposure mode of the biological material in the reducing agent solution is static contact or dynamic contact.
  • the reduction-treated biological material is subjected to the (d) dehydration or drying treatment.
  • the dehydration adopts an ethanol dehydration method; and the drying adopts freeze drying.
  • a crease-resistant dehydrated cross-linked biological material prepared by the method.
  • the maximum breaking force n of the dehydrated and cross-linked biological material is 25-30N.
  • the maximum breaking force of the dehydrated cross-linked biomaterial is tested after hydration.
  • the method for determining the maximum breaking force is as follows: after the dehydrated cross-linked biological material is hydrated, it is cut into 1cm*5cm film strips, and measured by a universal stretching machine.
  • the hydration of the dehydrated and cross-linked biological material can be understood as the re-absorption of water by the dehydrated and cross-linked biological material, such as being immersed in a physiological saline solution.
  • the dehydrated cross-linked biomaterial is folded and kept for at least 72 hours and then flattened without creases.
  • the dehydrated and cross-linked biological material is folded and compressed into a 1ml syringe cylinder with a 3*3cm membrane sheet, placed at a constant temperature of 40°C for 3 days, and then released into a physiological saline solution, flattened without creases.
  • the biological tissue prepared in the present application can be used for intervening biological valves, such as through minimally invasive intervention, and can also be used for surgical biological valves, such as surgical implantation.
  • a biological valve comprises a stent and a valve mounted on the stent, wherein the valve is the anti-crease dehydration cross-linked biological material described in the application.
  • the biological valve can be an interventional valve through minimally invasive intervention, or an implanted valve through surgical implantation.
  • An interventional system includes a heart valve and a delivery catheter, the heart valve is folded and delivered by the delivery catheter, and the heart valve is the biological valve described in this application.
  • the present application has at least one of the following beneficial effects:
  • the sugar groups in the biomaterial are opened in situ and form part of the cross-linked network.
  • the elasticity and water absorption of the obtained cross-linked biomaterial are significantly improved. crease.
  • the mucopolysaccharide molecule is a part of the biomaterial, the in situ opening and forming part of the cross-linked network not only solves the problem of crease recovery, but also solves the problem of immunogenicity.
  • the residual active groups between the first cross-linking agent and the second cross-linking agent can be mutually consumed, and the step of blocking the residual active groups is omitted.
  • the end-capping substance adopts a polyamino substance, and the polyamino substance comprises at least two amino groups, a part of the amino group reacts with the residual aldehyde group, and the remaining part forms a residual amino group, and the amino content in the polyamino substance increases, and the capping
  • the residual amino group content after the end treatment is high, and the cross-linking sites with the third cross-linking agent are many in the post-cross-linking treatment step, and the formed cross-linked network can further enhance the elasticity of the biological material.
  • the oxidant treatment step oxidizes the adjacent hydroxyl groups of the mucopolysaccharides in the pericardium to aldehyde groups, and the polysaccharide compounds are connected to the collagen fibers; It forms an integrated cross-linked structure with protein fibers.
  • Polyethylene glycol glycidyl ether acts as a third cross-linking agent to react with residual amino groups, and polyethylene glycol forms a cross-linked network.
  • the high symmetry of the polyethylene glycol chain enhances the high elasticity of biological tissues, and water is polyethylene glycol.
  • a benign solvent for glycol it exhibits high elasticity after immersion in water, further improving the crease resistance of biomaterials.
  • Fig. 1 is the schematic diagram of oxidation reaction step
  • FIG. 2 is a schematic diagram of the cross-linking treatment steps of the first cross-linking agent and the second cross-linking agent
  • FIG. 3 is a schematic diagram of the whole process of oxidation, cross-linking by the first cross-linking agent and cross-linking by the second cross-linking agent in sequence.
  • Fig. 4 is a flow chart showing that (a) oxidation treatment, (b) first crosslinking treatment, (c) second crosslinking treatment and (d) dehydration treatment are sequentially performed.
  • Fig. 5 is a flow chart showing that (c) the second crosslinking treatment, (a) the oxidation treatment, (b) the first crosslinking treatment and (d) the dehydration treatment are sequentially performed.
  • Fig. 6 is a flow chart showing that (a) oxidation treatment, b) first crosslinking treatment, (c) second crosslinking treatment and (d) dehydration treatment are sequentially performed.
  • FIG. 7 is a flowchart of Embodiment 1.
  • FIG. 8 is a comparison diagram of the maximum breaking force of the cross-linked film prepared in Example 1 and the conventional glutaraldehyde cross-linked film.
  • FIG. 9 is a graph showing the result of the anti-crease test of the cross-linked film prepared in Example 1.
  • FIG. 10 is a comparison diagram of the maximum breaking force of the cross-linked film prepared in Example 2 and the conventional glutaraldehyde cross-linked film.
  • FIG. 11 is a graph showing the results of the anti-crease test of the cross-linked film prepared in Example 2.
  • FIG. 12 is a comparison diagram of the maximum breaking force of the cross-linked film prepared in Example 4 and the conventional glutaraldehyde cross-linked film.
  • FIG. 13 is a graph showing the results of the anti-crease test of the cross-linked film prepared in Example 4.
  • the biological material is subjected to oxidation treatment, cross-linking treatment and dehydration treatment in sequence, and the cross-linking treatment includes the first cross-linking treatment and the second cross-linking treatment carried out in sequence.
  • the adjacent hydroxyl groups of mucopolysaccharides are oxidized to form aldehyde groups, and the polysaccharide compounds are connected to the collagen fibers; then through the cross-linking treatment, a cross-linked network structure is formed between the polysaccharides and the polysaccharides and between the protein fibers, and the final network structure is formed.
  • Cross-links are formed between polysaccharides and polysaccharides, between polysaccharides and collagen fibers, and between collagen fibers.
  • end-capping treatment blocks the residual active groups of the second cross-linking agent
  • post-cross-linking treatment cross-links the residual active groups of the end-capping substance
  • reduction treatment replaces the schiff-base chemical bond with a stable carbon-nitrogen single bond.
  • the biological material is subjected to oxidation treatment, first crosslinking treatment, second crosslinking treatment, end-capping treatment, post-crosslinking treatment, reduction treatment and dehydration treatment in sequence.
  • the pericardium is porcine pericardium or bovine pericardium. After cutting the hanging board, it is exposed to the oxidant solution, and the exposure method can be immersed or sprayed.
  • the oxidant can be selected from sodium periodate, the mass percentage concentration of the oxidant solution is 0.1%-1%, and the oxidation treatment time is 1-12 hours.
  • the adjacent hydroxyl groups of the mucopolysaccharides in the pericardium are oxidized into aldehyde groups, and the aldehyde groups react with the amino groups of collagen fibers to connect the mucopolysaccharides to the collagen fibers.
  • the first cross-linking treatment is the first cross-linking treatment:
  • the mucopolysaccharides are connected to the collagen fibers, the mucopolysaccharides are cross-linked with each other through aldehyde groups.
  • the first cross-linking agent needs to have a group that reacts with the aldehyde group, preferably a bisamino compound. linked polysaccharides.
  • the bisamino compound can be selected from lysine, ethylenediamine or hexamethylenediamine; the mass percentage concentration of the bisamino compound in the bisamino compound solution is 0.05% to 5%; the exposure time of the biological material in the bisamino compound solution 0.5 ⁇ 12h; the exposure method can be immersed or sprayed.
  • the collagen fibers of the biomaterial are cross-linked by a second cross-linking agent, and the second cross-linking agent can be selected from glutaraldehyde.
  • the biomaterial is exposed to the glutaraldehyde solution, and the exposure method can be soaked or sprayed.
  • the mass percentage concentration of glutaraldehyde in the glutaraldehyde solution is 0.05%-25%
  • the cross-linking time is 6h-3 weeks
  • the cross-linking temperature is 0-37°C.
  • the blocking treatment is performed by an end-capping substance.
  • the end-capping substance is preferably a polyamino substance, and the polyamino substance can be from polyethyleneimine, polylysine acid, ⁇ -polylysine, lysine, hexamethylenediamine, etc.
  • the biological material is exposed to the polyamino substance solution, and the mass percentage concentration of the polyamino substance is 0.5%-10%; the exposure time is 0.5h-12h; the exposure method can be immersion or spraying.
  • polyamino substances are used as end-capping substances, and the residual amino groups are cross-linked by the third cross-linking agent in this step.
  • the third cross-linking agent is selected from polyethylene glycol diglycidyl ether, polyethylene glycol
  • the cross-linked network formed by glycol enhances the high elasticity of the pericardium.
  • the high symmetry of the polyethylene glycol chain enhances the high elasticity of the biological tissue, which can completely restore the elastic deformation ability of the biological material;
  • water is a A benign solvent for polyethylene glycol, it exhibits high elasticity after immersion in water, further improving the crease resistance of biomaterials.
  • the mass percentage concentration of the third cross-linking agent in the third cross-linking agent solution is 0.01%-2%; the cross-linking time is 10-48h.
  • the biological material is exposed to a reducing agent solution
  • the reducing agent can be selected from sodium cyanoborohydride or sodium borohydride; the mass percentage concentration of the reducing agent is 0.01% to 2%; the exposure time is 1 to 12 hours;
  • the method can be immersed or sprayed.
  • the biological material is subjected to the second cross-linking treatment, oxidation treatment, first cross-linking treatment and dehydration treatment in sequence.
  • the adjacent hydroxyl groups of the mucopolysaccharides in the biomaterial are oxidized to form aldehyde groups, and the polysaccharide compounds are connected to the collagen fibers, and then the first cross-linking treatment is performed to form a cross-link between the polysaccharides and the polysaccharides.
  • a cross-linked network structure is formed between collagen fibers and collagen fibers, between polysaccharides and collagen fibers, and between polysaccharides and polysaccharides.
  • post-cross-linking treatment and reduction treatment may also be included, and then dehydration treatment is performed, and no end-capping treatment is required.
  • the post-crosslinking treatment crosslinks the residual active groups of the first crosslinking agent, and the reduction treatment replaces the schiff-base chemical bond with a stable carbon-nitrogen single bond.
  • the biological material is sequentially subjected to the second oxidation treatment, oxidation treatment, first cross-linking treatment, post-cross-linking treatment, reduction treatment and dehydration treatment.
  • the collagen fibers of the biomaterial are cross-linked by a second cross-linking agent, and the second cross-linking agent can be selected from glutaraldehyde.
  • the biomaterial is exposed to the glutaraldehyde solution, and the exposure method can be soaked or sprayed.
  • the mass percentage concentration of glutaraldehyde in the glutaraldehyde solution is 0.05%-25%
  • the cross-linking time is 6h-3 weeks
  • the cross-linking temperature is 0-37°C.
  • the pericardium is porcine pericardium or bovine pericardium. After cutting the hanging board, it is exposed to the oxidant solution, and the exposure method can be immersed or sprayed.
  • the oxidant can be selected from sodium periodate, the mass percentage concentration of the oxidant solution is 0.1%-1%, and the oxidation treatment time is 1-12 hours.
  • the adjacent hydroxyl groups of the mucopolysaccharides in the pericardium are oxidized to aldehyde groups, and the aldehyde groups react with the amino groups of the collagen fibers to connect the mucopolysaccharides to the collagen fibers.
  • the first cross-linking treatment is the first cross-linking treatment:
  • the mucopolysaccharides are cross-linked with each other through the aldehyde groups.
  • the first cross-linking agent needs to have a group that reacts with the aldehyde groups, preferably polyamino substances, such as lysine, ethylene glycol, etc. It is selected from amine, hexamethylenediamine, polyethyleneimine, polylysine or ⁇ -polylysine, and the mucopolysaccharide is linked together by the reaction of amino group and aldehyde group.
  • the mass percentage concentration of the polyamino substance in the polyamino substance solution is 0.5% to 10%; the exposure time of the biological material in the polyamino substance solution is 0.5 to 12h; the exposure method can be immersion or spray.
  • a polyamino substance is used as the first cross-linking agent, and the residual amino group is cross-linked by the third cross-linking agent in this step.
  • the third cross-linking agent is polyethylene glycol diglycidyl.
  • the cross-linked network formed by ether and polyethylene glycol enhances the high elasticity of the pericardium.
  • the high symmetry of the polyethylene glycol chain enhances the high elasticity of biological tissues, which can completely restore the elastic deformation ability of biological materials;
  • water is a benign solvent for polyethylene glycol, and it exhibits high elasticity after immersion in water, which further improves the crease resistance of biomaterials.
  • the mass percentage concentration of the third cross-linking agent in the third cross-linking agent solution is 0.01%-2%; the cross-linking time is 10-48h.
  • the biological material is exposed to a reducing agent solution
  • the reducing agent can be selected from sodium cyanoborohydride or sodium borohydride; the mass percentage concentration of the reducing agent is 0.01% to 2%; the exposure time is 1 to 12 hours;
  • the method can be immersed or sprayed.
  • the biological material is subjected to oxidation treatment, second cross-linking treatment, first cross-linking treatment and dehydration treatment in sequence.
  • the carbohydrate compound is connected to the collagen fibers
  • the second cross-linking treatment cross-links the collagen fibers of the biomaterial to each other
  • the first cross-linking treatment makes the polysaccharides form cross-links between the polysaccharides.
  • a cross-linked network structure is formed between collagen fibers and collagen fibers, between polysaccharides and collagen fibers, and between polysaccharides and polysaccharides.
  • post-cross-linking treatment and reduction treatment may also be included, and then dehydration treatment is performed, and no end-capping treatment is required.
  • the post-crosslinking treatment crosslinks the residual active groups of the first crosslinking agent, and the reduction treatment replaces the schiff-base chemical bond with a stable carbon-nitrogen single bond.
  • the biological material is subjected to oxidation treatment, second oxidation treatment, first cross-linking treatment, post-cross-linking treatment, reduction treatment and dehydration treatment in sequence.
  • the pericardium is porcine pericardium or bovine pericardium. After cutting the hanging board, it is exposed to the oxidant solution, and the exposure method can be immersed or sprayed.
  • the oxidant can be selected from sodium periodate, the mass percentage concentration of the oxidant solution is 0.1%-1%, and the oxidation treatment time is 1-12 hours.
  • the adjacent hydroxyl groups of the mucopolysaccharides in the pericardium are oxidized to aldehyde groups, and the aldehyde groups react with the amino groups of the collagen fibers to connect the mucopolysaccharides to the collagen fibers.
  • the collagen fibers of the biomaterial are cross-linked by a second cross-linking agent, and the second cross-linking agent can be selected from glutaraldehyde.
  • the biomaterial is exposed to the glutaraldehyde solution, and the exposure method can be soaked or sprayed.
  • the mass percentage concentration of glutaraldehyde in the glutaraldehyde solution is 0.05%-25%
  • the cross-linking time is 6h-3 weeks
  • the cross-linking temperature is 0-37°C.
  • the first cross-linking treatment is the first cross-linking treatment:
  • the mucopolysaccharides are cross-linked with each other through the aldehyde groups.
  • the first cross-linking agent needs to have a group that reacts with the aldehyde groups, preferably polyamino substances, such as lysine, ethylene glycol, etc. It is selected from amine, hexamethylenediamine, polyethyleneimine, polylysine or ⁇ -polylysine, and the mucopolysaccharide is linked together by the reaction of amino group and aldehyde group.
  • the mass percentage concentration of the polyamino substance in the polyamino substance solution is 0.5% to 10%; the exposure time of the biological material in the polyamino substance solution is 0.5 to 12h; the exposure method can be immersion or spray.
  • a polyamino substance is used as the first cross-linking agent, and the residual amino group is cross-linked by the third cross-linking agent in this step.
  • the third cross-linking agent is polyethylene glycol diglycidyl.
  • the cross-linked network formed by ether and polyethylene glycol enhances the high elasticity of the pericardium.
  • the high symmetry of the polyethylene glycol chain enhances the high elasticity of biological tissues, which can completely restore the elastic deformation ability of biological materials;
  • water is a benign solvent for polyethylene glycol, and it exhibits high elasticity after immersion in water, which further improves the crease resistance of biomaterials.
  • the mass percentage concentration of the third cross-linking agent in the third cross-linking agent solution is 0.01%-2%; the cross-linking time is 10-48h.
  • the biological material is exposed to a reducing agent solution
  • the reducing agent can be selected from sodium cyanoborohydride or sodium borohydride; the mass percentage concentration of the reducing agent is 0.01% to 2%; the exposure time is 1 to 12 hours;
  • the method can be immersed or sprayed.
  • the solvent of the solution used is a conventional solvent for processing biological materials, which will not negatively affect the results of biological materials, for example, water, PBS or physiological saline can be selected.
  • concentrations expressed in percentages represent the mass percentage concentrations unless otherwise specified; the solutions mentioned in the following examples are all configured in PBS unless otherwise specified.
  • 1% sodium periodate solution was soaked in porcine pericardium at room temperature for 2h; 2% lysine aqueous solution was soaked for 2h; then, cross-linked in 0.05% glutaraldehyde solution for 5 hours day, washed with 0.9% normal saline; soaked in 2% ⁇ -polylysine aqueous solution for 2 hours; 2% polyethylene glycol diglycidyl ether aqueous solution was added with 0.1% catalyst tetramethylethylenediamine to react for 24 hours. Washed 3 times with 0.9% physiological saline, soaked in 1% sodium cyanoborohydride aqueous solution for 2h. After washing three times, it was soaked in a 40% glycerin aqueous solution, and then freeze-dried.
  • Breaking force test After the dry film is hydrated, it is cut into 1*5cm film strips, and the maximum breaking force is measured by a universal stretching machine; the conventional glutaraldehyde cross-linked film is used as a control. The results are shown in FIG. 8 , the breaking force of the membrane prepared in Example 1 is 25-30 N, which is obviously better than that of the conventional glutaraldehyde cross-linked membrane.
  • the porcine pericardium was cross-linked in 0.05% glutaraldehyde solution for 5 days, and washed with 0.9% normal saline; the porcine pericardium was soaked in 1% sodium periodate solution at room temperature for 2h ; Then, soak in 2% ⁇ -polylysine solution for 2h. 2% polyethylene glycol diglycidyl ether was added with 0.1% catalyst to react for 24h. Wash with 0.9% normal saline for 3 times and soak in 1% sodium cyanoborohydride solution for 2h. After washing 3 times, soak in 40% isopropanol solution, then blow dry or freeze dry.
  • the breaking force experiment is the same as that of Example 1. The results are shown in Figure 10. The breaking force of the membrane prepared in Example 2 is obviously better than that of the conventional glutaraldehyde cross-linked membrane.
  • Porcine pericardium was soaked in 1% sodium periodate solution at room temperature for 2 h. Soak in 2% lysine solution for 2 hours; then, cross-link in 0.05% glutaraldehyde solution for 5 days, and wash with 0.9% normal saline. Soak in 2% bis-aminated polyethylene glycol solution to react for 24h. Wash with 09% normal saline for 3 times and soak in 1% sodium cyanoborohydride solution for 2h. After washing three times, it was soaked in a 40% glycerin aqueous solution, and then freeze-dried.
  • Porcine pericardium was soaked in 1% sodium periodate solution at room temperature for 2 h. Cross-linked in 0.05% glutaraldehyde solution for 5 days and rinsed with 0.9% saline. Soak in 2% polyethyleneimine solution for 2 hours; then, soak in 2% bis-amino polyethylene glycol aqueous solution for 24 hours. Wash with 0.9% normal saline for 3 times and soak in 1% sodium cyanoborohydride solution for 2h. After washing 3 times, it was soaked in a 60% glycerin aqueous solution, and then air-dried.
  • the breaking force experiment is the same as that of Example 1. The results are shown in Figure 12. The breaking force of the membrane prepared in Example 4 is obviously better than that of the conventional glutaraldehyde cross-linked membrane.

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Abstract

本申请公开一种抗折痕的脱水交联生物材料及其制备方法，制备方法包括对生物材料进行(a)氧化处理、(b)第一交联处理、(c)第二交联处理和(d)脱水处理制得；其中，(b)在(a)之后进行且(a)(b)(c)均在(d)之前进行；其中氧化处理时采用能够致使羟基转化为醛基的氧化剂，所述羟基来自于所述生物材料中的粘多糖，第一交联处理时采用能够致使粘多糖的醛基间相互交联的第一交联剂，第二交联处理时采用能够致使所述生物材料中胶原纤维间相互交联的第二交联剂。本申请还提供一种基于所述脱水交联生物材料的心脏瓣膜和介入***。本申请通过原位打开生物材料的粘多糖并将其形成交联网络的一部分，解决干膜瓣膜在输送器中长时间压握后重新复水折痕不能回复的问题。

Description

抗折痕的脱水交联生物材料及其制备方法和应用 技术领域
本申请涉及假体生物材料技术领域，具体涉及一种抗折痕的脱水交联生物材料及其制备方法和应用。
背景技术
近年来，介入生物材料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。现阶段介入瓣膜保存于戊二醛溶液中，在手术现场使用时，需要经过反复清洗和装载，增加了手术的时间和附加风险；其次，生物瓣膜残留的戊二醛易增加生物瓣膜的钙化和毒性。生物瓣膜通过脱水和预装载的方式能够完美地解决上述问题。然而，脱水和预装载的方式由于在上时间压握后再水合，要求生物瓣膜具有更好的弹性和韧性，解决长时间压握之后的折痕问题。现阶段，生物瓣仍然不能满足长时间压握之后的折痕问题，折痕的存在不仅影响瓣膜的流体力学性能而且在折痕处更易发生钙化和疲劳撕裂。
发明内容
本申请提供一种抗折痕的脱水交联生物材料及其制备方法和应用，解决干膜瓣膜在输送器中长时间压握后重新复水折痕不能回复的问题。
一种复合交联制备抗折痕的脱水生物材料的方法，其特征在于，对生物材料进行(a)氧化处理、(b)第一交联处理、(c)第二交联处理和(d)脱水处理制得；其中，(b)在(a)之后进行且(a)(b)(c)均在(d)之前进行；
其中氧化处理时采用能够致使羟基转化为醛基的氧化剂，所述羟基来自于所述生物材料中的粘多糖，第一交联处理时采用能够致使粘多糖的醛基间相互交联的第一交联剂，第二交联处理时采用能够致使所述生物材料中胶原纤维间相互交联的第二交联剂。
可选的，所述生物材料的胶原含量为60％～90％。
可选的，所述生物材料的生物来源为猪、牛、马或羊；所述生物材料的来源部位为心包膜、心脏瓣膜、血管、韧带、肌肉、肠或皮肤。
进一步可选的，所述生物材料包括猪心包膜或牛心包膜。
可选的，所述氧化处理包括将生物材料暴露于含氧化剂的溶液中。
可选的，所述羟基为粘多糖的邻羟基。
可选的，所述氧化剂为高碘酸钠。
可选的，所述含氧化剂的溶液中氧化剂的质量百分比浓度为0.1％～1％；配置氧化剂溶液的溶剂为处理生物材料的常规溶剂，不会破坏生物材料的结构，例如水、PBS或生理盐水等。
可选的，生物材料在含氧化剂的溶液中的暴露时间为1～12h。
可选的，生物材料在含氧化剂溶液中的暴露方式为静态接触或动态接触。
静态接触可以理解为氧化处理过程中，生物材料与含氧化剂的溶液之间保持相对静止， 例如浸泡。动态接触可以理解为氧化处理过程中，生物材料与含氧化剂溶液之间相对运动，例如向所述生物材料循环喷淋含氧化剂溶液，或在生物材料浸泡于含氧化剂溶液时搅拌或摇动反应体系。以下提到的静态接触或动态接触中，如无特殊说明，也可同此理解。
生物材料暴露于含氧化剂的溶液中，氧化剂用于将生物材料中粘多糖的邻羟基氧化成醛基，粘多糖通过其氧化后的醛基与所述生物材料中的胶原纤维连接；氧化处理后的生物材料暴露于第一交联剂中进行交联处理，生物材料中粘多糖之间通过第一交联剂交联连接；生物材料暴露于第二交联剂中进行交联处理，生物材料中胶原纤维之间通过第二交联剂交联连接；交联处理后的生物材料经脱水或干燥处理。
生物材料暴露于含氧化剂的溶液中，在该处理步骤中，一方面氧化剂将生物材料中的粘多糖分子的邻羟基氧化成醛基，另一方面，氧化后的醛基与生物材料中胶原纤维的氨基发生交联。在进一步的交联处理中，粘多糖分子与粘多糖分子之间、胶原纤维与胶原纤维之间由对应的交联剂交联形成网状结构。生物材料中本身的糖基原位打开，并形成交联网络的一部分，得到的交联生物材料其弹性和吸水性明显提高，在输送器中长期压握后，重新复水基本无折痕。同时由于粘多糖分子为生物材料的一部分，原位打开并形成交联网络的一部分在解决折痕回复问题的同时还解决了免疫原性的问题。
氧化步骤中发生的主要反应如图1所示。在按(a)(b)(c)次序、(a)(c)(b)次序或(c)(a)(b)次序进行的制备方式中，氧化处理步骤的操作均相同。
一种可选的处理次序：对生物材料依次进行(a)氧化处理、(b)第一交联处理和(c)第二交联处理：将氧化处理后的生物材料依次暴露于第一交联剂溶液和第二交联剂溶液中。
可选的，所述第一交联剂具有至少两个与醛基反应的活性基团，所述第二交联剂与第一交联剂为不同物质且第二交联剂能与第一交联剂的活性基团反应。
该处理方式中先进行粘多糖的交联再进行胶原纤维的交联。氧化处理中，生物材料中粘多糖的邻羟基被氧化为醛基，一部分醛基与生物材料中胶原纤维的氨基反应，致使粘多糖与胶原纤维之间形成交联；第一交联剂的活性基团与粘多糖的剩余部分醛基反应，致使粘多糖之间交联连接；第一交联剂交联粘多糖醛基的同时也会引入部分游离活性基团，第二交联剂主要与胶原纤维的氨基或羧基之间反应，致使胶原纤维之间相互连接，同时，前一步交联引入的游离活性基团也被第二交联剂交联。可省去第一交联剂残余活性基团封闭的步骤。
可选的，所述第一交联剂为至少具有两个氨基的小分子物质。
至少具有两个氨基的小分子物质包括氨基酸、二元胺等；其中氨基酸可选择赖氨酸；二元胺为含有二个氨基的氨基化合物，例如，乙二胺、丙二胺、己二胺、对苯二胺等。
可选的，所述第一交联剂为赖氨酸、乙二胺或己二胺。
可选的，所述第一交联剂溶液中第一交联剂的质量百分比浓度为0.05％～5％；配置第一交联剂溶液的溶剂为处理生物材料的常规溶剂，不会破坏生物材料的结构，例如水、PBS或生理盐水等。
可选的，生物材料暴露于第一交联剂溶液中的时间为0.5～12h。
可选的，所述第二交联剂为戊二醛；所述第二交联剂溶液中第二交联剂的质量百分比浓度为0.05％～25％，配置第二交联剂溶液的溶剂为处理生物材料的常规溶剂，不会破坏生物材 料的结构，例如水、PBS或生理盐水等；交联时间为6h～3周；交联温度为0～37℃。
可选的，生物材料在第一交联剂溶液和第二交联剂溶液中的暴露方式均为静态接触或动态接触。
该交联处理方式中，以第一交联剂为二元胺、第二交联剂为戊二醛为例，反应原理如图2所示，二元胺交联粘多糖分子，戊二醛交联胶原纤维。
以第一交联剂为二元胺、第二交联剂为戊二醛为例，氧化及两步交联的整个反应原理如图3所示：在氧化步骤中，一方面氧化剂将生物材料中的粘多糖分子的邻羟基氧化成醛基，另一方面，氧化后的醛基与生物材料中胶原纤维的氨基发生交联。在第一交联剂交联步骤中，第一交联剂如二元胺通过氨基与醛基的反应连接生物材料中的粘多糖分子。在第二交联剂交联处理步骤中，第二交联剂如戊二醛通过醛基与氨基或羧基的反应连接生物材料的胶原纤维，最终形成交联网络。交联网络中，粘多糖分子与胶原纤维连接、粘多糖分子之间相互连接、胶原纤维之间相互连接。生物材料本身的粘多糖分子形成交联网络的一部分，多糖基具有很好的亲水性能，改善生物材料的亲水性，在生物材料吸水后快速展平。
第一交联剂二元胺交联处理后，来自第一交联剂的残余氨基在第二交联剂处理过程中也被第二交联剂交联，可省去处理第一交联剂残余氨基的步骤。
可选的，还包括将交联处理后的生物材料暴露于含封端物质溶液中封闭残余活性基团的封端处理步骤；该残余活性基团来自于第二交联剂。
所述交联剂活性基团可以理解为来自于对应交联剂的活性基团，该方式中，交联活性基团为第二交联剂的活性基团。
可选的，所述封端物质为多氨基物质。
可选的，所述多氨基物质为聚乙烯亚胺、聚赖氨酸、ε-聚赖氨酸、赖氨酸或己二胺。
可选的，配置所述含封端物质溶液的溶剂为处理生物材料的常规溶剂，不会破坏生物材料的结构，例如水、PBS或生理盐水等；封端物质的质量百分比浓度为0.5％～10％。
可选的，生物材料在含封端物质溶液中的暴露时间为0.5h～12h。
可选的，生物材料在含封端物质溶液中的暴露方式为静态接触或动态接触。
可选的，还包括将封端处理后生物材料暴露于第三交联剂溶液中交联残余活性基团的后交联处理步骤；该残余活性基团来自于所述封端物质。
在封端处理步骤中，封端物质采用多氨基物质，多氨基物质包括至少两个氨基，一部分氨基与残余醛基反应、剩余部分形成残余氨基，多氨基物质中氨基含量增多，封端处理后残余氨基含量多，在该后交联处理步骤中与第三交联剂的交联位点多，形成的交联网络可进一步增强生物材料的弹性。
可选的，所述第三交联剂溶液中的第三交联剂至少具有一个与氨基反应的基团和一个亲水基团。
可选的，所述与氨基反应的基团包括酯键或环氧基团；所述亲水基团包括醇羟基或醚键。
可选的，所述第三交联剂为聚乙二醇缩水甘油醚。
可选的，配置所述第三交联剂溶液的溶剂为处理生物材料的常规溶剂，不会破坏生物材料的结构，例如水、PBS或生理盐水等；第三交联剂的质量百分比浓度为0.01％～2％。
可选的，生物材料在第三交联剂溶液中的暴露时间为2～48h。
可选的，生物材料在第三交联剂溶液中的暴露方式为静态接触或动态接触。
另一种可选的处理次序：对生物材料依次进行(c)第二交联处理、(a)氧化处理和(b)第一交联处理：将生物材料依次暴露于第二交联剂溶液、含氧化剂溶液和第一交联剂溶液中。
另一种可选的处理次序：对生物材料依次进行(a)氧化处理、(c)第二交联处理和(b)第一交联处理：将生物材料依次暴露于含氧化剂溶液、第二交联剂溶液和第一交联剂溶液中。
可选的，所述第二交联剂具有至少两个与氨基或羧基反应的活性基团，所述第一交联剂与第二交联剂为不同物质且第一交联剂能与第二交联剂的活性基团反应。
该处理方式中先进行胶原纤维交联再进行粘多糖的交联。氧化处理中，生物材料中粘多糖的邻羟基被氧化为醛基，一部分醛基与生物材料中胶原纤维的氨基交联；第二交联剂与胶原纤维的氨基或羧基反应，将胶原纤维之间相互交联；第二交联剂交联胶原纤维的同时也会引入部分游离活性基团，第一交联剂主要与粘多糖的醛基反应，将粘多糖之间相互交联，同时，第二交联剂交联步骤中引入的游离活性基团也被第一交联剂交联。可省去第一交联剂残余活性基团封闭的步骤。
可选的，所述第一交联剂为至少具有两个氨基的小分子物质。
至少具有两个氨基的小分子物质包括氨基酸、多元胺等；其中氨基酸可选择赖氨酸、聚赖氨酸或ε-聚赖氨酸等；多元胺可采用二元胺，二元胺为含有二个氨基的氨基化合物，例如，乙二胺、丙二胺、己二胺、对苯二胺等；多元胺还可采用聚乙烯亚胺。
可选的，所述第一交联剂为赖氨酸、乙二胺、己二胺、聚乙烯亚胺、聚赖氨酸或ε-聚赖氨酸。
可选的，第一交联剂溶液中第一交联剂的质量百分比浓度为0.5％～10％；配置第一交联剂溶液溶剂为处理生物材料的常规溶剂，不会破坏生物材料的结构，例如水、PBS或生理盐水等。
可选的，生物材料暴露于第一交联剂溶液中的时间为0.5～12h。
可选的，所述第二交联剂为戊二醛；所述第二交联剂溶液中第二交联剂的质量百分比浓度为0.05％～25％；配置的第二交联剂溶液的溶剂为处理生物材料的常规溶剂，不会破坏生物材料的结构，例如水、PBS或生理盐水等；交联时间为6h～3周；交联温度为0～37℃。
可选的，生物材料在第一交联剂溶液和第二交联剂溶液中的暴露方式均为静态接触或动态接触。
可选的，还包括将第一交联剂处理后的生物材料暴露于第三交联剂溶液中交联残余活性基团的后交联处理步骤；所述残余活性基团来自于第一交联剂。
该处理方式中，当第一交联剂采用双氨基小分子物质时，第一交联剂处理后残余活性基团为氨基，可直接进行聚乙二醇的后交联处理步骤。
可选的，所述第三交联剂溶液中的第三交联剂至少具有一个与氨基反应的基团和一个亲水基团。
可选的，所述与氨基反应的基团包括酯键或环氧基；所述亲水基团包括醇羟基或醚键。
可选的，所述第三交联剂为聚乙二醇缩水甘油醚。
后交联处理过程中也可加入催化剂以加速反应的进程，催化剂可选择甲基咪唑、三乙胺、 四甲基乙二胺。
聚乙二醇缩水甘油醚作为第三交联剂与残余氨基反应，通过聚乙二醇形成交联网络，一方面，聚乙二醇链高度对称性增强生物组织的高弹性，可完全恢复生物材料弹性变形的能力；另一方面，水是聚乙二醇的良性溶剂，浸水之后表现出高弹性，进一步改善生物材料的抗折痕性能。
可选的，配置所述第三交联剂溶液的溶剂为处理生物材料的常规溶剂，不会破坏生物材料的结构，例如水、PBS或生理盐水等；第三交联剂的质量百分比浓度为0.01％～2％。
可选的，生物材料在第三交联剂溶液中的暴露时间为2～48h。
可选的，生物材料在第三交联剂溶液中的暴露方式为静态接触或动态接触。
可选的，上上述的所有不同处理次序的方案中，均还包括将后交联处理后的生物材料暴露于还原剂溶液中的还原步骤。氨基和醛反应形成schiff-base化学键，但是schiff-base具有可逆的特点，通过还原反应转换成稳定的炭氮单键，避免压握过程中的化学键交换反应。
可选的，所述还原剂溶液中的还原剂为氰基硼氢化钠或者硼氢化钠；配置还原剂溶液的溶剂为处理生物材料的常规溶剂，不会破坏生物材料的结构，例如水、PBS或生理盐水等；还原剂的质量百分比浓度为0.01％～2％；生物材料在还原剂溶液中的暴露时间为1～12h；生物材料在还原剂溶液中的暴露方式为静态接触或动态接触。
可选的，还原处理后的生物材料进行所述的(d)脱水或干燥处理。
可选的，所述脱水采用乙醇脱水方法；所述干燥采用冷冻干燥。
一种如所述方法制备得到的抗折痕的脱水交联生物材料。
可选的，所述脱水交联生物材料的最大断裂力n为25～30N。
可选的，所述脱水交联生物材料的最大断裂力是在水合后测试。
可选的，所述最大断裂力的测定方法为：所述脱水交联生物材料水合后，切割成1cm*5cm膜条，通过万能拉伸机测得。
所述的脱水交联生物材料水合可以理解为脱水交联生物材料重新吸水例如浸泡于生理盐水水溶液中。
可选的，所述脱水交联生物材料折叠并至少保持72小时后展平无折痕。
可选的，所述脱水交联生物材料以3*3cm膜片折叠压缩于1ml注射器圆筒中，40℃恒温放置3天后释放到生理盐水溶液中，展平无折痕。
本申请制备的生物组织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。
一种生物瓣膜，包括支架和安装在支架上的瓣膜，所述瓣膜为本申请所述的抗折痕的脱水交联生物材料。
该生物瓣膜可以是通过微创介入的介入瓣膜，也可以是通过外科植入的植入瓣膜。
一种介入***，包括心脏瓣膜和输送导管，所述心脏瓣膜折叠后由输送导管输送，所述心脏瓣膜为本申请所述的生物瓣膜。
与现有技术相比，本申请至少具有如下有益效果之一：
(1)生物材料中本身的糖基原位打开，并形成交联网络的一部分，得到的交联生物材料 其弹性和吸水性明显提高，在输送器中长期压握后，重新复水基本无折痕。同时由于粘多糖分子为生物材料的一部分，原位打开并形成交联网络的一部分在解决折痕回复问题的同时还解决了免疫原性的问题。
(2)第一交联剂和第二交联剂之间的残余活性基团可以互相消耗，省略封闭残余活性基团的步骤。
(3)在封端处理步骤中，封端物质采用多氨基物质，多氨基物质包括至少两个氨基，一部分氨基与残余醛基反应、剩余部分形成残余氨基，多氨基物质中氨基含量增多，封端处理后残余氨基含量多，在该后交联处理步骤中与第三交联剂的交联位点多，形成的交联网络可进一步增强生物材料的弹性。
(4)氧化剂处理步骤把心包膜中的粘多糖的邻羟基氧化成醛基，多糖类化合物连接到胶原蛋白纤维上；再通过多胺类化合物交联，使得多糖与多糖之间以及多糖与蛋白纤维之间形成一体的交联网状结构。
(5)聚乙二醇缩水甘油醚作为第三交联剂与残余氨基反应，聚乙二醇形成交联网络，聚乙二醇链高度对称性增强生物组织的高弹性，且水是聚乙二醇的良性溶剂，浸水之后表现出高弹性，进一步改善生物材料的抗折痕性能。
附图说明
图1为氧化反应步骤的原理图；
图2为第一交联剂及第二交联剂交联处理步骤的原理图；
图3为氧化、第一交联剂交联及第二交联剂交联依次处理整过程的原理图。
图4为(a)氧化处理、(b)第一交联处理、(c)第二交联处理和(d)脱水处理依次进行的流程图。
图5为(c)第二交联处理、(a)氧化处理、(b)第一交联处理和(d)脱水处理依次进行的流程图。
图6为(a)氧化处理、b)第一交联处理、(c)第二交联处理和(d)脱水处理依次进行的流程图。
图7为实施例1的流程图。
图8为实施例1制备得到的交联膜与常规戊二醛交联膜的最大断裂力对比图。
图9为实施例1制备得到的交联膜抗折痕实验结果图。
图10为实施例2制备得到的交联膜与常规戊二醛交联膜的最大断裂力对比图。
图11为实施例2制备得到的交联膜抗折痕实验结果图。
图12为实施例4制备得到的交联膜与常规戊二醛交联膜的最大断裂力对比图。
图13为实施例4制备得到的交联膜抗折痕实验结果图。
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。
第一种实施方式中，对生物材料依次进行氧化处理、交联处理和脱水处理，交联处理包括依次进行的第一交联处理和第二交联处理，氧化剂氧化过程中将生物材料中的粘多糖的邻羟基氧化成醛基，多糖类化合物连接到胶原蛋白纤维上；再通过交联处理使得多糖与多糖之间、蛋白纤维之间形成一体的交联网状结构，最终形成的网络结构中多糖与多糖之间、多糖与胶原纤维之间、胶原纤维之间均形成交联。
第二交联处理后还可包括封端处理、后交联处理和还原处理，再进行脱水处理。封端处理封闭第二交联剂的残余活性基团，后交联处理交联封端物质的残余活性基团，还原处理将schiff-base化学键换成稳定的炭氮单键。
该实施方式的流程图可参照图4，依次对生物材料进行氧化处理、第一交联处理、第二交联处理、封端处理、后交联处理、还原处理和脱水处理。
氧化处理：
取新鲜的心包膜，裁剪挂板；心包膜为猪心包膜或牛心包膜。裁剪挂板后暴露于氧化剂溶液中，暴露方式可采用浸泡或喷淋。氧化剂可选择高碘酸钠，氧化剂溶液的质量百分浓度为0.1％-1％，氧化处理时间为1-12小时。
氧化处理后，心包膜中的粘多糖的邻羟基被氧化成醛基，醛基与胶原纤维的氨基反应，将粘多糖连接在胶原纤维上。
第一交联处理：
粘多糖连接在胶原纤维上后，粘多糖之间通过醛基相互交联，第一交联剂需具备与醛基反应的基团，优选采用双氨基化合物，通过氨基与醛基的反应将粘多糖之连接在一起。
双氨基化合物可从赖氨酸、乙二胺或己二胺中选择；双氨基化合物溶液中双氨基化合物的质量百分浓度为0.05％～5％；生物材料在双氨基化合物溶液中的暴露时间为0.5～12h；暴露方式可采用浸泡或喷淋。
第二交联处理：
生物材料的胶原纤维之间通过第二交联剂交联，第二交联剂可选择戊二醛，该步骤中，生物材料暴露于戊二醛溶液中，暴露方式可采用浸泡或喷淋。戊二醛溶液中戊二醛的质量百分浓度为0.05％～25％，交联时间为6h～3周，交联温度为0～37℃。
封端处理：
戊二醛交联处理后，生物材料中存在残余醛基，在该步骤中，通过封端物质进行封闭处理，封端物质优选多氨基物质，多氨基物质可从聚乙烯亚胺，聚赖氨酸，ε-聚赖氨酸，赖氨酸，己二胺等中选择。
该步骤中，生物材料暴露于多氨基物质溶液中，多氨基物质的质量百分比浓度为 0.5％～10％；暴露时间为0.5h～12h；暴露方式可采用浸泡或喷淋。
第三交联处理：
在前步封端处理步骤中，采用多氨基物质作为封端物质，残余氨基再经该步骤的第三交联剂交联，第三交联剂选用聚乙二醇二缩水甘油醚，聚乙二醇形成的交联网络增强心包膜的高弹性，一方面，聚乙二醇链高度对称性增强生物组织的高弹性，能够可完全恢复生物材料弹性变形的能力；另一方面，水是聚乙二醇的良性溶剂，浸水之后表现出高弹性，进一步改善生物材料的抗折痕性能。
第三交联剂溶液中第三交联剂的质量百分比浓度为0.01％～2％；交联时间为10～48h。
还原处理：
氨基和醛反应形成schiff-base化学键，但是schiff-base具有可逆的特点。通过还原反应转换成稳定的炭氮单键，避免压握过程中的化学键交换反应。
该步骤中，生物材料暴露于还原剂溶液中，还原剂可选择氰基硼氢化钠或者硼氢化钠；还原剂的质量百分浓度为0.01％～2％；暴露时间为1～12小时；暴露方式可采用浸泡或喷淋。
脱水处理：
甘油浸泡处理后采用乙醇脱水或冷冻干燥。
第二种实施方式中，对生物材料依次进行第二交联处理、氧化处理、第一交联处理和脱水处理，第二交联处理将生物材料的胶原纤维之间相互交联，氧化剂氧化过程中将生物材料中的粘多糖的邻羟基氧化成醛基，多糖类化合物连接到胶原蛋白纤维上，再通过第一交联处理使得多糖与多糖之间形成交联。最终形成胶原纤维与胶原纤维之间、多糖与胶原纤维之间、多糖与多糖之间的交联网状结构。
第一交联处理后还可包括后交联处理和还原处理，再进行脱水处理，无需进行封端处理。后交联处理交联第一交联剂的残余活性基团，还原处理将schiff-base化学键换成稳定的炭氮单键。
该实施方式的流程图可参照图5，依次对生物材料进行第二氧化处理、氧化处理、第一交联处理、后交联处理、还原处理和脱水处理。
第二交联处理：
生物材料的胶原纤维之间通过第二交联剂交联，第二交联剂可选择戊二醛，该步骤中，生物材料暴露于戊二醛溶液中，暴露方式可采用浸泡或喷淋。戊二醛溶液中戊二醛的质量百分浓度为0.05％～25％，交联时间为6h～3周，交联温度为0～37℃。
氧化处理：
取新鲜的心包膜，裁剪挂板；心包膜为猪心包膜或牛心包膜。裁剪挂板后暴露于氧化剂溶液中，暴露方式可采用浸泡或喷淋。氧化剂可选择高碘酸钠，氧化剂溶液的质量百分浓度为0.1％-1％，氧化处理时间为1-12小时。
氧化处理后，心包膜中的粘多糖的邻羟基被氧化成醛基，醛基与胶原纤维的氨基反应，将粘多糖连接在胶原纤维上。
第一交联处理：
粘多糖连接在胶原纤维上后，粘多糖之间通过醛基相互交联，第一交联剂需具备与醛基反应的基团，优选采用多氨基物质，例如可从赖氨酸、乙二胺、己二胺、聚乙烯亚胺、聚赖氨酸或ε-聚赖氨酸中选择，通过氨基与醛基的反应将粘多糖之连接在一起。
多氨基物质溶液中多氨基物质的质量百分浓度为0.5％～10％；生物材料在多氨基物质溶液中的暴露时间为0.5～12h；暴露方式可采用浸泡或喷淋。
后交联处理：
在前步第一交联处理步骤中，采用多氨基物质作为第一交联剂，残余氨基再经该步骤的第三交联剂交联，第三交联剂选用聚乙二醇二缩水甘油醚，聚乙二醇形成的交联网络增强心包膜的高弹性，一方面，聚乙二醇链高度对称性增强生物组织的高弹性，能够可完全恢复生物材料弹性变形的能力；另一方面，水是聚乙二醇的良性溶剂，浸水之后表现出高弹性，进一步改善生物材料的抗折痕性能。
第三交联剂溶液中第三交联剂的质量百分比浓度为0.01％～2％；交联时间为10～48h。
还原处理：
氨基和醛反应形成schiff-base化学键，但是schiff-base具有可逆的特点。通过还原反应转换成稳定的炭氮单键，避免压握过程中的化学键交换反应。
该步骤中，生物材料暴露于还原剂溶液中，还原剂可选择氰基硼氢化钠或者硼氢化钠；还原剂的质量百分浓度为0.01％～2％；暴露时间为1～12小时；暴露方式可采用浸泡或喷淋。
脱水处理：
甘油浸泡处理后采用乙醇脱水或冷冻干燥。
第三种实施方式中，对生物材料依次进行氧化处理、第二交联处理、第一交联处理和脱水处理，氧化剂氧化过程中将生物材料中的粘多糖的邻羟基氧化成醛基，多糖类化合物连接到胶原蛋白纤维上，第二交联处理将生物材料的胶原纤维之间相互交联，再通过第一交联处理使得多糖与多糖之间形成交联。最终形成胶原纤维与胶原纤维之间、多糖与胶原纤维之间、多糖与多糖之间的交联网状结构。
第一交联处理后还可包括后交联处理和还原处理，再进行脱水处理，无需进行封端处理。后交联处理交联第一交联剂的残余活性基团，还原处理将schiff-base化学键换成稳定的炭氮单键。
该实施方式的流程图可参照图6，依次对生物材料进行氧化处理、第二氧化处理、第一交联处理、后交联处理、还原处理和脱水处理。
氧化处理：
取新鲜的心包膜，裁剪挂板；心包膜为猪心包膜或牛心包膜。裁剪挂板后暴露于氧化剂溶液中，暴露方式可采用浸泡或喷淋。氧化剂可选择高碘酸钠，氧化剂溶液的质量百分浓度为0.1％-1％，氧化处理时间为1-12小时。
氧化处理后，心包膜中的粘多糖的邻羟基被氧化成醛基，醛基与胶原纤维的氨基反应，将粘多糖连接在胶原纤维上。
第二交联处理：
生物材料的胶原纤维之间通过第二交联剂交联，第二交联剂可选择戊二醛，该步骤中，生物材料暴露于戊二醛溶液中，暴露方式可采用浸泡或喷淋。戊二醛溶液中戊二醛的质量百分浓度为0.05％～25％，交联时间为6h～3周，交联温度为0～37℃。
第一交联处理：
粘多糖连接在胶原纤维上后，粘多糖之间通过醛基相互交联，第一交联剂需具备与醛基反应的基团，优选采用多氨基物质，例如可从赖氨酸、乙二胺、己二胺、聚乙烯亚胺、聚赖氨酸或ε-聚赖氨酸中选择，通过氨基与醛基的反应将粘多糖之连接在一起。
多氨基物质溶液中多氨基物质的质量百分浓度为0.5％～10％；生物材料在多氨基物质溶液中的暴露时间为0.5～12h；暴露方式可采用浸泡或喷淋。
后交联处理：
在前步第一交联处理步骤中，采用多氨基物质作为第一交联剂，残余氨基再经该步骤的第三交联剂交联，第三交联剂选用聚乙二醇二缩水甘油醚，聚乙二醇形成的交联网络增强心包膜的高弹性，一方面，聚乙二醇链高度对称性增强生物组织的高弹性，能够可完全恢复生物材料弹性变形的能力；另一方面，水是聚乙二醇的良性溶剂，浸水之后表现出高弹性，进一步改善生物材料的抗折痕性能。
第三交联剂溶液中第三交联剂的质量百分比浓度为0.01％～2％；交联时间为10～48h。
还原处理：
氨基和醛反应形成schiff-base化学键，但是schiff-base具有可逆的特点。通过还原反应转换成稳定的炭氮单键，避免压握过程中的化学键交换反应。
该步骤中，生物材料暴露于还原剂溶液中，还原剂可选择氰基硼氢化钠或者硼氢化钠；还原剂的质量百分浓度为0.01％～2％；暴露时间为1～12小时；暴露方式可采用浸泡或喷淋。
脱水处理：
甘油浸泡处理后采用乙醇脱水或冷冻干燥。
上述三种实施方式中，配置所用溶液的溶剂为处理生物材料的常规溶剂，不对生物材料的结果造成负面影响，例如可选择水、PBS或生理盐水等。
以下以具体实施例进行说明：
以下实施例中，以百分比表示的浓度如无特殊说明均表示质量百分比浓度；以下实施例中所说的溶液如无明确说明均采用PBS配置。
实施例1
参照图7所示流程图：以1％高碘酸钠溶液在室温条件下浸泡猪心包膜2h；2％的赖氨酸水溶液浸泡2h；随后，在0.05％戊二醛溶液中交联5天，用0.9％生理盐水清洗干净；2％的ε-聚赖氨酸水溶液浸泡2h；2％的聚乙二醇二缩水甘油醚水溶液加入0.1％的催化剂四甲基乙二胺反应24h。用0.9％生理盐水清洗3次，1％氰基硼氢化钠水溶液浸泡2h。清洗3次后，浸泡40％的甘油水溶液，之后冷冻干燥。
以实施例1制备得到的膜进行断裂力实验和吸水展平实验。
断裂力实验：干膜水合后，切割成1*5cm膜条，通过万能拉伸机测得最大断裂力；以常规戊二醛交联膜作为对照。结果如图8所示，实施例1制备得到的膜的断裂力在25～30N，明显优于常规戊二醛交联膜。
吸水展平实验：3*3cm膜片折叠压缩到1ml注射器圆筒里如(如图9中A所示)，在40℃的烘箱里放置3天；而后释放到生理盐水溶液中，观察膜片的展平情况。结果如图9中B和C所示，其中B为常规戊二醛交联干膜，膜片有大量折痕；C为HE干膜(实施例1制备)，释放浸水后，无明显可见折痕。
实施例2
参照图5所示流程图猪心包膜在0.05％戊二醛溶液中交联5天，用0.9％生理盐水清洗干净；以1％高碘酸钠溶液在室温条件下浸泡猪心包膜2h；随后，浸泡2％的ε-聚赖氨酸溶液2h。2％的聚乙二醇二缩水甘油醚加入0.1％的催化剂反应24h。用0.9％生理盐水清洗3次，浸泡1％氰基硼氢化钠溶液2h。清洗3次后，浸泡40％异丙醇溶液，之后鼓风干燥或者冷冻干燥。
断裂力实验同实施例1，结果如图10所示，实施例2制备得到的膜的断裂力明显优于常规戊二醛交联膜。
吸水展平实验：3*3cm膜片折叠压缩到1ml注射器圆筒里如(如图11中A所示)，在40℃的烘箱里放置3天；而后释放到生理盐水溶液中，观察膜片的展平情况。结果如图10中B和C所示，其中B为常规戊二醛交联干膜，膜片有大量折痕；C为HE干膜(实施例2制备)，释放浸水后，无明显可见折痕。
实施例3
以1％高碘酸钠溶液在室温条件下浸泡猪心包膜2h。浸泡2％的赖氨酸溶液2h；随后，在0.05％戊二醛溶液中交联5天，用0.9％生理盐水清洗干净。浸泡2％的双氨基化聚乙二醇溶液反应24h。用09％生理盐水清洗3次，浸泡1％氰基硼氢化钠溶液2h。清洗3次后，浸泡40％的甘油水溶液，之后冷冻干燥。
实施例4
以1％高碘酸钠溶液在室温条件下浸泡猪心包膜2h。在0.05％戊二醛溶液中交联5天，用0.9％生理盐水清洗干净。浸泡2％的聚乙烯亚胺溶液2h；随后，浸泡2％的双氨基化聚乙二醇水溶液反应24h。用0.9％生理盐水清洗3次，浸泡1％氰基硼氢化钠溶液2h。清洗3次后，浸泡60％的甘油水溶液，之后鼓风干燥。
断裂力实验同实施例1，结果如图12所示，实施例4制备得到的膜的断裂力明显优于常规戊二醛交联膜。
吸水展平实验：3*3cm膜片折叠压缩到1ml注射器圆筒里如(如图13中A所示)，在40℃的烘箱里放置3天；而后释放到生理盐水溶液中，观察膜片的展平情况。结果如图10中B和C所示，其中B为常规戊二醛交联干膜，膜片有大量折痕；C为HE干膜(实施例4制 备)，释放浸水后，无明显可见折痕。
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发明专利范围的限制。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 (48)

  1. 一种复合交联制备抗折痕的脱水生物材料的方法，其特征在于，对生物材料进行(a)氧化处理、(b)第一交联处理、(c)第二交联处理和(d)脱水处理制得；其中，(b)在(a)之后进行且(a)(b)(c)均在(d)之前进行；
    其中氧化处理时采用能够致使羟基转化为醛基的氧化剂，所述羟基来自于所述生物材料中的粘多糖，第一交联处理时采用能够致使粘多糖的醛基间相互交联的第一交联剂，第二交联处理时采用能够致使所述生物材料中胶原纤维间相互交联的第二交联剂。
  2. 根据权利要求1所述的方法，其特征在于，所述生物材料的胶原含量为60％～90％。
  3. 根据权利要求1所述的方法，其特征在于，所述生物材料的生物来源为猪、牛、马或羊；所述生物材料的来源部位为心包膜、心脏瓣膜、血管、韧带、肌肉、肠或皮肤。
  4. 根据权利要求1所述的方法，其特征在于，所述氧化处理包括将生物材料暴露于含氧化剂的溶液中。
  5. 根据权利要求4所述的方法，其特征在于，所述羟基为粘多糖的邻羟基。
  6. 根据权利要求4所述的方法，其特征在于，所述氧化剂为高碘酸钠。
  7. 根据权利要求4所述的方法，其特征在于，所述含氧化剂的溶液中氧化剂的质量百分比浓度为0.1％～1％。
  8. 根据权利要求4所述的方法，其特征在于，生物材料在含氧化剂的溶液中的暴露时间为1～12h。
  9. 根据权利要求4所述的方法，其特征在于，生物材料在含氧化剂溶液中的暴露方式为静态接触或动态接触。
  10. 根据权利要求1所述的方法，其特征在于，对生物材料依次进行(a)氧化处理、(b)第一交联处理和(c)第二交联处理：将氧化处理后的生物材料依次暴露于第一交联剂溶液和第二交联剂溶液中。
  11. 根据权利要求10所述的方法，其特征在于，所述第一交联剂具有至少两个与醛基反应的活性基团，所述第二交联剂与第一交联剂为不同物质且第二交联剂能与第一交联剂的活性基团反应。
  12. 根据权利要求11所述的方法，其特征在于，所述第一交联剂为至少具有两个氨基的小分子物质。
  13. 根据权利要求12所述的方法，其特征在于，所述第一交联剂为赖氨酸、乙二胺或己二胺。
  14. 根据权利要求10所述的方法，其特征在于，所述第一交联剂溶液中第一交联剂的质量百分比浓度为0.05％～5％；。
  15. 根据权利要求10所述的方法，其特征在于，生物材料暴露于第一交联剂溶液中的时间为0.5～12h。
  16. 根据权利要求10所述的方法，其特征在于，所述第二交联剂为戊二醛；所述第二交联剂溶液中第二交联剂的质量百分比浓度为0.05％～25％；交联时间为6h～3周；交联温度为0～37℃。
  17. 根据权利要求10所述的方法，其特征在于，生物材料在第一交联剂溶液和第二交联 剂溶液中的暴露方式均为静态接触或动态接触。
  18. 根据权利要求10所述的方法，其特征在于，还包括将交联处理后的生物材料暴露于含封端物质溶液中封闭残余活性基团的封端处理步骤；该残余活性基团来自于第二交联剂。
  19. 根据权利要求18所述的方法，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所述的方法，其特征在于，所述多氨基物质为聚乙烯亚胺、聚赖氨酸、ε-聚赖氨酸、赖氨酸或己二胺；所述含封端物质溶液中封端物质的质量百分比浓度为0.5％～10％；生物材料在含封端物质溶液中的暴露时间为0.5h～12h；生物材料在含封端物质溶液中的暴露方式为静态接触或动态接触。
  21. 根据权利要求18所述的方法，其特征在于，还包括将封端处理后生物材料暴露于第三交联剂溶液中交联残余活性基团的后交联处理步骤；该残余活性基团来自于所述封端物质。
  22. 根据权利要求21所述的方法，其特征在于，所述第三交联剂溶液中的第三交联剂至少具有一个与氨基反应的基团和一个亲水基团。
  23. 根据权利要求22所述的方法，其特征在于，所述与氨基反应的基团为酯键或环氧基团；所述亲水基团为醇羟基或醚键。
  24. 根据权利要求23所述的方法，其特征在于，所述第三交联剂为聚乙二醇缩水甘油醚。
  25. 根据权利要求24所述的方法，其特征在于，所述第三交联剂溶液中第三交联剂的质量百分比浓度为0.01％～2％；生物材料在第三交联剂溶液中的暴露时间为2h～48h；生物材料在第三交联剂溶液中的暴露方式为静态接触或动态接触。
  26. 根据权利要求1所述的方法，其特征在于，对生物材料依次进行(c)第二交联处理、(a)氧化处理和(b)第一交联处理：将生物材料依次暴露于第二交联剂溶液、含氧化剂溶液和第一交联剂溶液中。
  27. 根据权利要求1所述的方法，其特征在于，对生物材料依次进行(a)氧化处理、(c)第二交联处理和(b)第一交联处理：将生物材料依次暴露于含氧化剂溶液、第二交联剂溶液和第一交联剂溶液中。
  28. 根据权利要求26或27所述的方法，其特征在于，所述第二交联剂具有至少两个与氨基或羧基反应的活性基团，所述第一交联剂与第二交联剂为不同物质且第一交联剂能与第二交联剂的活性基团反应。
  29. 根据权利要求28所述的方法，其特征在于，所述第一交联剂为至少具有两个氨基的小分子物质。
  30. 根据权利要求29所述的方法，其特征在于，所述第一交联剂为赖氨酸、乙二胺、己二胺、聚乙烯亚胺、聚赖氨酸或ε-聚赖氨酸。
  31. 根据权利要求26或27所述的方法，其特征在于，第一交联剂溶液中第一交联剂的质量百分比浓度为0.5％～10％。
  32. 根据权利要求26或27所述的方法，其特征在于，生物材料暴露于第一交联剂溶液中的时间为0.5～12h。
  33. 根据权利要求26或27所述的方法，其特征在于，所述第二交联剂为戊二醛；所述第二交联剂溶液中第二交联剂的质量百分比浓度为0.05％～25％；交联时间为6h～3周；交联 温度为0～37℃。
  34. 根据权利要求26或27所述的方法，其特征在于，生物材料在第一交联剂溶液和第二交联剂溶液中的暴露方式均为静态接触或动态接触。
  35. 根据权利要求26或27所述的方法，其特征在于，还包括将第一交联剂处理后的生物材料暴露于第三交联剂溶液中交联残余活性基团的后交联处理步骤；所述残余活性基团来自于第一交联剂。
  36. 根据权利要求35所述的方法，其特征在于，所述第三交联剂溶液中的第三交联剂至少具有一个与氨基反应的基团和一个亲水基团。
  37. 根据权利要求36所述的方法，其特征在于，所述与氨基反应的基团包括酯键或环氧基团；所述亲水基团包括醇羟基或醚键。
  38. 根据权利要求37所述的方法，其特征在于，所述第三交联剂为聚乙二醇缩水甘油醚。
  39. 根据权利要求35所述的方法，其特征在于，所述第三交联剂溶液中第三交联剂的质量百分比浓度为0.01％～2％；生物材料在第三交联剂溶液中的暴露时间为10～48h2～48h；生物材料在第三交联剂溶液中的暴露方式为静态接触或动态接触。
  40. 根据权利要求21所述的方法，其特征在于，还包括将后交联处理后的生物材料暴露于还原剂溶液中的还原步骤。
  41. 根据权利要求35所述的方法，其特征在于，还包括将后交联处理后的生物材料暴露于还原剂溶液中的还原步骤。
  42. 根据权利要求40或41所述的方法，其特征在于，所述还原剂溶液中的还原剂为氰基硼氢化钠或者硼氢化钠；还原剂的质量百分比浓度为0.01％～2％；生物材料在还原剂溶液中的暴露时间为1～12h；生物材料在还原剂溶液中的暴露方式为静态接触或动态接触。
  43. 一种如权利要求1～42任一项权利要求所述方法制备得到的抗折痕的脱水交联生物材料。
  44. 根据权利要求43所述的脱水交联生物材料，其特征在于，所述脱水交联生物材料的最大断裂力n为25～30N。
  45. 根据权利要求44所述的脱水交联生物材料，其特征在于，所述脱水交联生物材料的最大断裂力是在水合后测试。
  46. 根据权利要求43所述的脱水交联生物材料，其特征在于，所述脱水交联生物材料折叠并至少保持72小时后展平无折痕。
  47. 一种生物瓣膜，包括支架和安装在支架上的瓣膜，其特征在于，所述瓣膜为权利要求43～46任一项权利要求所述的抗折痕的脱水交联生物材料。
  48. 一种介入***，包括心脏瓣膜和输送导管，所述心脏瓣膜折叠后由输送导管输送，其特征在于，所述心脏瓣膜为权利要求47所述的生物瓣膜。
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