CN110193095B - A kind of dry biological tissue material and preparation method thereof - Google Patents
A kind of dry biological tissue material and preparation method thereof Download PDFInfo
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- CN110193095B CN110193095B CN201810164768.0A CN201810164768A CN110193095B CN 110193095 B CN110193095 B CN 110193095B CN 201810164768 A CN201810164768 A CN 201810164768A CN 110193095 B CN110193095 B CN 110193095B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/10—Preservation of living parts
-
- 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
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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
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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
- A61L27/3683—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
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- A—HUMAN NECESSITIES
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- 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
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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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
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Abstract
本发明提供了一种干态生物组织材料及其制备方法，制备方法包括如下步骤：S1：清洗经过交联处理的生物组织；S2：将S1处理后的生物组织利用玻璃化溶液进行平衡；S3：将S2处理后的生物组织进行冷冻干燥处理；S4：将S3处理后的生物组织密封包装后进行灭菌，得到干态生物组织材料。本发明提供了一种干态生物组织材料及其制备方法，通过玻璃化溶液平衡及冷冻干燥的处理方式，使得生物组织材料中的水溶液在冷却处理时保持玻璃态，不会形成冰晶，避免了对胶原纤维结构的破坏，对组织的三维结构有较好的保护作用，该方法得到的生物组织形态和抗拉强度不会发生显著变化。
The invention provides a dry biological tissue material and a preparation method thereof. The preparation method includes the following steps: S1: cleaning the biological tissue after cross-linking treatment; S2: balancing the biological tissue after the S1 treatment with a vitrification solution; S3 : freeze-drying the biological tissue processed by S2; S4: sterilize the biological tissue processed by S3 in a sealed package to obtain a dry biological tissue material. The invention provides a dry-state biological tissue material and a preparation method thereof. Through vitrification solution equilibration and freeze-drying treatment methods, the aqueous solution in the biological tissue material can be kept in a glassy state during cooling treatment, and ice crystals will not be formed, thereby avoiding the need for The destruction of the collagen fiber structure has a good protective effect on the three-dimensional structure of the tissue, and the biological tissue morphology and tensile strength obtained by this method will not change significantly.
Description
Technical Field
The invention belongs to the field of medical instruments, and relates to a dry biological tissue material and a preparation method thereof.
Background
Currently, biological tissue materials for biological tissues used clinically, such as biological valves, biological patches, and the like, are generally subjected to a cross-linking fixation treatment using a chemical agent such as glutaraldehyde and/or formaldehyde, and stored in a diluted aqueous solution containing glutaraldehyde and/or formaldehyde so as to maintain a sterile environment and a hydrated state. However, a number of studies have demonstrated that biological tissues cross-linked with glutaraldehyde have the following drawbacks: glutaraldehyde has high toxicity, even low residual quantity can generate great influence on human body, and is not beneficial to the formation of biological tissue endothelialization; after the biological tissue is implanted into a human body, residual glutaraldehyde of the biological tissue can cause an immune response of the biological tissue, and the immune response is one of important reasons for the decay of the biological tissue. Therefore, the biological tissue with residual glutaraldehyde reagent must be washed several times before being implanted into the human body. This procedure also brings great inconvenience to the surgeon, while also exposing the medical staff to the glutaraldehyde environment.
In addition, preoperative medical personnel should minimize the preparation of medical instruments exposed to the body during implantation of biological tissue materials or bioprostheses made therefrom, since the use of "ready-to-use" tissue and prostheses not only reduces the chance of contamination or error, but also reduces implantation time. Therefore, the development of a dry biological tissue material not only has wide development prospect, but also has important practical value.
Patent document CN1245222C describes a method for preparing a compliant dehydrated tissue for implantation by first immersing the tissue in a gradient-increasing polar organic solution and then treating it with a solution containing glycerol or an aqueous solution of glycerol or a low molecular weight (<1000D) polyethylene glycol and 6000-15000D polyethylene glycol and heparin. The tissue is then simply immersed in an aqueous heparin solution and frozen and dried. However, this dehydration process significantly reduces the overall size of the tissue and the chemicals used (e.g., acetonitrile, acetone, etc.) are toxic. In addition, the biological tissue of this dehydration process cannot be successfully rehydrated and restored to its original dimensions.
Patent document CN101626682B relates to a biological tissue for surgical implantation, which is contacted with a non-aqueous treatment solution comprising a polyhydric alcohol and a C1-C3 alcohol and a part of the treatment solution is removed from the solution-treated biological tissue, leaving the tissue in a substantially dry state. However, this method requires a long treatment time and a large amount of solution, which is disadvantageous in simplifying the operation and reducing the cost. In addition, the residual moisture content in the "substantially dry" tissue cannot be controlled well by using a single alcohol dehydration treatment, and if the moisture content is more than 30%, the EO sterilization (ethylene oxide sterilization) effect is affected, so that the sterilization is incomplete.
The Pitombo subject group of the university of Saint Paul, Brazil discloses a low-temperature freeze drying treatment method for biological tissues, namely, water molecules in animal source tissues are firstly frozen to-80 ℃ to-50 ℃ to form ice crystals, then, the ice crystals are vacuumized, the frozen ice crystals are sublimated into gas state under the condition of keeping low pressure, the animal source tissues become dry, and meanwhile, glutaraldehyde residues are reduced in the process. However, since the density of ice is higher than that of water, when water is frozen into ice crystals during the freezing process, the volume of the ice becomes larger, and the collagen fiber tissue structure is destroyed. Therefore, the mechanical properties of the treated biological tissue are poor.
In conclusion, the existing biological tissue preparation and storage methods (such as aldehyde solution preservation) have the defects of short service life of the biological tissue, influence on the working environment of medical workers when the biological tissue is used, inconvenience in operation, increase in operation time and the like. Although the dry biological tissue material obtained by the dehydration treatment process of polyhydric alcohols such as glycerin has certain advantages, the dry biological tissue material still needs to be further improved in the aspects of biological tissue morphological change, residual reagent, rehydration performance, control of tissue water content and the like.
Disclosure of Invention
The invention aims to provide a dry biological tissue material and a preparation method thereof, and aims to solve at least one of the problems that the biological tissue prepared in the prior art is inconvenient to store and use, the water content is difficult to control, the biological safety is low, the mechanical property of the treated biological tissue material is reduced, and the like.
In order to solve at least one of the above technical problems, the present invention provides a method for preparing a dry biological tissue material, comprising:
step S1: cleaning the crosslinked biological tissue;
step S2: balancing the biological tissue treated in the step S1 by using a vitrification solution;
step S3: freeze-drying the biological tissue processed in the step S2;
step S4: and (4) sealing and packaging the biological tissue processed in the step S3, and then sterilizing to obtain the dry biological tissue material.
Further, the biological tissue subjected to the cross-linking treatment in step S1 is a xenogeneic or allogeneic biological tissue cross-linked by a cross-linking agent.
Further, the biological tissue subjected to the cross-linking treatment is pericardium, heart valve, tectorial membrane, pleura, small intestine submucosa, dura mater, ligament or skin.
Further, in the step S1, the crosslinking agent used in the crosslinking treatment is selected from one or more of glutaraldehyde, genipin, procyanidine, carbodiimide, and N-hydroxysuccinimide.
Further, the step S1 specifically includes:
step S11: cleaning the biological tissue subjected to the cross-linking treatment by using a cleaning solution;
step S12: and cleaning the biological tissue cleaned in the S11 by using a vitrification solution.
Further, the cleaning solution is selected from one or a combination of more of normal saline, phosphate buffer solution with the pH value of 6.8-8.6 and D-Hanks solution with the pH value of 6.8-8.6.
Further, in the step S2, the equilibration time for equilibration with the vitrification solution is 2 to 24 hours.
Further, the vitrification solution is an aqueous solution of a cryoprotectant with the mass percentage of 50-80%.
Further, the cryoprotectant comprises one or a combination of two of an osmotic cryoprotectant and an impermeable cryoprotectant.
Further, the osmotic cryoprotectant is selected from one or more of glycerol, dimethyl sulfoxide, ethylene glycol, propylene glycol, acetamide and methanol.
Further, the non-permeable cryoprotectant is selected from one or more of polyvinylpyrrolidone, sucrose, trehalose, polyethylene glycol, dextran, albumin and hydroxyethyl starch.
Further, the step S3 specifically includes:
step S31: cooling the biological tissue processed in step S2 by cooling in a refrigerant;
step S32: and (4) performing freeze drying treatment on the biological tissue processed in the step S31.
Further, the refrigerant is a dry ice bath, a liquid nitrogen bath, or a low temperature bath formed by mixing dry ice or liquid nitrogen with volatile liquid.
Further, the volatile liquid is selected from one or a combination of more of ethanol, acetone, diethyl ether, chloroform and acetonitrile.
Further, in step S31, when the biological tissue is cooled by being put into a coolant, the cooling temperature is-120 ℃ to-70 ℃.
Further, the step S32 specifically includes: vacuumizing the biological tissue processed in the step S31, wherein the vacuum degree is 0.5-3 kpa; heating to-40-0 ℃, wherein the heating rate is 0-40 ℃/h, and the vacuum degree is controlled to be 0.01-0.1 kpa; the whole vacuum drying time is 4-12 h; the temperature is restored to the room temperature, the temperature restoring speed is 40 ℃/h-80 ℃/h, and the air pressure is restored to the normal pressure.
Furthermore, the time required for vacuumizing to the vacuum degree of 0.5-3 kpa is 15-45 min, the time required for vacuumizing to the vacuum degree of 0.01-0.1 kpa is 20-50 min, and the time required for returning to the normal pressure is 0.5-10 h.
Further, in step S4, the biological tissue processed in step S3 is placed in a dry and clean container in an environment with a relative humidity of less than 30% or an inert environment for sealing and packaging.
Further, in step S4, the sterilization mode is ethylene oxide sterilization, electron beam radiation sterilization or gamma ray radiation sterilization.
The invention also provides a dry biological tissue material which is prepared by the preparation method.
Compared with the prior art, the invention provides a dry biological tissue material and a preparation method thereof, by adopting the processing modes of vitrification solution balance and freeze drying, the water solution in the biological tissue material keeps a glass state during cooling treatment, ice crystals can not be formed, the damage to a collagen fiber structure is avoided, the three-dimensional structure of the tissue is well protected, and the biological tissue shape and the tensile strength obtained by the method can not be obviously changed.
In addition, the dry biological tissue material prepared by the method has good biocompatibility, high biological safety and no toxic reagent residue, reduces the risk of tissue calcification, controls the water content of the tissue to be between 15 and 30 percent, and can be sterilized by a conventional sterilization method subsequently.
In addition, the preparation method of the dry biological tissue material is simple and rapid, and the prepared dry biological tissue and the corresponding prosthesis can be used as soon as being taken, so that the contamination or error probability can be reduced, and the operation time can be shortened. When in clinical operation, the rehydration speed in the physiological saline is high, and the original hydration state can be recovered within about 15 to 20 minutes generally.
Drawings
FIG. 1 is a schematic flow chart of a method for preparing a dry biological tissue material according to an embodiment of the present invention;
FIG. 2 is a staining chart of dried bovine pericardium tissue prepared in example 1 according to the present invention after histological staining experiments;
FIG. 3 is a staining pattern of dried porcine pericardium tissue prepared in example 2 of the present invention after histological staining experiments.
Detailed Description
The dry biological tissue material and the preparation method thereof according to the present invention will be further described in detail with reference to the accompanying drawings and specific examples. Advantages and features of the present invention will become apparent from the following description and from the claims. It is to be noted that the drawings are in a very simplified form and are provided solely for the purpose of facilitating and distinctly facilitating the description of the embodiments of the present invention.
The features of the inventive concept and methods of accomplishing the same may be understood more readily by reference to the following detailed description of the embodiments and the accompanying drawings. The inventive concept may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
FIG. 1 is a schematic flow chart of a method for preparing a dry biological tissue material according to an embodiment of the present invention. Referring to fig. 1, a method for preparing a dry biological tissue material according to the present application includes:
step S1: cleaning the crosslinked biological tissue;
step S2: balancing the biological tissue treated in the step S1 by using a vitrification solution;
step S3: freeze-drying the biological tissue processed in the step S2;
step S4: and (4) sealing and packaging the biological tissue processed in the step S3, and then sterilizing to obtain the dry biological tissue material.
In the present invention, the biological tissue subjected to the cross-linking treatment in step S1 is a xenogenic or allogeneic biological tissue, such as pericardium, heart valve, covering membrane, pleura, small intestine submucosa, dura mater, ligament or skin, which is subjected to the cross-linking treatment by the cross-linking agent.
In the present invention, the crosslinking agent used in the crosslinking treatment in step S1 is not particularly limited. Preferably, the cross-linking agent may be selected from one or more combinations of glutaraldehyde, genipin, procyanidins, carbodiimide, and N-hydroxysuccinimide.
In some preferred embodiments, the step S1 may specifically include: step S11: cleaning the biological tissue subjected to the cross-linking treatment by using a cleaning solution; step S12: and cleaning the biological tissue cleaned in the step S11 by using a vitrification solution.
The cleaning solution can be one or a combination of more of normal saline, phosphate buffer solution with the pH value of 6.8-8.6 and D-Hanks solution with the pH value of 6.8-8.6. When cleaning is carried out by adopting the cleaning solution, the biological tissue is cleaned for 3-5 times, and each time is cleaned for 3-5 minutes. When the vitrification solution is adopted for cleaning, the biological tissue is cleaned for 3-5 times, and each time is cleaned for 1-10 minutes.
In some preferred embodiments, the step S2, the equilibration time with the vitrification solution is 2h to 24 h.
In a preferred embodiment of the present invention, the step S12 is performed by using a vitrification solution for cleaning, and the step S2 is performed by using a vitrification solution for balancing, so that the cryoprotectant in the vitrification solution can more fully penetrate into the biological tissue, and the formation of ice crystals in the aqueous solution inside the biological tissue during the subsequent cooling process can be avoided.
In the invention, the vitrification solution is an aqueous solution of cryoprotectant with the mass percent of 50-80%. Wherein, the cryoprotectant can be selected from one or two combinations of osmotic cryoprotectant and non-osmotic cryoprotectant. Preferably, the osmotic cryoprotectant is selected from the group consisting of glycerol, DMSO (dimethyl sulfoxide), ethylene glycol, propylene glycol, acetamide, and methanol in one or more combinations. Preferably, the non-osmotic cryoprotectant is selected from one or more combinations of polyvinylpyrrolidone (PVP), sucrose, trehalose, polyethylene glycol, dextran, albumin and hydroxyethyl starch. The present invention is not particularly limited with respect to the ratio between the plurality of cryoprotectants in the aqueous solution containing the plurality of cryoprotectants.
In the freeze-drying process according to some preferred embodiments of the present invention, the step S3 specifically includes: step S31: putting the biological tissue treated in the step S2 into a refrigerant to cool for 1-5 min; step S32: and (4) performing freeze drying treatment on the biological tissue processed in the step S31. The refrigerant is preferably a dry ice bath, a liquid nitrogen bath, or a low-temperature bath in which dry ice or liquid nitrogen is mixed with a volatile liquid. Further preferably, the volatile liquid is selected from one or a combination of ethanol, acetone, diethyl ether, chloroform and acetonitrile. Preferably, in step S31, the cooling temperature is-120 ℃ to-70 ℃ when the biological tissue is put into the coolant to be cooled. In practice, a low temperature thermometer may be used to monitor the cooling temperature.
After the rapid cooling process of step S31, the aqueous solution contained in the biological tissue material is in a glass state between liquid state and solid state, and the glass state aqueous solution can maintain its own form but cannot flow like liquid, and has the following characteristics: the molecules are amorphous and not arranged according to a lattice structure. The physical properties of the glassy state are different from those of the solid state and the liquid state, so that the damage of cooling to tissues can be avoided or reduced.
In step S32, when the biological tissue cooled in step S31 is freeze-dried, the biological tissue is first vacuumized, and the degree of vacuum (i.e., the difference between the standard atmospheric pressure and the absolute pressure) is controlled to be 0.5kpa to 3kpa, preferably 15 to 45 min; then heating to-40-0 ℃, wherein the heating rate is 0-40 ℃/h, the vacuum degree is controlled to be maintained at 0.01-0.1 kpa, and the preferential vacuum-pumping time is 20-50 min. The whole vacuum drying time is 4-12 h. After drying, controlling the temperature of the freeze drying system to re-warm to room temperature, wherein the re-warming rate is 40 ℃/h-80 ℃/h, the air pressure is slowly restored to normal pressure, and the time required by re-pressing is preferably 0.5 h-10 h. The water content of the biological tissue after the drying is finished can be controlled between 15 percent and 30 percent.
In some preferred embodiments of the present invention, a freeze-drying machine may be used to perform freeze-drying treatment, the freeze-drying machine is pre-cooled to-100 ℃ to-40 ℃, and then the temperature change rate, vacuum degree and drying time of the biological tissue are controlled by controlling the program of the freeze-drying machine, and the rewarming rate is increased as much as possible, so that the water content of the tissue is controlled to be 15% -30%, and an optimal drying state is achieved.
The inventors have found that biological tissue undergoes "devitrification" by absorbing heat from the surroundings during the warming process after freeze-drying. And the damage of ice crystals generated in the process of devitrification to tissues is larger than the damage of ice crystals formed in the process of cooling to cells and tissues, so that the ice crystals must be reheated at a certain rate to avoid devitrification in the process of reheating. After a great deal of experiments, the inventor finds that the higher rewarming rate can increase the temperature for removing the glass so as to inhibit the growth of the ice crystal, and the high rewarming rate can even completely avoid the occurrence of the ice crystal.
In some preferred embodiments of the present invention, in step S4, the sealing and packaging process is as follows: the biological tissue processed in step S3 is placed in a dry and clean container (e.g., a bottle or a packaging bag) in an environment with a relative humidity of less than 30% or an inert environment for sealed packaging. The sterilization mode in step S4 may be EO sterilization (ethylene oxide sterilization), electron beam radiation sterilization, or gamma ray radiation sterilization.
The invention also provides a dry biological tissue material which is prepared by the preparation method.
For a further understanding of the present invention, preferred embodiments of the present invention will be described below with reference to more detailed embodiments to highlight the features and characteristics of a dry biological tissue material and a method for preparing the same provided by the present invention. The description is only intended to illustrate the features and advantages of the method of the invention, and not to limit the scope of protection of the invention. In the following examples, unless otherwise specified, "solutions" refer to aqueous solutions.
Example 1
In this embodiment, the biological tissue material is selected from bovine pericardial tissue, and the preparation method of the dry biological tissue material is specifically as follows:
step 1.1: bovine pericardial tissue was harvested at a local slaughter house, stripped of fat, trimmed, and washed, and then fixed for 5 days by cross-linking with 0.625% glutaraldehyde (Sigma-Aldrich co.llc.) solution. Cutting the cross-linked and fixed bovine pericardium into small pieces (15mm x 15mm), washing the small pieces of the bovine pericardium tissue at room temperature by using normal saline (Huaren pharmaceutical industry Co., Ltd.), and washing for 3 times, wherein each time is 3-5 minutes.
Step 1.2: immersing the bovine pericardium in a glycerol solution with the mass percent of 50% to clean for 3 times, wherein each time lasts for 5-10 minutes;
step 1.3: taking out the tissue treated in the step 1.2, and transferring the tissue into a trehalose solution with the mass percent of 60% for balancing for 12 hours;
step 1.4: taking out the tissue treated in the step 1.3, putting the tissue into a dry ice-ether bath for cooling for 3min, and monitoring by using a low-temperature thermometer at the temperature of about-100 +/-5 ℃;
step 1.5: taking out the tissue treated in the step 1.4, and performing freeze-drying treatment by using a freeze dryer (a trial freeze dryer Epsilon 2-6D in Christ, Germany); the initial environment in the freeze-dryer was 20 ℃ at room temperature and atmospheric pressure. Precooling before use, reducing the ambient temperature in a dryer to-40 ℃, then quickly putting the biological tissue treated in the step 1.4 into a freeze dryer for vacuumizing, keeping the temperature at (-40 ℃) for 2 hours, controlling the vacuum degree in the freeze dryer to be 0.832 +/-0.2 kpa, and ensuring that the time for reaching the vacuum degree through vacuumizing at normal pressure is about 30 min. Then heating to-20 deg.C at a rate of 10 deg.C/h, maintaining the heating process for 2h, simultaneously vacuumizing while maintaining the vacuum degree of 0.021 + -0.005 kpa in the freeze dryer, and vacuumizing for 40min to reach the vacuum degree. The total vacuum drying time was 4 h. Then, the temperature is raised to 20 ℃ at the speed of 40 ℃/h, the pressure in the freeze dryer is gradually changed to the normal pressure, and the re-pressing time is 10 h.
Step 1.6: placing the bovine pericardium into a dialysis bag in a dry and clean environment
Dupont china group ltd), sealing, EO sterilizing.
The indexes of the dry bovine pericardium tissue prepared by the method, such as water content, rehydration time, cytotoxicity and the like, are measured to evaluate the performance indexes. Wherein the prepared dry bovine pericardial tissue has a water content of 28.3 + -1.5% (measured by Mettler-Tollido Karl Fischer moisture meter, the same applies below), a rehydration time of 16.07 + -0.55 minutes, and a cytotoxicity of grade 1.
The dried bovine pericardial tissue prepared in this example was hydrated, and the shape and size, hot-crimping temperature, maximum tensile strength and other indicators after hydration were compared with those before treatment, and the results are shown in table 1 below.
TABLE 1 comparison of relevant indices before and after bovine pericardial treatment
The bovine pericardial tissue prepared by the method is subjected to a Histological (HE) staining experiment, as shown in fig. 2, the result shows that the fibers in the bovine pericardial tissue are relatively consistent in direction, good in continuity and wavy, and the collagen fiber structure in the pericardium is not damaged. Therefore, the dry bovine pericardium obtained by the preparation method in example 1 can maintain the original structure and state of the tissue.
Example 2
In this embodiment, the biological tissue material is selected from porcine pericardium tissue, and the preparation method of the dry biological tissue material is specifically as follows:
step 2.1: obtaining porcine pericardium tissues in a local slaughter house, peeling off fat, trimming and cleaning the porcine pericardium tissues, then crosslinking and fixing the porcine pericardium tissues by using N-hydroxysuccinimide, specifically, preparing a crosslinking solution by using PBS (phosphate buffer solution) with the pH value of 5.5, wherein the concentration of the N-hydroxysuccinimide is 0.1mol/L, and the porcine pericardium and the crosslinking solution are mixed in a ratio of 8:1 (W/V); crosslinking for 16h at 20 ℃ with shaking at 400 r/min; then washed with shaking for 10min 8 times. Cutting the fixed pig heart bags into small pieces (30mm x 50mm), and then cleaning the cross-linked and fixed pig heart bags with physiological saline at room temperature for 3-5 times, wherein the cleaning time is 3-5 minutes each time.
Step 2.2: immersing the pig heart bag in 65% glycol solution by mass, and cleaning for 5 times, wherein each time is 5-10 minutes;
step 2.3: taking out the tissue treated in the step 2.2, and transferring the tissue into a trehalose solution with the mass percent of 30% and a sucrose solution with the mass percent of 50% for balancing for 6 hours;
step 2.4: taking out the tissue processed in the step 3, putting the tissue into liquid nitrogen-ethanol for cooling for 2.5min, and monitoring by adopting a low-temperature thermometer at the temperature of about-110 +/-5 ℃;
step 2.5: taking out the tissue treated in the step 2.4, and performing freeze-drying treatment by using a freeze dryer (a trial freeze dryer Epsilon 2-6D in Christ, Germany); the initial environment in the freeze-dryer was 22 ℃ at room temperature and atmospheric pressure. Precooling before use, reducing the ambient temperature in a dryer to-70 ℃, then quickly putting the biological tissue processed by S4 into a freeze dryer for vacuumizing, keeping the temperature at (-70 ℃) for 4 hours, controlling the vacuum degree in the freeze dryer to be 1.512 +/-0.5 kpa, and ensuring that the time for reaching the vacuum degree through vacuumizing at normal pressure is about 20 min. Then heating to-40 deg.C at a rate of 20 deg.C/h, maintaining the heating process for 1.5h, simultaneously vacuumizing while maintaining the vacuum degree of 0.065 + -0.005 kpa in the freeze dryer, and vacuumizing for 30min to reach the vacuum degree. The whole system is maintained for 3 hours at the temperature of minus 40 ℃ and the vacuum degree of 0.065 +/-0.005 kpa. Therefore, the total vacuum drying time was 8.5 h. Then, the temperature is raised to 20 ℃ at the speed of 60 ℃/h, the pressure in the freeze dryer is gradually changed to the normal pressure, and the re-pressing time is 8 h. Step 2.6: placing the biological tissue pig heart bag into a dialysis bag in a dry nitrogen-containing environment, sealing, and sterilizing EO.
The indexes of water content, rehydration time, cytotoxicity and the like of the dry-state pig heart tissue prepared by the method are measured so as to evaluate the performance indexes of the dry-state pig heart tissue. Wherein, the water content of the prepared dry porcine pericardium tissue is 29.3 +/-2.1%, the rehydration time is 15.07 +/-0.69 minutes, and the cytotoxicity is grade 1.
The dried porcine pericardium tissue prepared in this example was hydrated and the shape and size, hot-wrinkle temperature, maximum tensile strength and other indicators after hydration were compared with those before treatment, and the results are shown in table 2 below.
TABLE 2 comparison of relevant indices of swine pericardium before and after treatment and hydration
The porcine pericardium tissue prepared by the method is subjected to a Histological (HE) staining experiment, as shown in fig. 3, the result shows that the fiber directions in the dry porcine pericardium tissue are relatively consistent, the continuity is good, the dry porcine pericardium tissue is wavy, and the collagen fiber structure in the dry porcine pericardium is not damaged. Therefore, the dry porcine pericardium obtained by the preparation method in the embodiment 2 can keep the original structure and state of the tissue.
Example 3
In this embodiment, the biological tissue material is selected from bovine pericardial tissue, and the preparation method of the dry biological tissue material is specifically as follows:
step 3.1: obtaining bovine pericardium tissues in a local slaughter house, peeling, trimming and cleaning the bovine pericardium tissues, then crosslinking and fixing the bovine pericardium tissues by using carbodiimide, and specifically, preparing a crosslinking solution by using PBS (phosphate buffer solution) with the pH value of 5.5, wherein the concentration of the carbodiimide is 0.1mol/L, and the porcine small intestine mucosa and the crosslinking solution are mixed according to the proportion of 8:1 (W/V); crosslinking for 16h at 20 ℃ with shaking at 400 r/min; then washed with shaking for 10min 8 times. Cutting the fixed bovine pericardial tissue into small pieces (30mm x 50mm), and then cleaning the crosslinked and fixed bovine pericardial tissue with physiological saline at room temperature for 3-5 times, wherein each time is 3-5 minutes.
Step 3.2: immersing the bovine pericardium tissue in a mixed solution of 50% by mass of glycol and 30% by mass of glycerol, and cleaning for 3 times, wherein each time is 5-10 minutes;
step 3.3: taking out the tissue treated in the step 3.2, and transferring the tissue into a sucrose solution with the mass percent of 70% for balancing for 2 hours;
step 3.4: taking out the tissue treated in the step 3.3, putting the tissue into dry ice-acetone for cooling for 3.5min, and monitoring by using a low-temperature thermometer at the temperature of about-78 +/-5 ℃;
step 3.5: taking out the tissue treated in the step 3.4, and performing freeze-drying treatment by using a freeze dryer (a trial freeze dryer Epsilon 2-6D in Christ, Germany); the initial environment in the freeze-dryer was 22 ℃ at room temperature and atmospheric pressure. Precooling before use, reducing the ambient temperature in a dryer to-100 ℃, then quickly putting the biological tissue processed by S4 into a freeze dryer for vacuumizing, keeping the temperature at (-100 ℃) for 1h, controlling the vacuum degree in the freeze dryer to be 2.802 +/-0.5 kpa, and ensuring that the time for reaching the vacuum degree through vacuumizing at normal pressure is about 20 min. Then heating to-10 ℃ at the speed of 40 ℃/h, continuously heating for 2.25h, simultaneously vacuumizing in the heating process, keeping the vacuum degree in the freeze dryer at 0.091 +/-0.005 kpa, and keeping the time for reaching the vacuum degree by vacuumizing for 35 min. The vacuum is kept for 8h under the conditions of the temperature and the pressure (-10 ℃, 0.091 +/-0.005 kpa). The total vacuum drying time was 11.25 h. Then, the temperature is raised to 20 ℃ at the speed of 60 ℃/h, the pressure in the freeze dryer is gradually changed to the normal pressure, and the re-pressing time is 7 h.
Step 3.6: in a dry nitrogen-containing environment, the bovine pericardium tissue of the biological tissue is put into a dialysis bag, sealed and sterilized by EO.
The indexes of the dry bovine pericardium tissue prepared by the method, such as water content, rehydration time, cytotoxicity and the like, are measured to evaluate the performance indexes. Wherein, the water content of the prepared dry bovine pericardium tissue is 25.3 +/-1.5%, the rehydration time is 18.07 +/-0.61 minutes, and the cytotoxicity is grade 1.
The dried bovine pericardial tissue prepared in this example was hydrated, and the shape and size, hot-crimping temperature, maximum tensile strength and other indicators after hydration were compared with those before treatment, and the results are shown in table 3 below.
TABLE 3 comparison of relevant indices before and after bovine pericardial treatment
In conclusion, the invention provides a preparation method of a dry biological tissue material, which is characterized in that the solution in the biological tissue material keeps a glass state during cooling through a vitrification solution balance and freeze drying treatment mode, so that ice crystals are not formed, the damage to a collagen fiber structure is avoided, the three-dimensional structure of the tissue is well protected, and the biological tissue shape and the tensile strength obtained by the method are not remarkably changed.
In addition, the dry biological tissue material prepared by the method has good biocompatibility, high biological safety and no toxic reagent residue, reduces the risk of tissue calcification, controls the water content of the tissue to be between 15 and 30 percent, and is beneficial to subsequent sterilization.
In addition, the dry biological tissue material and the corresponding prosthesis prepared by the invention can be used as soon as being taken, so that the probability of contamination or error can be reduced, and the operation time can be shortened. When in clinical operation, the rehydration speed in the physiological saline is high, and the original hydration state can be recovered within about 15 to 20 minutes generally.
It should be noted that, in the present specification, the embodiments are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments may be referred to each other.
The above description is only for the purpose of describing the preferred embodiments of the present invention, and is not intended to limit the scope of the present invention, and any variations and modifications made by those skilled in the art based on the above disclosure are within the scope of the appended claims.
Claims (19)
1. A method of preparing a dry biological tissue material, comprising:
step S1: cleaning the crosslinked biological tissue;
step S2: balancing the biological tissue treated in the step S1 by using a vitrification solution;
step S3: freeze-drying the biological tissue processed in the step S2;
step S4: sealing and packaging the biological tissue processed in the step S3, and then sterilizing to obtain the dry biological tissue material;
the step S3 specifically includes:
step S31: cooling the biological tissue processed in step S2 by cooling in a refrigerant;
step S32: freeze-drying the biological tissue processed in the step S31;
in step S32, after the drying is finished, the temperature of the freeze drying system is controlled to re-warm to room temperature, and the re-warming rate is 40 ℃/h to 80 ℃/h.
2. The method according to claim 1, wherein the biological tissue after cross-linking treatment in step S1 is xenogeneic or allogeneic biological tissue after cross-linking treatment with a cross-linking agent.
3. The method for preparing a dry biological tissue material according to claim 2, wherein the biological tissue subjected to the cross-linking treatment is pericardium, heart valve, tectorial membrane, pleura, small intestine submucosa, dura mater, ligament or skin.
4. The method for preparing a dry biological tissue material according to claim 1, wherein the step S1 is performed by using a cross-linking agent selected from one or more of glutaraldehyde, genipin, procyanidins, carbodiimide, and N-hydroxysuccinimide.
5. The method for preparing a dry biological tissue material according to claim 1, wherein the step S1 specifically comprises:
step S11: cleaning the biological tissue subjected to the cross-linking treatment by using a cleaning solution;
step S12: and cleaning the biological tissue cleaned in the S11 by using a vitrification solution.
6. The method for preparing a dry biological tissue material according to claim 5, wherein the cleaning solution is selected from one or more of normal saline, phosphate buffer solution with pH value of 6.8-8.6 and D-Hanks solution with pH value of 6.8-8.6.
7. The method of claim 1, wherein the step S2, the equilibration time with vitrification solution is 2-24 h.
8. The method for preparing a dry biological tissue material according to any one of claims 1 to 7, wherein the vitrification solution is an aqueous solution of cryoprotectant in an amount of 50 to 80% by mass.
9. The method of claim 8, wherein the cryoprotectant comprises one or a combination of two of a permeable cryoprotectant and an impermeable cryoprotectant.
10. The method of claim 9, wherein the osmotic cryoprotectant is selected from the group consisting of glycerol, dimethyl sulfoxide, ethylene glycol, propylene glycol, acetamide, and methanol.
11. The method of claim 9, wherein the non-osmotic cryoprotectant is selected from the group consisting of polyvinylpyrrolidone, sucrose, trehalose, polyethylene glycol, dextran, albumin and hydroxyethyl starch.
12. The method of claim 1, wherein the refrigerant is a dry ice bath, a liquid nitrogen bath, or a low temperature bath of dry ice or a mixture of liquid nitrogen and a volatile liquid.
13. The method according to claim 12, wherein the volatile liquid is selected from one or more of ethanol, acetone, diethyl ether, chloroform and acetonitrile.
14. The method for preparing a dry biological tissue material according to claim 1, wherein the cooling temperature is-120 ℃ to-70 ℃ when the biological tissue is cooled by being put into the refrigerant in step S31.
15. The method for preparing a dry biological tissue material according to claim 1, wherein the step S32 specifically comprises:
vacuumizing the biological tissue processed in the step S31, wherein the vacuum degree is 0.5-3 kPa;
heating to-40-0 ℃, wherein the heating rate is 0-40 ℃/h, and the vacuum degree is controlled to be 0.01-0.1 kPa;
the whole vacuum drying time is 4-12 h;
after drying, controlling the temperature of the freeze drying system to re-warm to room temperature, wherein the re-warming rate is 40 ℃/h-80 ℃/h, and the air pressure returns to normal pressure.
16. The method according to claim 15, wherein the time required for vacuuming to a vacuum degree of 0.5 to 3kPa is 15 to 45min, the time required for vacuuming to a vacuum degree of 0.01 to 0.1kPa is 20 to 50min, and the time required for returning to normal pressure is 0.5 to 10 hours.
17. The method as claimed in claim 1, wherein in step S4, the biological tissue processed in step S3 is sealed in a dry and clean container under an environment with a relative humidity of less than 30% or an inert environment.
18. The method for preparing a dry biological tissue material according to claim 1, wherein the sterilization manner in step S4 is ethylene oxide sterilization, electron beam irradiation sterilization or gamma ray irradiation sterilization.
19. A dry biological tissue material produced by the method of any one of claims 1 to 18.
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| CN111281604B (en) | 2020-01-22 | 2022-04-22 | 沛嘉医疗科技(苏州)有限公司 | Artificial biological valve and its treatment method |
| JP2023550257A (en) * | 2020-10-21 | 2023-12-01 | ティシュー テスティング テクノロジーズ エルエルシー | Minimizing immunogenicity of decellularized tissue |
| CN112957530B (en) * | 2021-02-05 | 2022-09-20 | 上海翰凌医疗器械有限公司 | Dry biological tissue material and preparation method thereof |
| CN114177358B (en) * | 2021-12-13 | 2022-07-19 | 天新福（北京）医疗器材股份有限公司 | Biological scaffold, and forming method and application thereof |
| CN115212352B (en) * | 2022-08-08 | 2023-06-13 | 科凯(南通)生命科学有限公司 | Preparation method of biological dry valve and biological dry valve |
| CN115337456B (en) * | 2022-08-08 | 2023-09-15 | 科凯(南通)生命科学有限公司 | High permeability solution and application thereof |
| CN115887767A (en) * | 2022-11-28 | 2023-04-04 | 广州希倍医疗科技有限公司 | Non-crosslinked active collagen matrix particles and preparation method and application thereof |
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