WO2024001217A1 - 一种生物组织的处理方法及医用材料 - Google Patents
一种生物组织的处理方法及医用材料 Download PDFInfo
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- WO2024001217A1 WO2024001217A1 PCT/CN2023/076496 CN2023076496W WO2024001217A1 WO 2024001217 A1 WO2024001217 A1 WO 2024001217A1 CN 2023076496 W CN2023076496 W CN 2023076496W WO 2024001217 A1 WO2024001217 A1 WO 2024001217A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- capping
- treatment
- biological tissue
- solution
- cleaning
- Prior art date
Links
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Classifications
-
- 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
- A61L27/3691—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 physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/18—Modification of implant surfaces in order to improve biocompatibility, cell growth, fixation of biomolecules, e.g. plasma treatment
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
Definitions
- the invention relates to the technical field of medical devices, and in particular to a biological tissue processing method and medical materials.
- biomedical materials As one of the effective tools for centuries to fight against diseases, have received widespread attention and application.
- biomedical materials can be used in skeletal-muscular systems such as bones, teeth, joints, and tendons; in soft tissues such as skin, breast esophagus, respiratory tract, and bladder; and in cardiovascular systems such as artificial heart valves, blood vessels, and cardiovascular intubations.
- skeletal-muscular systems such as bones, teeth, joints, and tendons
- soft tissues such as skin, breast esophagus, respiratory tract, and bladder
- cardiovascular systems such as artificial heart valves, blood vessels, and cardiovascular intubations.
- Biomedical materials are the basis for the study of artificial organs and medical devices, and have become an important branch of contemporary materials science.
- Biomedical materials can be divided into biomedical metal materials, biomedical inorganic materials, biomedical polymer materials, biomedical composite materials, and biologically derived materials according to the composition and properties of the materials.
- biologically derived materials are formed from specially processed natural biological tissues and are also called biologically regenerated materials.
- Biological tissues can be obtained from animals of the same or heterogeneous species.
- Special processing includes fixation, sterilization and minor processing to eliminate antigenicity to maintain the original configuration of biological tissues, as well as dismantling the original configuration and reconstructing a new physical form. Handled intensely. Since the processed biological tissues have lost their vitality, biologically derived materials are nonviable materials. However, because biologically derived materials have a configuration and function similar to natural tissues, or their composition is similar to natural tissues, they are not suitable for use in the dynamic processes of the human body. Plays an important role in repair and replacement.
- biologically derived materials commonly used in clinical fields such as artificial heart valves and biological patches are derived from animal-derived collagen fiber tissue. Such products are prone to calcification after implantation, and calcification can cause hardening or tearing of biological tissue materials. , resulting in failure of biological tissue materials.
- Many biological tissue materials are stored using glutaraldehyde and/or formaldehyde, or are cross-linked and fixed using chemical reagents. A large number of studies have proven that after cross-linking of tissues, residual amine groups, aldehyde groups, and carboxyl groups (oxidized by aldehyde groups Form) and other reactive groups. When the treated biological tissue is implanted into the human body, the reactive groups form calcified binding points, resulting in Calcification.
- the purpose of the present invention is to provide a biological tissue processing method and medical materials, aiming to improve the anti-calcification performance of the treated biological tissue.
- the present invention provides a biological tissue processing method, which includes the following steps:
- Cross-linking the biological tissue, and the surface of the cross-linked biological tissue has active groups
- the end-capping biological tissue is subjected to antioxidant and sterilization treatments.
- the cross-linking agent includes glutaraldehyde
- the reactive groups include amine groups, free aldehyde groups and free carboxyl groups.
- the processing method further includes:
- the biological tissue is cleaned for the first time.
- the cleaning agent for the first cleaning includes physiological saline, phosphate buffer with a pH of 6.8-8.6, D-Hanks solution with a pH of 6.8-8.6, or a pH of 6.8-8.6 and a mass percentage of 0.1 Either %-2% glutaraldehyde solution.
- the step of end-capping treatment includes:
- the biological tissue that has been subjected to the second end-capping treatment is subjected to a third end-capping treatment using a third end-capping solution to block at least part of the free carboxyl groups.
- the first capping solution includes a first capping agent, the first capping agent includes at least one of heparin, mannose, formaldehyde, and galactose; and/or,
- the second end-capping solution includes a second end-capping agent, the second end-capping agent includes at least one of sodium borohydride, taurine, polyvinyl alcohol, and amino sulfate; and/or,
- the third end-capping solution includes a third end-capping agent, and the third end-capping agent includes N-hydroxysulfosuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbondioxide At least one of imine hydrochloride and amine compound.
- the pH of the first capping solution is 5.0-9.0, and the mass percentage of any component of the first capping agent in the first capping solution is 0.01%-10%,
- the temperature of the first end-capping treatment is 0-50°C, and the treatment time is 1h-72h;
- the pH of the second capping solution is 5.0-9.0, and in the second capping solution, the mass percentage of any component of the second capping agent is 0.01%-10%.
- the temperature of the secondary end-capping treatment is 0-50°C and the treatment time is 1h-72h;
- the pH of the third end-capping solution is 5.0-9.0, and in the third end-capping solution, the mass percentage of any component of the third end-capping agent is 0.01%-10%.
- the temperature of the secondary end-capping treatment is 0-50°C and the treatment time is 1h-72h.
- the step of end-capping further includes treating the organism that has undergone the first end-capping treatment.
- the tissue undergoes a second cleaning;
- the end-capping step further includes performing a third step on the biological tissue that has been subjected to the second end-capping treatment. cleaning;
- the end-capping step further includes subjecting the biological tissue to a fourth time. Clean.
- the cleaning agent used in the second cleaning is a glutaraldehyde solution, and the mass percentage of the glutaraldehyde solution is 0.01%-2%, and the pH is 6.8-8.6; and/or,
- the cleaning agent used in the third cleaning is the second end-capping agent; and/or,
- the cleaning agent used in the fourth cleaning is the third end-capping agent.
- a mixed solution of a sterilizing agent and an antioxidant is used to perform antioxidant and sterilizing treatment on the end-capping biological tissue.
- the sterilizing agent includes either glutaraldehyde or formaldehyde, so describe Antioxidants include at least one of vitamin C, vitamin E, and reducing sugar, and the mass percentage of the antioxidants in the mixed solution is 0.01%-10%, and the sterilizing agent is in the mixed solution. The mass percentage is 0.01%-10%, and the pH of the mixed solution is 6.8-8.6.
- the temperature of the antioxidant and sterilization treatment is 0-50°C, and the time is 1h-72h.
- the biological tissue includes any one of pericardium, heart valve, pleura, small intestinal submucosa, dura mater, dura mater, ligament, and animal skin.
- the present invention also provides a medical material, which is obtained by processing biological tissue through the biological tissue processing method as described in the preceding item.
- the biological tissue processing method and medical materials of the present invention have the following advantages:
- the aforementioned biological tissue processing method includes the following steps: cross-linking the biological tissue, and the surface of the cross-linked biological tissue has active groups; performing end-capping treatment on the cross-linked biological tissue. , so as to block at least part of the active groups; and, perform antioxidant and sterilization treatment on the end-blocked biological tissue.
- end-capping treatment the active group is blocked and loses its activity, so that it cannot serve as a calcification binding point. Therefore, when the treated biological tissue is implanted into the human body, it is difficult to calcify and has good anti-calcification properties.
- Figure 1 is a comparison chart of the experimental results of Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3 of the present invention
- Figure 2 shows the medical material provided in Example 2 of the present invention, the medical material provided in Comparative Example 4, the medical material provided in Comparative Example 5, and the medical material provided in Comparative Example 6 after being implanted in the subcutaneous tissue of rats for 8 weeks. , a diagram comparing the calcium content of various medical materials;
- Figure 3 shows the medical materials provided in Embodiment 3 of the present invention, the medical materials provided in Comparative Example 7, the medical materials provided in Comparative Example 8, and the medical materials provided in Comparative Example 9 after being implanted in the subcutaneous tissue of rats for 8 weeks.
- Figure 3 shows the medical materials provided in Embodiment 3 of the present invention, the medical materials provided in Comparative Example 7, the medical materials provided in Comparative Example 8, and the medical materials provided in Comparative Example 9 after being implanted in the subcutaneous tissue of rats for 8 weeks.
- Figure 4 shows the medical material provided in Embodiment 4 of the present invention, the medical material provided in Comparative Example 10, the medical material provided in Comparative Example 11, and the medical material provided in Comparative Example 12 when implanted in rats. Comparison diagram of calcium content of various medical materials in subcutaneous tissue after 8 weeks;
- Figure 5 shows the medical material provided in Embodiment 5 of the present invention, the medical material provided in Comparative Example 13, the medical material provided in Comparative Example 14, and the medical material provided in Comparative Example 15 when implanted into the subcutaneous tissue of rats. After 8 weeks, a comparison diagram of the calcium content of each medical material;
- Figure 6 shows the medical material provided in Embodiment 6 of the present invention, the medical material provided in Comparative Example 16, the medical material provided in Comparative Example 17, and the medical material provided in Comparative Example 18 when implanted under the skin of a rat. Comparison of the calcium content of various medical materials after 8 weeks of organization.
- each embodiment described below has one or more technical features, but this does not mean that the inventor must implement all the technical features in any embodiment at the same time, or can only implement them in different embodiments separately. some or all of the technical features.
- those skilled in the art can selectively implement some or all of the technical features in any embodiment based on the disclosure of the present invention and design specifications or implementation requirements, or Selectively implement a combination of some or all of the technical features in multiple embodiments, thereby increasing the flexibility of the implementation of the present invention.
- One of the objects of the present invention is to provide a biological tissue processing method to treat biological tissue taken from animals.
- the biological tissue is processed, and the processed biological tissue is formed into biologically derived materials, which can be used in the medical field.
- the biological tissues include, but are not limited to, pericardium, heart valves, membranes, pleura, small intestinal submucosa, dura mater, dura mater, ligaments, skin, etc. from animals.
- the animals mentioned here are, for example, cows, pigs, rabbits, etc.
- the processing method includes the following steps:
- Step S10 Cross-link the biological tissue.
- the surface of the cross-linked biological tissue has active groups.
- Step S20 Perform end-capping treatment on the cross-linked biological tissue to block at least part of the active groups.
- step S30 perform anti-oxidation and sterilization treatment on the end-capped biological tissue to form a biologically derived material.
- the biological tissue can be cross-linked with an aldehyde compound.
- an aldehyde compound in a non-limiting example, a glutaraldehyde solution is used to cross-link the biological tissue.
- glutaraldehyde solution is used to cross-link the biological tissue.
- each glutaraldehyde molecule has an aldehyde group at both ends, this may cause the aldehyde group at one end to be cross-linked with the amine group, and the aldehyde group at the other end to form a free aldehyde group, and the cross-linking is often insufficient. This prevents the amine groups in biological tissues from being completely combined with the aldehyde groups, resulting in residual amine groups on the biological tissues.
- free aldehyde groups may be oxidized to form free carboxyl groups. Therefore, the active groups present on the surface of the cross-linked biological tissue at least include amine groups, free aldehyde groups and free carboxyl groups.
- the amine groups, free aldehyde groups and free carboxyl groups on the surface of the biological tissue will form calcification binding points, causing the biological tissue to easily calcify and reduce its service life.
- the treatment method further includes step S20 to block the amine groups, free aldehyde groups and free carboxyl groups on the surface of the cross-linked biological tissue, so that At least part of the reactive groups loses reactivity.
- step S20 the amine group, free aldehyde group and free carboxyl group can be blocked in a suitable order.
- the step S20 includes the following steps S21, S22 and S23.
- the step S21 is: using a first capping solution to perform a first capping process on the cross-linked biological tissue, so that At least partially blocked amine groups.
- the step S22 is: using a second end-capping solution to perform a second end-capping treatment on the biological tissue that has been subjected to the first end-capping treatment, so as to block at least part of the free aldehyde groups.
- the step S23 is: using a third end-capping solution to perform a third end-capping treatment on the biological tissue that has been subjected to the second end-capping treatment, so as to block at least part of the free carboxyl groups.
- the first capping solution includes a first capping agent
- the first capping agent includes but is not limited to at least one of heparin, mannose, formaldehyde, and galactose, that is, the first capping agent
- the terminal agent includes a single component, or may include two or more components.
- the pH of the first capping solution is 5.0-9.0, and in the first capping solution, the mass percentage of any component of the first capping agent is 0.01%-10%.
- the temperature of the secondary end-capping treatment is 0-50°C, and the treatment time is 1h-72h.
- the second end-capping solution includes a second end-capping agent, and the second end-capping agent includes at least one of sodium borohydride, taurine, polyvinyl alcohol, and amino sulfate, that is, the second end-capping agent
- the terminal agent may include a single component or two or more components.
- the pH of the second capping solution is 5.0-9.0, and in the second capping solution, the mass percentage of any component of the second capping agent is 0.01%-10%.
- the temperature of the secondary end-capping treatment is 0-50°C and the treatment time is 1h-72h.
- the third end-capping solution includes a third end-capping agent, and the third end-capping agent includes N-hydroxysulfosuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbondioxide At least one of imine hydrochloride and amine compound, that is to say, the third end-capping agent may include a single component, or may include two or more components.
- the pH of the third end-capping solution is 5.0-9.0, and in the third end-capping solution, the mass percentage of any component of the third end-capping agent is 0.01%-10%.
- the temperature of the secondary end-capping treatment is 0-50°C and the treatment time is 1h-72h. It should be noted that the pH mentioned in this article is the pH at room temperature.
- the processing method also includes a first cleaning step. Specifically, after the execution of step S10 and before the execution of step S21, a third cleaning step is performed on the cross-linked biological tissue. One wash.
- the end-capping step also includes multiple cleaning steps. Specifically, after step S21 is executed and before step S22 is executed, the biological tissue that has been subjected to the first end-capping process is cleaned for a second time. After step S22 is performed and before step S23 is performed, the biological tissue that has been subjected to the second end-capping process is cleaned for a third time. After step S23 is executed and before step S30 is executed, the biological tissue that has been subjected to the third end-capping process is cleaned for the fourth time.
- the cleaning agent for the first cleaning includes physiological saline, phosphate buffer with a pH of 6.8-8.6, D-Hanks solution with a pH of 6.8-8.6, or a mass percentage of 0.1%-2% and a pH of Any one of the glutaraldehyde solutions of 6.8-8.6.
- the first cleaning is performed at room temperature, and the number of cleanings is 3-5 times, each time for 3-5 minutes.
- glutaraldehyde solution as the cleaning agent during the first cleaning is that the biological tissue after cross-linking treatment can be supplemented with cross-linking, so that the amine groups in the biological tissue can further interact with the aldehydes of glutaraldehyde. base combination to reduce residual amine groups.
- the cleaning agent for the second cleaning is glutaraldehyde solution, and the concentration of the glutaraldehyde solution is 0.01%-2%, and the pH is 6.8-8.6.
- the second cleaning is performed at room temperature, and the number of cleanings is 3-5 times, each time for 3-5 minutes.
- the purpose of this cleaning is to use glutaraldehyde to further react with the biological tissue after the first end-capping treatment to combine with the remaining amine groups as much as possible and form free aldehyde groups. At least part of these free aldehyde groups are It was blocked during the second sealing process.
- the cleaning agent for the third cleaning is the second end-capping agent.
- the third cleaning is performed at room temperature, and the number of cleanings is 3-5 times, each time for 3-5 minutes.
- the purpose of this cleaning is to further block free aldehyde groups. It should be noted that during the second end-capping treatment and the third cleaning process, at least part of the free aldehyde groups are oxidized into free carboxyl groups.
- the cleaning agent for the fourth cleaning is the third end-capping agent.
- the fourth cleaning is performed at room temperature, and the number of cleanings is 3-5 times, each time for 3-5 minutes.
- the purpose of this cleaning is to further block at least part of the free carboxyl groups.
- a mixed solution of a sterilant and an antioxidant is used to perform anti-oxidation and sterilization treatment on the end-capping biological tissue.
- Optional sterilizing agents include, but are not limited to, either glutaraldehyde or formaldehyde.
- Optional antioxidants include, but are not limited to, at least one of vitamin C, vitamin E, and reducing sugar.
- the reducing sugar includes but is not limited to at least one of glucose, fructose, maltose, and galactose.
- the pH of the mixed solution is 6.8-8.6, and the mass percentage of the antioxidant in the mixed solution is 0.01%-10%, and the mass percentage of the sterilizing agent in the mixed solution is 0.01%-10%.
- the temperature of anti-oxidation and sterilization treatment is 0-50°C, and the time is 1h-72h.
- the residual aldehyde groups in the obtained biologically derived material can no longer be oxidized, thereby preventing new calcification binding points from being generated, thereby improving the anti-calcification performance of the biologically derived material.
- embodiments of the present invention also provide a biologically derived material, which is obtained by processing biological tissue through the aforementioned biological tissue processing method.
- the biological tissue used in this example is bovine pericardium.
- the processing process of beef pericardium is as follows:
- the sample was cleaned for the first time using a glutaraldehyde solution with a mass percentage of 0.625% and a pH of 7.4 at room temperature.
- the number of cleanings was 3 times for 3 minutes each time.
- the sample after the first cleaning was subjected to the first end-capping treatment using the first end-capping solution at room temperature, and the treatment time was 24 hours.
- the pH of the first capping solution is 7.4, and the first capping agent is heparin, and the mass percentage of heparin is 0.5%.
- the "room temperature” mentioned in this article refers to the indoor temperature, which is usually 20°C ⁇ 5°C.
- the cleaning agent for the second cleaning is a glutaraldehyde solution with a mass percentage of 0.625% and a pH of 7.4.
- the cleaning times are three times, each time for 5 minutes.
- the sample after the second cleaning was subjected to a second end-capping treatment using a second end-capping solution at 5° C. for a treatment time of 72 hours.
- the pH of the second capping solution is 5.0
- the second capping agent is sodium borohydride
- the mass percentage of sodium borohydride is 1.5%.
- the cleaning agent for the third cleaning is the second end-capping solution, and the number of cleanings is 3 times, 5 minutes each time.
- the sample washed for the third time was subjected to a third end-capping treatment using a third end-capping solution at room temperature, and the treatment time was 72 hours.
- the pH of the third capping solution is 5.0
- the third capping agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 1-(3
- the mass percentage of -dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 3.4%.
- the sample that has been subjected to the third end-capping treatment is washed a fourth time at room temperature.
- the cleaning agent for the fourth cleaning is the third end-capping solvent, and the number of cleanings is 3 times, 3 minutes each time.
- the sample that has been cleaned for the fourth time is subjected to antioxidant and sterilization treatment at 5° C. using a mixed solution of antioxidant and sterilant to obtain biologically derived materials.
- the anti-oxidation and sterilization treatment time is 72 hours
- the pH value of the mixed solution is 7.4, in which the sterilant is glutaraldehyde, with a mass percentage of 0.625%
- the antioxidant is vitamin C, with a mass percentage of 0.5%.
- Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that only the bovine pericardium was cross-linked and washed for the first time, but the sample was not subjected to three end-capping treatments, the second cleaning, and the third cleaning. Three washes and said fourth wash.
- Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the bovine pericardium was subjected to cross-linking treatment, first cleaning, first end-capping treatment and the second cleaning, but the second end-capping treatment was not performed. , the third cleaning, the third end-capping treatment, the fourth cleaning, antioxidant and sterilization treatment.
- Comparative Example 3 The difference between Comparative Example 3 and Embodiment 1 is that the bovine pericardium was cross-linked, cleaned for the first time, capped for the first time, cleaned for the second time, capped for the second time, and cleaned for the third time.
- the third sealing treatment, fourth cleaning, antioxidant and sterilization treatment were not performed.
- Spectrophotometry was performed on the biologically derived materials obtained in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 to detect the contents of amino groups, free aldehyde groups, and free carboxyl groups on the surface of the biologically derived materials obtained in each example.
- the results are shown in Figure 1.
- the content of active groups on the surface of the biologically derived material obtained in Comparative Example 1 is 156 nmol/ml, of which the content of amine groups is 34 nmol/mg, the content of free aldehyde groups is 68 nmol/mg, and the content of free carboxyl groups is 156 nmol/ml.
- the content is 54nmol/mg.
- the content of active groups on the surface of the biologically derived material obtained in Comparative Example 2 is 107 nmol/mg, of which the content of amine groups is 8 nmol/mg, the content of free aldehyde groups is 51 nmol/mg, and the content of free carboxyl groups is 48 nmol/mg.
- the content of active groups on the surface of the biologically derived material obtained in Comparative Example 3 is 54 nmol/mg, of which the content of amine groups is 6 nmol/mg, the content of free aldehyde groups is 7 nmol/mg, and the content of free carboxyl groups is 41 nmol/mg.
- the content of active groups on the surface of the biologically derived material obtained in Example 1 is 17 nmol/mg, of which the content of amine groups is 4 nmol/mg, the content of free aldehyde groups is 8 nmol/mg, and the content of free carboxyl groups is 5 nmol/mg. That is to say, the amine group content on the surface of the biological tissue after the first end-capping treatment and the second cleaning is greatly reduced, and the amine group content on the surface of the biological tissue after the second end-capping treatment and the third cleaning is greatly reduced.
- the content of free aldehyde groups is greatly reduced, and the content of amine groups, free aldehyde groups and free carboxyl groups on the surface of the biological tissue after three end-capping treatments, four cleanings and anti-oxidation and sterilization treatments are all greatly reduced.
- the biological tissue used in this example is bovine pericardium.
- the processing process of beef pericardium is as follows:
- the sample was washed for the first time using a phosphate buffer solution with a pH of 7.4 at room temperature, five times for 5 minutes each time.
- the sample after the first cleaning was subjected to the first end-capping treatment using the first end-capping solution at room temperature, and the treatment time was 48 hours.
- the pH of the first capping solution is 6.2, and the first capping agent is formaldehyde, and the mass percentage of formaldehyde is 2%.
- the cleaning agent for the second cleaning is a glutaraldehyde solution with a mass percentage of 0.625% and a pH of 7.4.
- the number of cleanings is 3 times, 3 minutes each time.
- the sample after the second cleaning was subjected to a second end-capping treatment using a second end-capping solution at 39° C. for a treatment time of 12 hours.
- the pH of the second capping solution is 7.4, and the second capping agent is taurine, and the mass percentage of taurine is 0.5%.
- the cleaning agent for the third cleaning is the second end-capping solution, and the number of cleanings is 3 times, 5 minutes each time.
- the sample washed for the third time was subjected to a third end-capping treatment using a third end-capping solution at room temperature, and the treatment time was 72 hours.
- the pH of the third capping solution is 8.6, and the third capping agent is N-hydroxysulfosuccinimide, and the mass percentage of N-hydroxysulfosuccinimide is 4.5%.
- the sample that has been subjected to the third end-capping treatment is washed a fourth time at room temperature.
- the cleaning agent for the fourth cleaning is the third end-capping solution, and the number of cleanings is 3 times, 3 minutes each time.
- the sample that has been cleaned for the fourth time is subjected to antioxidant and sterilization treatment at 45° C. using a mixed solution of antioxidants and sterilants to obtain biologically derived materials.
- the anti-oxidation and sterilization treatment time is 10 hours
- the pH of the mixed solution is 8.3
- the sterilant is glutaraldehyde with a mass percentage of 2.0%
- the antioxidant is vitamin C with a mass percentage of 8.5%.
- Comparative Example 4 The difference between Comparative Example 4 and Example 2 is that only the bovine pericardium was cross-linked and cleaned for the first time, but the sample was not subjected to three end-capping processes, the second cleaning, and the third cleaning. Three washes and said fourth wash.
- Comparative Example 5 The difference between Comparative Example 5 and Example 2 is that the bovine pericardium was subjected to cross-linking treatment, first cleaning, first end-capping treatment and the second cleaning, but the second end-capping treatment was not performed. , the third cleaning, the third end-capping treatment, the fourth cleaning, antioxidant and sterilization treatment.
- Comparative Example 6 The difference between Comparative Example 6 and Example 2 is that the bovine pericardium was cross-linked, cleaned for the first time, capped for the first time, cleaned for the second time, capped for the second time and cleaned for the third time.
- the third sealing treatment, fourth cleaning, antioxidant and sterilization treatment were not performed.
- Example 2 The biologically derived materials obtained in Example 2, Comparative Example 4, Comparative Example 5 and Comparative Example 6 were respectively implanted under the skin of rats and taken out after 8 weeks, and the calcium content of each biologically derived material was measured using an atomic absorption spectrophotometer. , the results are shown in Figure 2. It can be seen from Figure 2 that the biologically derived material obtained without end-capping treatment (i.e., the biologically derived material obtained in Comparative Example 4) has the highest calcium content, and the biologically derived material obtained after the first end-capping treatment (i.e., Comparative Example 5) has the highest calcium content.
- the calcium content on the biologically derived material obtained was second, and the calcium content on the biologically derived material obtained after the first and second sealing treatments (i.e., the biologically derived material obtained in Comparative Example 6) was second, and the calcium content on the biologically derived material obtained after the third sealing treatment was third.
- the calcium content on the biologically derived material that is, the biologically derived material obtained in Example 2) obtained by end treatment, antioxidant and sterilization treatment is the lowest. In other words, after three times of end-capping treatment and antioxidant and sterilization treatment, the biologically derived material has the best anti-calcification performance.
- the biological tissue used in this example is bovine pericardium.
- the processing process of beef pericardium is as follows:
- the sample was washed for the first time using a phosphate buffer solution with a pH of 7.4 at room temperature, five times for 5 minutes each time.
- the sample after the first cleaning was subjected to the first end-capping treatment using the first end-capping solution at 48° C. for a treatment time of 6 hours.
- the pH of the first capping solution is 8.6, and the first capping agent is mannose, and the mass percentage of mannose is 1%.
- the cleaning agent for the second cleaning is a glutaraldehyde solution with a mass percentage of 0.625% and a pH of 7.4.
- the number of times is 3 times, 3 minutes each time.
- the sample after the second cleaning was subjected to a second end-capping treatment using a second end-capping solution at 50° C. for a treatment time of 72 hours.
- the pH of the second capping solution is 5.0
- the second capping agent is polyvinyl alcohol
- the mass percentage of polyvinyl alcohol is 1.2%.
- the cleaning agent for the third cleaning is the second end-capping solution, and the number of cleanings is 3 times, 5 minutes each time.
- the sample washed for the third time was subjected to a third end-capping treatment using a third end-capping solution at room temperature, and the treatment time was 72 hours.
- the pH of the third capping solution is 8.0
- the third capping agent is 2-hydroxyethylamine
- the mass percentage of 2-hydroxyethylamine is 0.5%.
- the sample that has been subjected to the third end-capping treatment is washed a fourth time at room temperature.
- the cleaning agent for the fourth cleaning is the third end-capping solution, and the number of cleanings is 3 times, 3 minutes each time.
- the sample that has been cleaned for the fourth time is subjected to antioxidant and sterilization treatment at 5° C. using a mixed solution of antioxidant and sterilant to obtain biologically derived materials.
- the anti-oxidation and sterilization treatment time is 72 hours
- the pH of the mixed solution is 6.8, in which the sterilant is glutaraldehyde, with a mass percentage of 0.625%
- the antioxidant is vitamin E, with a mass percentage of 2.3%.
- Comparative Example 7 The difference between Comparative Example 7 and Example 3 is that only the bovine pericardium was cross-linked and washed for the first time, but the sample was not subjected to three end-capping treatments, the second cleaning, and the third cleaning. Three washes and said fourth wash.
- Comparative Example 8 The difference between Comparative Example 8 and Example 3 is that the bovine pericardium was subjected to cross-linking treatment, first cleaning, first end-capping treatment and the second cleaning, but the second end-capping treatment was not performed. , the third cleaning, the third end-capping treatment, the fourth cleaning, antioxidant and sterilization treatment.
- Comparative Example 9 lies in the cross-linking treatment of bovine pericardium and the first clearing process. washing, first sealing treatment, second cleaning, second sealing treatment and third cleaning, but no third sealing treatment, fourth cleaning, antioxidant and sterilization treatment.
- Example 3 The biologically derived materials obtained in Example 3, Comparative Example 7, Comparative Example 8 and Comparative Example 9 were respectively implanted under the skin of rats and taken out after 8 weeks, and the calcium content of each biologically derived material was measured using an atomic absorption spectrophotometer. , the results are shown in Figure 3. It can be seen from Figure 3 that the biologically derived material obtained without end-capping treatment (i.e., the biologically derived material obtained in Comparative Example 7) has the highest calcium content, and the biologically derived material obtained after the first end-capping treatment (i.e., Comparative Example 8) has the highest calcium content.
- the calcium content on the biologically derived material obtained was second, and the calcium content on the biologically derived material obtained after the first and second sealing treatments (i.e., the biologically derived material obtained in Comparative Example 9) was second, after three sealing treatments.
- the calcium content on the biologically derived material that is, the biologically derived material obtained in Example 3 obtained by end treatment, antioxidant and sterilization treatment is the lowest. In other words, after three times of end-capping treatment and antioxidant and sterilization treatment, the biologically derived material has the best anti-calcification performance.
- the biological tissue used in this example is bovine pericardium.
- the processing process of beef pericardium is as follows:
- the sample was washed for the first time using a phosphate buffer solution with a pH of 7.4 at room temperature, five times for 5 minutes each time.
- the sample after the first cleaning was subjected to the first end-capping treatment at 50° C. using the first end-capping solution for a treatment time of 2 hours.
- the pH of the first capping solution is 8.6, and the first capping agent is galactose and formaldehyde, and the mass percentage of galactose is 9.5% and the mass percentage of formaldehyde is 3.3%.
- the cleaning agent for the second cleaning is a glutaraldehyde solution with a mass percentage of 2% and a pH of 7.4.
- the number of cleanings is 3 times, 3 minutes each time.
- the sample after the second cleaning was subjected to a second end-capping treatment using a second end-capping solution at 37° C. for a treatment time of 48 hours.
- the pH of the second capping solution is 8.4, and the second capping agent is glucosamine sulfate, and the mass percentage of glucosamine sulfate is 8.6%.
- the cleaning agent for the third cleaning is the second end-capping solution, and the number of cleanings is 5 times, 5 minutes each time.
- the sample washed for the third time was subjected to a third end-capping treatment using a third end-capping solution at room temperature, and the treatment time was 72 hours.
- the pH of the third capping solution is 8.0
- the third capping agent is 2-hydroxyethylamine
- the mass percentage of 2-hydroxyethylamine is 0.5%.
- the sample that has been subjected to the third end-capping treatment is washed a fourth time at room temperature.
- the cleaning agent for the fourth cleaning is the third end-capping solution, and the number of cleanings is 3 times, 3 minutes each time.
- the sample that has been cleaned for the fourth time is subjected to antioxidant and sterilization treatment at 5° C. using a mixed solution of antioxidant and sterilant to obtain biologically derived materials.
- the anti-oxidation and sterilization treatment time is 72 hours
- the pH of the mixed solution is 7.1
- the antioxidant is glutaraldehyde, with a mass percentage of 0.625%
- the sterilizing agent is vitamin E, with a mass percentage of 0.5%.
- Comparative Example 10 The difference between Comparative Example 10 and Example 4 is that only the bovine pericardium was cross-linked and cleaned for the first time, but the sample was not subjected to three end-capping processes, the second cleaning, and the third cleaning. Three washes and said fourth wash.
- Comparative Example 11 The difference between Comparative Example 11 and Example 4 is that the bovine pericardium was cross-linked, cleaned for the first time, capped for the first time, and cleaned for the second time, but the second capping was not performed. treatment, the third cleaning, the third end-capping treatment, the fourth cleaning, antioxidant and sterilization treatment.
- Comparative Example 12 lies in the cross-linking treatment of bovine pericardium, the first Cleaning, first sealing treatment, second cleaning, second sealing treatment and third cleaning, but no third sealing treatment, fourth cleaning, antioxidant and sterilization treatment.
- Example 4 The biologically derived materials obtained in Example 4, Comparative Example 10, Comparative Example 11 and Comparative Example 12 were respectively implanted under the skin of rats and taken out after 8 weeks, and the atomic absorption spectrophotometer was used to measure the properties of each biologically derived material. Calcium content, the results are shown in Figure 4. It can be seen from Figure 4 that the biologically derived material obtained without end-capping treatment (i.e., the biologically derived material obtained in Comparative Example 10) has the highest calcium content, and the biologically derived material obtained after the first end-capping treatment (i.e., Comparative Example 10) has the highest calcium content.
- the calcium content on the biologically derived material obtained after the first and second end-capping treatments is second.
- Three times of end-capping treatment and antioxidant and sterilization treatment resulted in the lowest calcium content in the biologically derived material (ie, the biologically derived material obtained in Example 3).
- the biologically derived material has the best anti-calcification performance.
- the biological tissue used in this example is bovine pericardium.
- the processing process of beef pericardium is as follows:
- the sample was washed for the first time using a phosphate buffer solution with a pH of 7.4 at room temperature, five times for 5 minutes each time.
- the sample after the first cleaning was subjected to the first end-capping treatment at 50° C. using the first end-capping solution for a treatment time of 2 hours.
- the pH of the first capping solution is 8.6, and the first capping agent is galactose and formaldehyde, and the mass percentage of galactose is 9.5% and the mass percentage of formaldehyde is 3.3%.
- the cleaning agent for the second cleaning is a glutaraldehyde solution with a mass percentage of 2% and a pH of 7.4.
- the number of cleanings is 3 times, 3 minutes each time.
- the sample after the second cleaning was subjected to a second end-capping treatment using a second end-capping solution at 10° C. for a treatment time of 72 hours.
- the pH of the second capping solution is 7.4, and the second capping agent is glucosamine sulfate and sodium borohydride.
- the mass percentage of glucosamine sulfate is 8.6%, and the mass percentage of sodium borohydride is 2.1%. .
- the cleaning agent for the third cleaning is the second end-capping solution, and the number of cleanings is 5 times, 5 minutes each time.
- the sample washed for the third time was subjected to a third end-capping treatment using a third end-capping solution at room temperature, and the treatment time was 72 hours.
- the pH of the third capping solution is 8.0
- the third capping agent is 2-hydroxyethylamine
- the mass percentage of 2-hydroxyethylamine is 0.5%.
- the sample that has been subjected to the third end-capping treatment is washed a fourth time at room temperature.
- the cleaning agent for the fourth cleaning is the third end-capping solution, and the number of cleanings is 3 times, 3 minutes each time.
- the sample that has been cleaned for the fourth time is subjected to antioxidant and sterilization treatment at 5° C. using a mixed solution of antioxidant and sterilant to obtain biologically derived materials.
- the anti-oxidation and sterilization treatment time is 72 hours
- the pH of the mixed solution is 6.8, in which the sterilant is glutaraldehyde, with a mass percentage of 0.625%
- the antioxidant is vitamin E, with a mass percentage of 2.3%.
- Comparative Example Thirteen and Example Five The difference between Comparative Example Thirteen and Example Five is that only the bovine pericardium was cross-linked and washed for the first time, but the sample was not subjected to three end-capping treatments, the second cleaning, and the The third wash and said fourth wash.
- Comparative Example 14 The difference between Comparative Example 14 and Example 5 is that the bovine pericardium was cross-linked, washed for the first time, capped for the first time, and washed for the second time, but the second capping was not performed. treatment, the third cleaning, the third end-capping treatment, the fourth cleaning, antioxidant and sterilization treatment.
- Comparative Example 15 The difference between Comparative Example 15 and Embodiment 5 is that the bovine pericardium was cross-linked, washed for the first time, blocked for the first time, washed for the second time, blocked for the second time, and washed for the third time.
- the third sealing treatment, fourth cleaning, antioxidant and sterilization treatments were not performed.
- Example 5 The biologically derived materials obtained in Example 5, Comparative Example 13, Comparative Example 14 and Comparative Example 15 were respectively implanted under the skin of rats and taken out after 8 weeks, and each biologically derived material was measured using an atomic absorption spectrophotometer. The calcium content, the results are shown in Figure 5. It can be seen from Figure 5 that the biologically derived material obtained without end-capping treatment (i.e., the biologically derived material obtained in Comparative Example 13) has the highest calcium content, and the biologically derived material obtained after the first end-capping treatment (i.e., Comparative Example 13) has the highest calcium content.
- the calcium content on the biologically derived material obtained in Comparative Example 14) was second, and the calcium content on the biologically derived material obtained after the first and second end-capping treatments (i.e., the biologically derived material obtained in Comparative Example 15) was third.
- the biologically derived material (that is, the biologically derived material obtained in Example 3) has the lowest calcium content. In other words, after three times of end-capping treatment and antioxidant and sterilization treatment, the biologically derived material has the best anti-calcification performance.
- the biological tissue used in this example is bovine pericardium.
- the processing process of beef pericardium is as follows:
- the sample was washed for the first time using a phosphate buffer solution with a pH of 7.4 at room temperature, five times for 5 minutes each time.
- the sample after the first cleaning was subjected to the first end-capping treatment at 50° C. using the first end-capping solution for a treatment time of 2 hours.
- the pH of the first capping solution is 8.6, and the first capping agent is galactose and formaldehyde, and the mass percentage of galactose is 9.5% and the mass percentage of formaldehyde is 3.3%.
- the cleaning agent for the second cleaning is a glutaraldehyde solution with a mass percentage of 2% and a pH of 7.4.
- the number of cleanings is 3 times, 3 minutes each time.
- the sample after the second cleaning was subjected to a second end-capping treatment using a second end-capping solution at 37° C. for a treatment time of 48 hours.
- the pH of the second capping solution is 8.4, and the second capping agent includes glucosamine sulfate and sodium borohydride.
- the mass percentage of glucosamine sulfate is 8.6% and the mass percentage of sodium borohydride is 2.1%. .
- the cleaning agent for the third cleaning is the second end-capping solution, and the number of cleanings is 5 times, 5 minutes each time.
- the sample washed for the third time was subjected to a third end-capping treatment using a third end-capping solution at room temperature, and the treatment time was 72 hours.
- the pH of the third capping solution is 8.0
- the third capping agent includes 2-hydroxyethylamine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2 -The mass percentage of hydroxyethylamine is 0.5%, and the mass percentage of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 7.3%.
- the sample that has been subjected to the third end-capping treatment is washed a fourth time at room temperature.
- the cleaning agent for the fourth cleaning is the third end-capping solution, and the number of cleanings is 3 times, 3 minutes each time.
- the sample that has been cleaned for the fourth time is subjected to antioxidant and sterilization treatment at 5° C. using a mixed solution of antioxidant and sterilant to obtain biologically derived materials.
- the anti-oxidation and sterilization treatment time is 72 hours
- the pH of the mixed solution is 7.4, in which the sterilant is glutaraldehyde, with a mass percentage of 0.625%
- the antioxidant is vitamin E, with a mass percentage of 0.5%.
- Comparative Example 16 The difference between Comparative Example 16 and Example 6 is that only the bovine pericardium was subjected to cross-linking treatment and the first cleaning, but the sample was not subjected to three end-capping treatments, the second cleaning, and the The third wash and said fourth wash.
- Comparative Example 17 The difference between Comparative Example 17 and Example 6 is that the bovine pericardium was cross-linked, washed for the first time, capped for the first time, and washed for the second time, but the second capping was not performed. treatment, the third cleaning, the third end-capping treatment, the fourth cleaning, antioxidant and sterilization treatment reason.
- Comparative Example 18 lies in the cross-linking treatment, first cleaning, first end-capping treatment, second cleaning, second end-capping treatment and third cleaning of the bovine pericardium.
- the third sealing treatment, fourth cleaning, antioxidant and sterilization treatments were not performed.
- Example 6 The biologically derived materials obtained in Example 6, Comparative Example 16, Comparative Example 17 and Comparative Example 18 were respectively implanted into the subcutaneous tissue of rats and taken out after 8 weeks, and each biologically derived material was measured using an atomic absorption spectrophotometer. The calcium content, the results are shown in Figure 6.
- the biologically derived material obtained without end-capping treatment i.e., the biologically derived material obtained in Comparative Example 16
- the biologically derived material obtained after the first end-capping treatment i.e., Comparative Example
- the calcium content on the biologically derived material obtained in Comparative Example 17) was second
- the calcium content on the biologically derived material obtained after the first and second end-capping treatments i.e., the biologically derived material obtained in Comparative Example 18
- the biologically derived material has the lowest calcium content.
- the biologically derived material has the best anti-calcification performance.
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Abstract
本发明提供了一种生物组织的处理方法及医用材料，所述处理方法包括如下步骤：对生物组织进行交联处理，经交联处理后的所述生物组织的表面具有活性基团；对经交联处理的所述生物组织进行封端处理，以使至少部分的所述活性基团封闭；以及，对经封端处理的所述生物组织进行抗氧化及灭菌处理。由生物组织经所述处理方法处理得到的医用材料的表面具有较少的活性基团，其在植入患者体内时，降低钙化结合点，提高医用材料的抗钙化性能。
Description
本发明涉及医疗器械技术领域，具体涉及一种生物组织的处理方法及医用材料。
随着经济发展和生活水平的日益提高，生物医用材料(Biomedical Materials)作为人类同疾病做斗争的有效工具之一，受到了广泛的重视和应用。具体地，生物医用材料可用于骨、牙、关节、肌腱等骨骼-肌肉系统，用于皮肤、乳食道、呼吸道、膀胱等软组织，用于人工心脏瓣膜、血管、心血管内插管等心血管系统，用于血液净化膜和分离膜、气体选择性透过门、角膜接触镜等医用膜材等等方面。生物医用材料是研究人工器官和医疗器械的基础，已成为当代材料学科的重要分支。
生物医用材料按照材料的组成和性质可以分为生物医用金属材料、生物医用无机材料、生物医用高分子材料、生物医用复合材料、以及生物衍生材料。其中，生物衍生材料由经过特殊处理的天然生物组织形成，也被成为生物再生材料。生物组织可取自同种或异种动物体，特殊处理包括维持生物组织原有构型而进行的固定、灭菌和消除抗原性的轻微处理，以及拆散原有构型、重建新的物理形态的强烈处理。由于经过处理的生物组织已经失去生命力，因此生物衍生材料是无生命力的材料，但是由于生物衍生材料具有类似于自然组织的构型和功能，或其组成类似于自然组织，因此在人体动态过程的修复和替换中具有重要作用。
目前，临床上常用在人工心脏瓣膜、生物补片等领域的生物衍生材料取自动物源性胶原纤维组织，这类产品在植入物后易发生钙化，钙化会引起生物组织材料硬化或撕裂，从而导致生物组织材料失效。许多生物组织材料使用戊二醛和/或甲醛储存，或使用化学试剂进行交联固定处理，大量的研究证明，组织经交联后，其上残留胺基、醛基、羧基(由醛基氧化形成)等活性基团，当处理后的生物组织被植入人体时，活性基团形成钙化结合点，导致
钙化。
发明内容
本发明的目的在于提供一种生物组织的处理方法及医用材料，旨在提高经处理后的生物组织的抗钙化性能。
为实现上述目的，本发明提供了一种生物组织的处理方法，包括如下步骤：
对生物组织进行交联处理，经交联处理后的所述生物组织的表面具有活性基团；
对经交联处理的所述生物组织进行封端处理，以使至少部分的所述活性基团封闭；以及，
对经封端处理的所述生物组织进行抗氧化及灭菌处理。
可选地，所述交联剂包括戊二醛，所述活性基团包括胺基、游离醛基和游离羧基。
可选地，在对所述生物组织进行交联处理之后，以及在对经交联处理的所述生物组织进行封端处理之前，所述处理方法还包括：
对所述生物组织进行第一次清洗。
可选地，所述第一次清洗的清洗剂包括生理盐水、PH为6.8-8.6的磷酸盐缓冲液、PH为6.8-8.6的D-Hanks溶液、或PH为6.8-8.6且质量百分数为0.1％-2％的戊二醛溶液中的任一种。
可选地，所述封端处理的步骤包括：
采用第一封端溶液对经交联处理的所述生物组织进行第一次封端处理，以使至少部分的胺基封闭；
采用第二封端溶液对经第一次封端处理的所述生物组织进行第二次封端处理，以使至少部分的游离醛基封闭；以及，
采用第三封端溶液对经第二次封端处理的所述生物组织进行第三次封端处理，以使至少部分的游离羧基封闭。
可选地，所述第一封端溶液包括第一封端剂，所述第一封端剂包括肝素、甘露糖、甲醛、半乳糖中的至少一种；和/或，
所述第二封端溶液包括第二封端剂，所述第二封端剂包括硼氢化钠、牛磺酸、聚乙烯醇、氨基硫酸盐中的至少一种；和/或，
所述第三封端溶液包括第三封端剂，所述第三封端剂包括N-羟基硫代琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、胺基化合物中的至少一种。
可选地，所述第一封端溶液的PH为5.0-9.0，且在所述第一封端溶液中，所述第一封端剂的任意组份的质量百分数为0.01％-10％，所述第一次封端处理的温度为0-50℃，处理的时间为1h-72h；
所述第二封端溶液的PH为5.0-9.0，且在所述第二封端溶液中，所述第二封端剂的任意组份的质量百分数为0.01％-10％，所述第二次封端处理的温度为0-50℃处理时间为1h-72h；
所述第三封端溶液的PH为5.0-9.0，且在所述第三封端溶液中，所述第三封端剂的任意组份的质量百分数为0.01％-10％，所述第三次封端处理的温度为0-50℃处理时间为1h-72h。
可选地，在所述第一次封端处理之后，以及在所述第二次封端处理之前，所述封端处理的步骤还包括对经所述第一次封端处理的所述生物组织进行第二次清洗；
在所述第二次封端处理之后，以及在所述第三次封端处理之前，所述封端处理的步骤还包括对经所述第二次封端处理的所述生物组织进行第三次清洗；
在所述第三次封端处理之后，以及在对经封端处理的所述生物组织进行抗氧化及灭菌处理之前，所述封端处理的步骤还包括对所述生物组织进行第四次清洗。
可选地，所述第二次清洗时的清洗剂为戊二醛溶液，且戊二醛溶液的质量百分数为0.01％-2％，PH为6.8-8.6；和/或，
所述第三次清洗时的清洗剂为所述第二封端剂；和/或，
所述第四次清洗时的清洗剂为所述第三封端剂。
可选地，采用灭菌剂和抗氧化剂的混合溶液对经封端处理的所述生物组织进行抗氧化及灭菌处理，所述灭菌剂包括戊二醛或甲醛中的任一种，所述
抗氧化剂包括维生素C、维生素E、还原性糖中的至少一种，且所述抗氧化剂在所述混合溶液中的质量百分数为0.01％-10％，所述灭菌剂在所述混合溶液中的质量百分数为0.01％-10％，所述混合溶液的PH为6.8-8.6。
可选地，所述抗氧化及灭菌处理的温度为0-50℃，时间为1h-72h。
可选地，所述生物组织包括心包、心脏瓣膜、胸膜、小肠粘膜下层、硬脑膜、硬脊膜、韧带、动物皮肤中的任一种。
为实现上述目的，本发明还提供了一种医用材料，由生物组织经如前任一项所述的生物组织的处理方法处理得到。
与现有技术相比，本发明的生物组织的处理方法及医用材料具有如下优点：
前述的生物组织的处理方法包括如下步骤：对生物组织进行交联处理，经交联处理后的所述生物组织的表面具有活性基团；对经交联处理的所述生物组织进行封端处理，以使至少部分的所述活性基团封闭；以及，对经封端处理的所述生物组织进行抗氧化及灭菌处理。通过封端处理使得活性基团被封闭并失去活性，从而不能作为钙化结合点，因此当经处理后的所述生物组织植入人体后难以钙化，具有较好的抗钙化性能。
附图用于更好地理解本发明，不构成对本发明的不当限定。其中：
图1是本发明实施例一、对比例一、对比例二及对比例三的实验结果对照图；
图2是本发明实施例二所提供的医用材料、对比例四所提供的医用材料、对比例五所提供的医用材料、及对比例六所提供的医用材料在植入大鼠皮下组织8周后，各医用材料的钙含量的对照示意图；
图3是本发明实施例三所提供的医用材料、对比例七所提供的医用材料、对比例八所提供的医用材料、及对比例九所提供的医用材料在植入大鼠皮下组织8周后，各医用材料的钙含量的对照示意图；
图4是本发明实施例四所提供的医用材料、对比例十所提供的医用材料、对比例十一所提供的医用材料、及对比例十二所提供的医用材料在植入大鼠
皮下组织8周后，各医用材料的钙含量的对照示意图；
图5是本发明实施例五所提供的医用材料、对比例十三所提供的医用材料、对比例十四所提供的医用材料、及对比例十五所提供的医用材料在植入大鼠皮下组织8周后，各医用材料的钙含量的对照示意图；
图6是本发明实施例六所提供的医用材料、对比例十六所提供的医用材料、对比例十七所提供的医用材料、及对比例十八所提供的医用材料在植入大鼠皮下组织8周后，各医用材料的钙含量的对照示意图。
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。需要说明的是，本实施例中所提供的图示仅以示意方式说明本发明的基本构想，遂图式中仅显示与本发明中有关的组件而非按照实际实施时的组件数目、形状及尺寸绘制，其实际实施时各组件的型态、数量及比例可为一种随意的改变，且其组件布局型态也可能更为复杂。
另外，以下说明内容的各个实施例分别具有一或多个技术特征，然此并不意味着使用本发明者必需同时实施任一实施例中的所有技术特征，或仅能分开实施不同实施例中的一部或全部技术特征。换句话说，在实施为可能的前提下，本领域技术人员可依据本发明的公开内容，并视设计规范或实作需求，选择性地实施任一实施例中部分或全部的技术特征，或者选择性地实施多个实施例中部分或全部的技术特征的组合，借此增加本发明实施时的弹性。
如在本说明书中所使用的，单数形式“一”、“一个”以及“该”包括复数对象，复数形式“多个”包括两个以上的对象，除非内容另外明确指出外。如在本说明书中所使用的，术语“或”通常是以包括“和/或”的含义而进行使用的，除非内容另外明确指出外。对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本发明中的具体含义。
本发明的目的之一在于提供一种生物组织的处理方法，以对取自动物的
生物组织进行处理，并使得经处理后的生物组织形成为生物衍生材料，并能够应用于医疗领域。所述生物组织包括但不限于来自于动物的心包、心脏瓣膜、覆膜、胸膜、小肠粘膜下层、硬脑膜、硬脊膜、韧带、皮肤等。这里所述的动物例如是牛、猪、兔等。
所述处理方法包括如下步骤：
步骤S10：对所述生物组织进行交联处理。经交联处理后的所述生物组织的表面具有活性基团。
步骤S20：对经交联处理的所述生物组织进行封端处理，以使至少部分的所述活动基团封闭。
以及，步骤S30：对经封端处理的所述生物组织进行抗氧化及灭菌处理，以形成生物衍生材料。
通过对经交联处理的所述生物组织进行封端处理，使得处理后的所述生物组织表面残留的至少部分的活性基团失去反应活性。当所述生物衍生材料在植入患者体内时，失去反应活性的所述活性基团不能与钙结合，也就不能形成钙化点，减少甚至避免所述生物衍生材料发生钙化，提高所述生物衍生材料的抗钙化性能。
可选地，所述步骤S10中可通过醛类化合物对所述生物组织进行交联处理，在一个非限制性的实施例中，采用戊二醛溶液对所述生物组织进行交联处理。本领域技术人员知晓，所述生物组织中含有大量的蛋白质，而蛋白质由氨基酸组成，因此在利用戊二醛溶液对所述生物组织进行交联处理的过程中，戊二醛的醛基与氨基酸的胺基进行亲核加成-脱水反应，形成含有C＝N基团的Schiff碱。由于每个戊二醛分子的两端各具有一个醛基，这有可能会导致一端的醛基与胺基交联，另一端的醛基形成游离醛基，并且交联往往是不充分的，这使得生物组织中的胺基不能完全地与醛基结合，导致所述生物组织上残留胺基，此外，在交联过程中，还会存在游离醛基被氧化形成游离羧基的情况。因此，经交联处理后的所述生物组织的表面存在的活性基团至少包括胺基、游离醛基及游离羧基。若直接将经交联处理的所述生物组织植入患者体内，该生物组织表面的胺基、游离醛基和游离羧基形成钙化结合点，导致生物组织易钙化，降低使用寿命。
基于此，在所述步骤S10执行之后，所述处理方法还包括所述步骤S20，以对经交联处理后的所述生物组织的表面的胺基、游离醛基和游离羧基进行封闭，使得至少部分的所述活性基团失去反应活性。在执行所述步骤S20时，可以按照合适的顺序依次封闭胺基、游离醛基及游离羧基。如此，所述步骤S20包括如下的步骤S21、步骤S22和步骤S23，所述步骤S21为：采用第一封端溶液对经交联处理的所述生物组织进行第一次封端处理，以使至少部分的胺基封闭。所述步骤S22为：采用第二封端溶液对经第一次封端处理的所述生物组织进行第二次封端处理，以使至少部分的游离醛基封闭。所述步骤S23为：采用第三封端溶液对经所述第二次封端处理的所述生物组织进行第三次封端处理，以使至少部分的游离羧基封闭。
其中，所述第一封端溶液包括第一封端剂，所述第一封端剂包括但不限于肝素、甘露糖、甲醛、半乳糖中的至少一种，也即，所述第一封端剂包括单一组份，也可以包括两种以上的组份。所述第一封端溶液的PH为5.0-9.0，且在所述第一封端溶液中，所述第一封端剂的任意组份的质量百分数为0.01％-10％，所述第一次封端处理的温度为0-50℃，处理的时间为1h-72h。所述第二封端溶液包括第二封端剂，所述第二封端剂包括硼氢化钠、牛磺酸、聚乙烯醇、氨基硫酸盐中的至少一种，即，所述第二封端剂可包括单一组份，也可以包括两种以上的组份。所述第二封端溶液的PH为5.0-9.0，且在所述第二封端溶液中，所述第二封端剂的任意组份的质量百分数为0.01％-10％，所述第二次封端处理的温度为0-50℃处理时间为1h-72h。所述第三封端溶液包括第三封端剂，所述第三封端剂包括N-羟基硫代琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、胺基化合物中的至少一种，也就是说，所述第三封端剂可包括单一组份，也可以包括两种以上的组份。所述第三封端溶液的PH为5.0-9.0，且在所述第三封端溶液中，所述第三封端剂的任意组份的质量百分数为0.01％-10％，所述第三次封端处理的温度为0-50℃处理时间为1h-72h。需要说明的是，本文中所述及的PH均是室温下的PH。
进一步地，所述处理方法还包括第一次清洗的步骤，具体是，在所述步骤S10执行完毕之后，以及在执行所述步骤S21之前，对经交联处理后的所述生物组织进行第一次清洗。
再进一步地，所述封端处理的步骤中还包括多次清洗的步骤。具体是，在所述步骤S21执行完毕之后，以及在执行所述步骤S22之前，对经第一次封端处理的所述生物组织进行第二次清洗。在所述步骤S22执行完毕之后，以及在执行所述步骤S23之前，对经第二次封端处理的所述生物组织进行第三次清洗。在所述步骤S23执行完毕之后，以及在执行所述步骤S30之前，对经第三次封端处理的所述生物组织进行第四次清洗。
可选地，所述第一次清洗的清洗剂包括生理盐水、PH为6.8-8.6的磷酸盐缓冲液、PH为6.8-8.6的D-Hanks溶液、或者质量百分数为0.1％-2％且PH为6.8-8.6的戊二醛溶液中的任意一种。通常，所述第一次清洗在室温下进行，清洗的次数为3-5次，每次3-5min。采用戊二醛溶液作为所述第一次清洗时的清洗剂的好处是，可以对交联处理后的所述生物组织进行补充交联，使得生物组织中的胺基进一步与戊二醛的醛基结合，减少残留的胺基。
所述第二次清洗的清洗剂为戊二醛溶液，且戊二醛溶液的浓度为0.01％-2％，PH为6.8-8.6。所述第二次清洗在室温下进行，且清洗的次数为3-5次，每次3-5min。本次清洗的目的是利用戊二醛与第一次封端处理后的生物组织进一步反应，以尽可能地与残留的胺基结合，且形成游离醛基，这些游离醛基中的至少部分在第二次封端处理过程中被封闭。
所述第三次清洗的清洗剂为所述第二封端剂，所述第三次清洗在室温下进行，清洗次数为3-5次，每次3-5min。本次清洗的目的在于进一步地封闭游离醛基。需要说明的是，在所述第二次封端处理以及所述第三次清洗的过程中，至少部分的游离醛基被氧化为游离羧基。
所述第四次清洗的清洗剂为所述第三封端剂，所述第四次清洗在室温下进行，清洗次数为3-5次，每次3-5min。本次清洗的目的在于进一步地对至少部分的游离羧基进行封闭。
进一步地，在所述步骤S30中，采用灭菌剂及抗氧化剂的混合溶液对经封端处理的所述生物组织进行抗氧化及灭菌处理。可选的所述灭菌剂包括但不限于戊二醛或甲醛中的任一种。可选的抗氧化剂包括但不限于维生素C、维生素E、还原性糖中的至少一种。本领域技术人员知晓，所述还原性糖包括但不限于葡萄糖、果糖、麦芽糖、半乳糖中的至少一种。所述混合溶液的PH
为6.8-8.6，且所述抗氧化剂在所述混合溶液中的质量百分数为0.01％-10％，所述灭菌剂在所述混合溶液中的质量百分数为0.01％-10％。抗氧化及灭菌处理的温度为0-50℃，时间为1h-72h。经抗氧化及灭菌处理后，可以使得得到的生物衍生材料中残留的醛基不再被氧化，进而不产生新的钙化结合点，提高所述生物衍生材料的抗钙化性能。
进一步的，本发明实施例还提供了一种生物衍生材料，所述生物衍生材料由生物组织经前述的生物组织的处理方法处理得到。
为使本发明的目的、优点和特征更加清楚，以下结合具体实施例对本发明作进一步详细说明。需说明的是，附图均采用非常简化的形式且均使用非精准的比例，仅用以方便、明晰地辅助说明本发明实施例的目的。附图中相同或相似的附图标记代表相同或相似的部件。
<实施例一>
本实施例中所使用的生物组织为牛心包。对牛心包的处理过程如下：
首先，从屠宰厂获取新鲜的牛心包，经剥落、挑选和清洗后，采用质量百分数为0.625％的戊二醛溶液进行交联处理，处理时间为14天。之后，将经交联处理的牛心包切割(切割机的型号为Trotec Speed x-100)为50mmx50mm的样品。
之后，在室温下采用质量百分数为0.625％、PH为7.4的戊二醛溶液对所述样品进行第一次清洗，清洗次数为3次，每次3min。
接着，在室温下采用第一封端溶液对经第一次清洗后的所述样品进行第一次封端处理，处理时间为24h。所述第一封端溶液的PH为7.4，且第一封端剂为肝素，肝素的质量百分数为0.5％。需要说明的是，本文中所述及的“室温”是指室内温度，其通常为20℃±5℃。
之后，在室温下对经第一次封端处理的所述样品进行第二次清洗。所述第二次清洗的清洗剂为质量百分数为0.625％、PH为7.4的戊二醛溶液，清洗次数为三次，每次5min。
接着，在5℃下采用第二封端溶液对经第二次清洗后的所述样品进行第二次封端处理，处理时间为72h。所述第二封端溶液的PH为5.0，且第二封端剂为硼氢化钠，硼氢化钠的质量百分数为1.5％。
随后，在室温下对经第二次封端处理的所述样品进行第三次清洗。所述第三次清洗的清洗剂为所述第二封端溶液，清洗次数为3次，每次5min。
接着，在室温下采用第三封端溶液对经第三次清洗的所述样品进行第三次封端处理，处理时间为72h。所述第三封端溶液的PH为5.0，且第三封端剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的质量百分数为3.4％。
之后，在室温下对经所述第三次封端处理的所述样品进行第四次清洗。所述第四次清洗的清洗剂为所述第三封端溶剂，清洗的次数为3次，每次3min。
最后，在5℃下采用抗氧化剂及灭菌剂的混合溶液对经所述第四次清洗的所述样品进行抗氧化及灭菌处理得到生物衍生材料。抗氧化及灭菌处理的时间为72h，所述混合溶液的PH值7.4，其中，灭菌剂为戊二醛，其质量百分数为0.625％，抗氧化剂为维生素C，其质量百分数为0.5％。
<对比例一>
对比例一与实施例一的区别之处在于仅对牛心包进行交联处理和所述第一次清洗，而未对所述样品进行三次封端处理及所述第二次清洗、所述第三次清洗及所述第四次清洗。
<对比例二>
对比例二与实施例一的区别之处在于对牛心包进行交联处理、第一次清洗、第一次封端处理和所述第二次清洗，而未进行所述第二次封端处理、所述第三次清洗、所述第三次封端处理、所述第四次清洗、抗氧化及灭菌处理。
<对比例三>
对比例三与实施例一的区别之处在于对牛心包进行交联处理、第一次清洗、第一次封端处理、第二次清洗、第二次封端处理和第三次清洗，而未进行第三次封端处理、第四次清洗和抗氧化及灭菌处理。
分别对实施例一、对比例一、对比例二、及对比例三得到生物衍生材料进行分光光度检测，以检测各例中得到的生物衍生材料表面的氨基、游离醛基和游离羧基的含量，结果如图1所示。由图1可看到，对比例一得到的生物衍生材料表面的活性基团的含量为156nmol/ml，其中胺基的含量为34nmol/mg、游离醛基的含量为68nmol/mg、游离羧基的含量为54nmol/mg。对比例二得到的生物衍生材料表面的活性基团的含量为107nmol/mg，其中胺基的含量为8nmol/mg、游离醛基的含量为51nmol/mg、游离羧基的含量为48nmol/mg。对比例三得到的生物衍生材料表面的活性基团的含量为54nmol/mg，其中，胺基的含量为6nmol/mg、游离醛基的含量为7nmol/mg、游离羧基的含量为41nmol/mg。实施例一得到的生物衍生材料表面的活性基团的含量为17nmol/mg，其中胺基的含量为4nmol/mg、游离醛基的含量为8nmol/mg、游离羧基的含量为5nmol/mg。也就是说，经第一次封端处理及第二次清洗后的所述生物组织表面的胺基含量大大降低，经第二次封端处理及第三次清洗后的所述生物组织表面的游离醛基的含量大大降低，而经过三次封端处理、四次清洗且经抗氧化及灭菌处理后的所述生物组织表面的胺基、游离醛基及游离羧基的含量都大大降低。
<实施例二>
本实施例中所使用的生物组织为牛心包。对牛心包的处理过程如下：
首先，从屠宰厂获取新鲜的牛心包，经剥落、挑选和清洗后，采用质量百分数为0.625％的戊二醛溶液进行交联处理，处理时间为14天。之后，将经交联处理的牛心包切割(切割机的型号为Trotec Speed x-100)为50mmx50mm的样品。
之后，在室温下采用PH为7.4的磷酸盐缓冲溶液对所述样品进行第一次清洗，清洗次数为5次，每次5min。
接着，在室温下采用第一封端溶液对经第一次清洗后的所述样品进行第一次封端处理，处理时间为48h。所述第一封端溶液的PH为6.2，且第一封端剂为甲醛，甲醛的质量百分数为2％。
之后，在室温下对经第一次封端处理的所述样品进行第二次清洗。所述
第二次清洗的清洗剂为质量百分数为0.625％、PH为7.4的戊二醛溶液，清洗次数为3次，每次3min。
接着，在39℃下采用第二封端溶液对经第二次清洗后的所述样品进行第二次封端处理，处理时间为12h。所述第二封端溶液的PH为7.4，且第二封端剂为牛磺酸，牛磺酸的质量百分数为0.5％。
随后，在室温下对经第二次封端处理的所述样品进行第三次清洗。所述第三次清洗的清洗剂为所述第二封端溶液，清洗次数为3次，每次5min。
接着，在室温下采用第三封端溶液对经第三次清洗的所述样品进行第三次封端处理，处理时间为72h。所述第三封端溶液的PH为8.6，且第三封端剂为N-羟基硫代琥珀酰亚胺，N-羟基硫代琥珀酰亚胺的质量百分数为4.5％。
之后，在室温下对经所述第三次封端处理的所述样品进行第四次清洗。所述第四次清洗的清洗剂为所述第三封端溶液，清洗的次数为3次，每次3min。
最后，在45℃下采用抗氧化剂及灭菌剂的混合溶液对经所述第四次清洗的所述样品进行抗氧化及灭菌处理得到生物衍生材料。抗氧化及灭菌处理的时间为10h，所述混合溶液的PH为8.3，其中，灭菌剂为戊二醛，其质量百分数为2.0％，抗氧化剂为维生素C，其质量百分数为8.5％。
<对比例四>
对比例四与实施例二的区别之处在于仅对牛心包进行交联处理和所述第一次清洗，而未对所述样品进行三次封端处理及所述第二次清洗、所述第三次清洗及所述第四次清洗。
<对比例五>
对比例五与实施例二的区别之处在于对牛心包进行交联处理、第一次清洗、第一次封端处理和所述第二次清洗，而未进行所述第二次封端处理、所述第三次清洗、所述第三次封端处理、所述第四次清洗、抗氧化及灭菌处理。
<对比例六>
对比例六与实施例二的区别之处在于对牛心包进行交联处理、第一次清洗、第一次封端处理、第二次清洗、第二次封端处理和第三次清洗，而未进行第三次封端处理、第四次清洗和抗氧化及灭菌处理。
将实施例二、对比例四、对比例五及对比例六得到的生物衍生材料分别植入大鼠的皮下，8周后取出，并通过原子吸收分光光度计分别测量各生物衍生材料的钙含量，结果如图2所示。通过图2看到，未经过封端处理得到的生物衍生材料(即对比例四得到的生物衍生材料)上的钙含量最多，经过第一次封端处理得到的生物衍生材料(即对比例五得到的生物衍生材料)上的钙含量次之，经过第一次及第二次封端处理得到的生物衍生材料(即对比例六得到的生物衍生材料)上的钙含量再次之，经过三次封端处理和抗氧化及灭菌处理得到生物衍生材料(即实施例二得到的生物衍生材料)上的钙含量最少。也就是说，经过三次封端处理和抗氧化及灭菌处理得到生物衍生材料的抗钙化性能最优。
<实施例三>
本实施例中所使用的生物组织为牛心包。对牛心包的处理过程如下：
首先，从屠宰厂获取新鲜的牛心包，经剥落、挑选和清洗后，采用质量百分数为0.625％的戊二醛溶液进行交联处理，处理时间为14天。之后，将经交联处理的牛心包切割(切割机的型号为Trotec Speed x-100)为50mmx50mm的样品。
之后，在室温下采用PH为7.4的磷酸盐缓冲溶液对所述样品进行第一次清洗，清洗次数为5次，每次5min。
接着，在48℃下采用第一封端溶液对经第一次清洗后的所述样品进行第一次封端处理，处理时间为6h。所述第一封端溶液的PH为8.6，且第一封端剂为甘露糖，甘露糖的质量百分数为1％。
之后，在室温下对经第一次封端处理的所述样品进行第二次清洗。所述第二次清洗的清洗剂为质量百分数为0.625％、PH为7.4的戊二醛溶液，清洗
次数为3次，每次3min。
接着，在50℃下采用第二封端溶液对经第二次清洗后的所述样品进行第二次封端处理，处理时间为72h。所述第二封端溶液的PH为5.0，且第二封端剂为聚乙烯醇，聚乙烯醇的质量百分数为1.2％。
随后，在室温下对经第二次封端处理的所述样品进行第三次清洗。所述第三次清洗的清洗剂为所述第二封端溶液，清洗次数为3次，每次5min。
接着，在室温下采用第三封端溶液对经第三次清洗的所述样品进行第三次封端处理，处理时间为72h。所述第三封端溶液的PH为8.0，且第三封端剂为2-羟基乙胺，2-羟基乙胺的质量百分数为0.5％。
之后，在室温下对经所述第三次封端处理的所述样品进行第四次清洗。所述第四次清洗的清洗剂为所述第三封端溶液，清洗的次数为3次，每次3min。
最后，在5℃下采用抗氧化剂及灭菌剂的混合溶液对经所述第四次清洗的所述样品进行抗氧化及灭菌处理得到生物衍生材料。抗氧化及灭菌处理的时间为72h，所述混合溶液的PH为6.8，其中，灭菌剂为戊二醛，其质量百分数为0.625％，抗氧化剂为维生素E，其质量百分数为2.3％。
<对比例七>
对比例七与实施例三的区别之处在于仅对牛心包进行交联处理和所述第一次清洗，而未对所述样品进行三次封端处理及所述第二次清洗、所述第三次清洗及所述第四次清洗。
<对比例八>
对比例八与实施例三的区别之处在于对牛心包进行交联处理、第一次清洗、第一次封端处理和所述第二次清洗，而未进行所述第二次封端处理、所述第三次清洗、所述第三次封端处理、所述第四次清洗、抗氧化及灭菌处理。
<对比例九>
对比例九与实施例三的区别之处在于对牛心包进行交联处理、第一次清
洗、第一次封端处理、第二次清洗、第二次封端处理和第三次清洗，而未进行第三次封端处理、第四次清洗和抗氧化及灭菌处理。
将实施例三、对比例七、对比例八及对比例九得到的生物衍生材料分别植入大鼠的皮下，8周后取出，并通过原子吸收分光光度计分别测量各生物衍生材料的钙含量，结果如图3所示。通过图3看到，未经过封端处理得到的生物衍生材料(即对比例七得到的生物衍生材料)上的钙含量最多，经过第一次封端处理得到的生物衍生材料(即对比例八得到的生物衍生材料)上的钙含量次之，经过第一次及第二次封端处理得到的生物衍生材料(即对比例九得到的生物衍生材料)上的钙含量再次之，经过三次封端处理和抗氧化及灭菌处理得到生物衍生材料(即实施例三得到的生物衍生材料)上的钙含量最少。也就是说，经过三次封端处理和抗氧化及灭菌处理得到生物衍生材料的抗钙化性能最优。
<实施例四>
本实施例中所使用的生物组织为牛心包。对牛心包的处理过程如下：
首先，从屠宰厂获取新鲜的牛心包，经剥落、挑选和清洗后，采用质量百分数为2％的戊二醛溶液进行交联处理，处理时间为14天。之后，将经交联处理的牛心包切割(切割机的型号为Trotec Speed x-100)为50mmx50mm的样品。
之后，在室温下采用PH为7.4的磷酸盐缓冲溶液对所述样品进行第一次清洗，清洗次数为5次，每次5min。
接着，在50℃下采用第一封端溶液对经第一次清洗后的所述样品进行第一次封端处理，处理时间为2h。所述第一封端溶液的PH为8.6，且第一封端剂为半乳糖和甲醛，且半乳糖的质量百分数为9.5％，甲醛的质量百分数为3.3％。
之后，在室温下对经第一次封端处理的所述样品进行第二次清洗。所述第二次清洗的清洗剂为质量百分数为2％、PH为7.4的戊二醛溶液，清洗次数为3次，每次3min。
接着，在37℃下采用第二封端溶液对经第二次清洗后的所述样品进行第二次封端处理，处理时间为48h。所述第二封端溶液的PH为8.4，且第二封端剂为胺基葡萄糖硫酸盐，胺基葡萄糖硫酸盐的质量百分数为8.6％。
随后，在室温下对经第二次封端处理的所述样品进行第三次清洗。所述第三次清洗的清洗剂为所述第二封端溶液，清洗次数为5次，每次5min。
接着，在室温下采用第三封端溶液对经第三次清洗的所述样品进行第三次封端处理，处理时间为72h。所述第三封端溶液的PH为8.0，且第三封端剂为2-羟基乙胺，2-羟基乙胺的质量百分数为0.5％。
之后，在室温下对经所述第三次封端处理的所述样品进行第四次清洗。所述第四次清洗的清洗剂为所述第三封端溶液，清洗的次数为3次，每次3min。
最后，在5℃下采用抗氧化剂及灭菌剂的混合溶液对经所述第四次清洗的所述样品进行抗氧化及灭菌处理得到生物衍生材料。抗氧化及灭菌处理的时间为72h，所述混合溶液的PH为7.1，其中，抗氧化剂为戊二醛，其质量百分数为0.625％，灭菌剂为维生素E，其质量百分数为0.5％。
<对比例十>
对比例十与实施例四的区别之处在于仅对牛心包进行交联处理和所述第一次清洗，而未对所述样品进行三次封端处理及所述第二次清洗、所述第三次清洗及所述第四次清洗。
<对比例十一>
对比例十一与实施例四的区别之处在于对牛心包进行交联处理、第一次清洗、第一次封端处理和所述第二次清洗，而未进行所述第二次封端处理、所述第三次清洗、所述第三次封端处理、所述第四次清洗、抗氧化及灭菌处理。
<对比例十二>
对比例十二与实施例四的区别之处在于对牛心包进行交联处理、第一次
清洗、第一次封端处理、第二次清洗、第二次封端处理和第三次清洗，而未进行第三次封端处理、第四次清洗和抗氧化及灭菌处理。
将实施例四、对比例十、对比例十一及对比例十二得到的生物衍生材料分别植入大鼠的皮下，8周后取出，并通过原子吸收分光光度计分别测量各生物衍生材料的钙含量，结果如图4所示。通过图4看到，未经过封端处理得到的生物衍生材料(即对比例十得到的生物衍生材料)上的钙含量最多，经过第一次封端处理得到的生物衍生材料(即对比例十一得到的生物衍生材料)上的钙含量次之，经过第一次及第二次封端处理得到的生物衍生材料(即对比例十二得到的生物衍生材料)上的钙含量再次之，经过三次封端处理和抗氧化及灭菌处理得到生物衍生材料(即实施例三得到的生物衍生材料)上的钙含量最少。也就是说，经过三次封端处理和抗氧化及灭菌处理得到生物衍生材料的抗钙化性能最优。
<实施例五>
本实施例中所使用的生物组织为牛心包。对牛心包的处理过程如下：
首先，从屠宰厂获取新鲜的牛心包，经剥落、挑选和清洗后，采用质量百分数为2％的戊二醛溶液进行交联处理，处理时间为14天。之后，将经交联处理的牛心包切割(切割机的型号为Trotec Speed x-100)为50mmx50mm的样品。
之后，在室温下采用PH为7.4的磷酸盐缓冲溶液对所述样品进行第一次清洗，清洗次数为5次，每次5min。
接着，在50℃下采用第一封端溶液对经第一次清洗后的所述样品进行第一次封端处理，处理时间为2h。所述第一封端溶液的PH为8.6，且第一封端剂为半乳糖和甲醛，且半乳糖的质量百分数为9.5％，甲醛的质量百分数为3.3％。
之后，在室温下对经第一次封端处理的所述样品进行第二次清洗。所述第二次清洗的清洗剂为质量百分数为2％、PH为7.4的戊二醛溶液，清洗次数为3次，每次3min。
接着，在10℃下采用第二封端溶液对经第二次清洗后的所述样品进行第二次封端处理，处理时间为72h。所述第二封端溶液的PH为7.4，且第二封端剂为胺基葡萄糖硫酸盐和硼氢化钠，胺基葡萄糖硫酸盐的质量百分数为8.6％，硼氢化钠的质量百分数为2.1％。
随后，在室温下对经第二次封端处理的所述样品进行第三次清洗。所述第三次清洗的清洗剂为所述第二封端溶液，清洗次数为5次，每次5min。
接着，在室温下采用第三封端溶液对经第三次清洗的所述样品进行第三次封端处理，处理时间为72h。所述第三封端溶液的PH为8.0，且第三封端剂为2-羟基乙胺，2-羟基乙胺的质量百分数为0.5％。
之后，在室温下对经所述第三次封端处理的所述样品进行第四次清洗。所述第四次清洗的清洗剂为所述第三封端溶液，清洗的次数为3次，每次3min。
最后，在5℃下采用抗氧化剂及灭菌剂的混合溶液对经所述第四次清洗的所述样品进行抗氧化及灭菌处理得到生物衍生材料。抗氧化及灭菌处理的时间为72h，所述混合溶液的PH为6.8，其中，灭菌剂为戊二醛，其质量百分数为0.625％，抗氧化剂为维生素E，其质量百分数为2.3％。
<对比例十三>
对比例十三与实施例五的区别之处在于仅对牛心包进行交联处理和所述第一次清洗，而未对所述样品进行三次封端处理及所述第二次清洗、所述第三次清洗及所述第四次清洗。
<对比例十四>
对比例十四与实施例五的区别之处在于对牛心包进行交联处理、第一次清洗、第一次封端处理和所述第二次清洗，而未进行所述第二次封端处理、所述第三次清洗、所述第三次封端处理、所述第四次清洗、抗氧化及灭菌处理。
<对比例十五>
对比例十五与实施例五的区别之处在于对牛心包进行交联处理、第一次清洗、第一次封端处理、第二次清洗、第二次封端处理和第三次清洗，而未进行第三次封端处理、第四次清洗和抗氧化及灭菌处理。
将实施例五、对比例十三、对比例十四及对比例十五得到的生物衍生材料分别植入大鼠的皮下，8周后取出，并通过原子吸收分光光度计分别测量各生物衍生材料的钙含量，结果如图5所示。通过图5看到，未经过封端处理得到的生物衍生材料(即对比例十三得到的生物衍生材料)上的钙含量最多，经过第一次封端处理得到的生物衍生材料(即对比例十四得到的生物衍生材料)上的钙含量次之，经过第一次及第二次封端处理得到的生物衍生材料(即对比例十五得到的生物衍生材料)上的钙含量再次之，经过三次封端处理和抗氧化及灭菌处理得到生物衍生材料(即实施例三得到的生物衍生材料)上的钙含量最少。也就是说，经过三次封端处理和抗氧化及灭菌处理得到生物衍生材料的抗钙化性能最优。
<实施例六>
本实施例中所使用的生物组织为牛心包。对牛心包的处理过程如下：
首先，从屠宰厂获取新鲜的牛心包，经剥落、挑选和清洗后，采用质量百分数为2％的戊二醛溶液进行交联处理，处理时间为14天。之后，将经交联处理的牛心包切割(切割机的型号为Trotec Speed x-100)为50mmx50mm的样品。
之后，在室温下采用PH为7.4的磷酸盐缓冲溶液对所述样品进行第一次清洗，清洗次数为5次，每次5min。
接着，在50℃下采用第一封端溶液对经第一次清洗后的所述样品进行第一次封端处理，处理时间为2h。所述第一封端溶液的PH为8.6，且第一封端剂为半乳糖和甲醛，且半乳糖的质量百分数为9.5％，甲醛的质量百分数为3.3％。
之后，在室温下对经第一次封端处理的所述样品进行第二次清洗。所述第二次清洗的清洗剂为质量百分数为2％、PH为7.4的戊二醛溶液，清洗次数
为3次，每次3min。
接着，在37℃下采用第二封端溶液对经第二次清洗后的所述样品进行第二次封端处理，处理时间为48h。所述第二封端溶液的PH为8.4，且第二封端剂包括胺基葡萄糖硫酸盐和硼氢化钠，胺基葡萄糖硫酸盐的质量百分数为8.6％，硼氢化钠的质量百分数为2.1％。
随后，在室温下对经第二次封端处理的所述样品进行第三次清洗。所述第三次清洗的清洗剂为所述第二封端溶液，清洗次数为5次，每次5min。
接着，在室温下采用第三封端溶液对经第三次清洗的所述样品进行第三次封端处理，处理时间为72h。所述第三封端溶液的PH为8.0，且第三封端剂包括2-羟基乙胺和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，2-羟基乙胺的质量百分数为0.5％，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的质量百分数为7.3％。
之后，在室温下对经所述第三次封端处理的所述样品进行第四次清洗。所述第四次清洗的清洗剂为所述第三封端溶液，清洗的次数为3次，每次3min。
最后，在5℃下采用抗氧化剂及灭菌剂的混合溶液对经所述第四次清洗的所述样品进行抗氧化及灭菌处理得到生物衍生材料。抗氧化及灭菌处理的时间为72h，所述混合溶液的PH为7.4，其中，灭菌剂为戊二醛，其质量百分数为0.625％，抗氧化剂为维生素E，其质量百分数为0.5％。
<对比例十六>
对比例十六与实施例六的区别之处在于仅对牛心包进行交联处理和所述第一次清洗，而未对所述样品进行三次封端处理及所述第二次清洗、所述第三次清洗及所述第四次清洗。
<对比例十七>
对比例十七与实施例六的区别之处在于对牛心包进行交联处理、第一次清洗、第一次封端处理和所述第二次清洗，而未进行所述第二次封端处理、所述第三次清洗、所述第三次封端处理、所述第四次清洗、抗氧化及灭菌处
理。
<对比例十八>
对比例十八与实施例六的区别之处在于对牛心包进行交联处理、第一次清洗、第一次封端处理、第二次清洗、第二次封端处理和第三次清洗，而未进行第三次封端处理、第四次清洗和抗氧化及灭菌处理。
将实施例六、对比例十六、对比例十七及对比例十八得到的生物衍生材料分别植入大鼠的皮下，8周后取出，并通过原子吸收分光光度计分别测量各生物衍生材料的钙含量，结果如图6所示。通过图6看到，未经过封端处理得到的生物衍生材料(即对比例十六得到的生物衍生材料)上的钙含量最多，经过第一次封端处理得到的生物衍生材料(即对比例十七得到的生物衍生材料)上的钙含量次之，经过第一次及第二次封端处理得到的生物衍生材料(即对比例十八得到的生物衍生材料)上的钙含量再次之，经过三次封端处理和抗氧化及灭菌处理得到生物衍生材料(即实施例六得到的生物衍生材料)上的钙含量最少。也就是说，经过三次封端处理和抗氧化及灭菌处理得到生物衍生材料的抗钙化性能最优。
虽然本发明披露如上，但并不局限于此。本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
Claims (13)
- 一种生物组织的处理方法，其特征在于，包括如下步骤：对生物组织进行交联处理，经交联处理后的所述生物组织的表面具有活性基团；对经交联处理的所述生物组织进行封端处理，以使至少部分的所述活性基团封闭；以及，对经封端处理的所述生物组织进行抗氧化及灭菌处理。
- 根据权利要求1所述的生物组织的处理方法，其特征在于，所述交联剂包括戊二醛，所述活性基团包括胺基、游离醛基和游离羧基。
- 根据权利要求1或2所述的生物组织的处理方法，其特征在于，在对所述生物组织进行交联处理之后，以及在对经交联处理的所述生物组织进行封端处理之前，所述处理方法还包括：对所述生物组织进行第一次清洗。
- 根据权利要求3所述的生物组织的处理方法，其特征在于，所述第一次清洗的清洗剂包括生理盐水、PH为6.8-8.6的磷酸盐缓冲液、PH为6.8-8.6的D-Hanks溶液、或PH为6.8-8.6且质量百分数为0.1％-2％的戊二醛溶液中的任一种。
- 根据权利要求2所述的生物组织的处理方法，其特征在于，所述封端处理的步骤包括：采用第一封端溶液对经交联处理的所述生物组织进行第一次封端处理，以使至少部分的胺基封闭；采用第二封端溶液对经第一次封端处理的所述生物组织进行第二次封端处理，以使至少部分的游离醛基封闭；以及，采用第三封端溶液对经第二次封端处理的所述生物组织进行第三次封端处理，以使至少部分的游离羧基封闭。
- 根据权利要求5所述的生物组织的处理方法，其特征在于，所述第一封端溶液包括第一封端剂，所述第一封端剂包括肝素、甘露糖、甲醛、半乳糖中的至少一种；和/或，所述第二封端溶液包括第二封端剂，所述第二封端剂包括硼氢化钠、牛磺酸、聚乙烯醇、氨基硫酸盐中的至少一种；和/或，所述第三封端溶液包括第三封端剂，所述第三封端剂包括N-羟基硫代琥珀酰亚胺、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、胺基化合物中的至少一种。
- 根据权利要求6所述的生物组织的处理方法，其特征在于，所述第一封端溶液的PH为5.0-9.0，且在所述第一封端溶液中，所述第一封端剂的任意组份的质量百分数为0.01％-10％，所述第一次封端处理的温度为0-50℃，处理的时间为1h-72h；所述第二封端溶液的PH为5.0-9.0，且在所述第二封端溶液中，所述第二封端剂的任意组份的质量百分数为0.01％-10％，所述第二次封端处理的温度为0-50℃处理时间为1h-72h；所述第三封端溶液的PH为5.0-9.0，且在所述第三封端溶液中，所述第三封端剂的任意组份的质量百分数为0.01％-10％，所述第三次封端处理的温度为0-50℃处理时间为1h-72h。
- 根据权利要求5所述的生物组织的处理方法，其特征在在于，在所述第一次封端处理之后，以及在所述第二次封端处理之前，所述封端处理的步骤还包括对经所述第一次封端处理的所述生物组织进行第二次清洗；在所述第二次封端处理之后，以及在所述第三次封端处理之前，所述封端处理的步骤还包括对经所述第二次封端处理的所述生物组织进行第三次清洗；在所述第三次封端处理之后，以及在对经封端处理的所述生物组织进行抗氧化及灭菌处理之前，所述封端处理的步骤还包括对所述生物组织进行第四次清洗。
- 根据权利要求8所述的生物组织的处理方法，其特征在于，所述第二次清洗时的清洗剂为戊二醛溶液，且戊二醛溶液的质量百分数为0.01％-2％，PH为6.8-8.6；和/或，所述第三次清洗时的清洗剂为所述第二封端剂；和/或，所述第四次清洗时的清洗剂为所述第三封端剂。
- 根据权利要求1所述的生物组织的处理方法，其特征在于，采用灭菌剂和抗氧化剂的混合溶液对经封端处理的所述生物组织进行抗氧化及灭菌处理，所述灭菌剂包括戊二醛或甲醛中的任一种，所述抗氧化剂包括维生素C、维生素E、还原性糖中的至少一种，且所述抗氧化剂在所述混合溶液中的质量百分数为0.01％-10％，所述灭菌剂在所述混合溶液中的质量百分数为0.01％-10％，所述混合溶液的PH为6.8-8.6。
- 根据权利要求10所述的生物组织的处理方法，其特征在于，所述抗氧化及灭菌处理的温度为0-50℃，时间为1h-72h。
- 根据权利要求1所述的生物组织的处理方法，其特征在于，所述生物组织包括心包、心脏瓣膜、胸膜、小肠粘膜下层、硬脑膜、硬脊膜、韧带、动物皮肤中的任一种。
- 一种医用材料，其特征在于，由生物组织经如权利要求1-12中任一项所述的生物组织的处理方法处理得到。
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| US20090164005A1 (en) * | 2007-12-21 | 2009-06-25 | Edwards Lifesciences Corporation | Capping Bioprosthetic Tissue to Reduce Calcification |
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| US20140288641A1 (en) * | 2011-07-11 | 2014-09-25 | The Children's Hospital Of Philadelphia | Oxidation resistant bioprosthetic tissues and preparation thereof |
| CN114209886A (zh) * | 2021-11-09 | 2022-03-22 | 江苏臻亿医疗科技有限公司 | 一种生物组织材料及其制备方法 |
| CN114652893A (zh) * | 2020-12-23 | 2022-06-24 | 上海微创心通医疗科技有限公司 | 生物假体组织的处理方法和生物假体心脏瓣膜 |
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| US20060217804A1 (en) * | 2005-03-25 | 2006-09-28 | Dove Jeffrey S | Treatment of bioprosthetic tissues to mitigate post implantation calcification |
| US20090164005A1 (en) * | 2007-12-21 | 2009-06-25 | Edwards Lifesciences Corporation | Capping Bioprosthetic Tissue to Reduce Calcification |
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| US20140288641A1 (en) * | 2011-07-11 | 2014-09-25 | The Children's Hospital Of Philadelphia | Oxidation resistant bioprosthetic tissues and preparation thereof |
| CN114652893A (zh) * | 2020-12-23 | 2022-06-24 | 上海微创心通医疗科技有限公司 | 生物假体组织的处理方法和生物假体心脏瓣膜 |
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