CN114848912B - Acellular dermis and preparation method thereof - Google Patents
Acellular dermis and preparation method thereof Download PDFInfo
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- CN114848912B CN114848912B CN202210449597.2A CN202210449597A CN114848912B CN 114848912 B CN114848912 B CN 114848912B CN 202210449597 A CN202210449597 A CN 202210449597A CN 114848912 B CN114848912 B CN 114848912B
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/362—Skin, e.g. dermal papillae
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
-
- 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
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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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dermatology (AREA)
- Medicinal Chemistry (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention relates to the field of biomedical materials, in particular to acellular dermis and a preparation method thereof. The invention provides a preparation method of acellular dermis, which comprises the following steps: s1, soaking raw leather by adopting a glycerol solution; s2, soaking the raw hide in the step S1 by adopting a low osmotic pressure solution; s3, repeating the steps S1-S2 for more than or equal to 0 times; the glycerol solution is combined with the low osmotic pressure solution to effectively remove cells and epidermis, and the structural shape of the basal lamina and the acellular dermis is ensured to be complete, so that the soft tissue repair product which has certain anti-infective capability and certain tensile strength, is free of bacterial virus pathogens, has good tissue compatibility and is easy for adhesion growth of autologous cells can be obtained.
Description
Technical Field
The invention relates to the field of biomedical materials, in particular to acellular dermis and a preparation method thereof.
Background
Tissue repair or wound healing refers to a series of pathophysiological processes in which local tissue is repaired by proliferation or regeneration after a tissue defect (e.g., wound surface, etc.) is caused by trauma or other diseases. Although different tissues have respective repair characteristics and laws after suffering from wounds, the repair process and laws of soft tissues, particularly body surface soft tissues, are most representative, skin is the largest tissue organ of the human body, and skin damage is most common both in the flat time and in the war time. Skin wound repair is a complex and progressive process, requires multiple synergistic effects, not only requires physiological repair inside the body, but also requires artificial provision of wound repair materials to cover the wound in time, and provides an environment favorable for wound healing. The decellularized dermis is formed by removing cell components in epidermis and dermis which possibly induce strong rejection reaction and dermis tissues left after skin appendages through a physical or chemical method, has a complete three-dimensional network structure and good biocompatibility, is closest to autologous skin tissue components, and has higher application value in soft tissue repair, in particular skin wound repair based on the advantages.
The prior soft tissue repair materials are mainly divided into non-absorbable (polyester patch, polypropylene patch, expanded polytetrafluoroethylene patch and the like); absorbable (polyglycolic acid, polylactic acid glycolic acid, etc.); composite materials and biomaterials (autologous skin, allogeneic skin, xenogeneic skin, amniotic membrane, decellularized dermal matrix, small intestinal submucosa, collagens, etc.).
Taking biological materials as an example, most biological materials are treated by adopting a decellularizing and crosslinking method in the treatment process at present, wherein the decellularizing method mainly comprises a repeated freeze thawing method, a hypertonic solution method, an enzyme, a detergent and the like, and specific reagents are as follows: dispase II, sodium dodecyl sulfate and DNase are combined, ultrasonic wave after freeze thawing, EDTA (0.01-0.20%), sodium dodecyl sulfate, vitamin C, DNAE enzyme, pancreatin, 0.25% -5% triton, glycerol alkali solution and the like. The individual biological materials may optionally be decellularized using reagents such as strong acids and strong bases. However, the above method has the following drawbacks:
(1) The biological material products mostly use cross-linking agents in the treatment process at present, so as to improve the mechanical property of the products, however, the cross-linked products can inhibit wound healing in the use process, the cytotoxicity of the materials is increased in a short period, local tissues are stimulated to generate long-time inflammatory reaction, and some cross-linking agents can release cross-linking agent monomers in long-term tissue repair to generate chronic toxicity to the local tissues and organisms;
(2) When the partial biological material products are prepared, reagents such as strong acid and alkali are used, the strong reaction greatly damages the structure of collagen, even damages the integrity of a basement membrane of allogenic skin playing an important role in repairing, so that the effect of the products is reduced in the use process, and even the tissue repairing effect cannot be achieved;
(3) The treatment process of partial biological material products has poor decellularization effect, can not thoroughly remove cells and reduce DNA residues causing immune rejection reaction, so that the immunogenicity of the products is poor;
(4) The use of individual enzymes may cause problems of excessive or insufficient handling of the material due to the concentration or insufficient handling time, and the excessive handling may cause the destruction or loosening of the collagen structure, and the destruction or absence of a part of the basement membrane; the lack of treatment results in cell residues and unsatisfactory immunogenicity.
Disclosure of Invention
Therefore, the technical problems to be solved by the invention are to overcome the defects of high immunogenicity, large rejection reaction, incomplete collagen structure and basement membrane, large toxicity of crosslinked dermis and the like in the prior art, thereby providing the decellularized dermis and the preparation method thereof, wherein the prepared decellularized dermis has no epidermis layer, no cell residue, low DNA residue, low immunogenicity and no immune rejection reaction, maintains the complete collagen structure and basement membrane, has low toxicity and meets the use requirements of biological material products.
For this purpose, the invention provides the following technical scheme:
a method of preparing decellularized dermis comprising:
s1, soaking raw leather by adopting a glycerol solution;
s2, soaking the raw hide in the step S1 by adopting a low osmotic pressure solution;
s3, repeating the steps S1-S2 for more than or equal to 0 times.
Optionally, the method further comprises:
in the step S1, the raw skin is soaked in glycerol solutions with different concentrations in sequence.
Optionally, in step S1, the raw hide is soaked in a glycerol solution with gradually increasing concentration gradient.
Optionally, in the step S1, the glycerol solution is a glycerol solution with a glycerol concentration of 10% -50% by volume, 50% -85% by volume and 85% -98% by volume in sequence.
Optionally, the condition of soaking the raw skin by adopting a glycerol solution with the glycerol concentration of 10-50% by volume is that the raw skin is soaked for 3-10 hours at room temperature.
Optionally, the condition of soaking the raw skin by adopting a glycerol solution with the glycerol concentration of 50-85% by volume is that the raw skin is soaked for 4-8 hours at room temperature.
Optionally, the condition of soaking the raw skin by adopting a glycerol solution with the glycerol concentration of 85-98% by volume is that the raw skin is soaked for 3-12h at the temperature of 33-37 ℃;
optionally, when the raw materials are soaked in glycerol solution with the glycerol concentration of 85-98% by volume, the liquid is changed after ultrasonic oscillation is carried out for 10-30min every time of soaking for 0.5-1.5h, and the ultrasonic condition is 20KHz-50KHz and 220W. The soaking of the raw skin under the condition is favorable for thorough dehydration of cells in tissues, the dehydrated cells fall off by ultrasonic oscillation, and in addition, the high-concentration glycerol has the effects of removing viruses and killing bacteria.
Optionally, the solvent of the glycerol solution is ethanol water solution or physiological saline. The ethanol aqueous solution and the physiological saline are both favorable for removing the epidermis, and the ethanol aqueous solution also has the effect of assisting in removing viruses and killing bacteria, and the glycerol has better effects of removing the epidermis and removing the viruses and killing bacteria under the cooperation of the solvent.
Optionally, when the solvent of the glycerol solution is ethanol water solution, the ethanol concentration of the solvent is 0.2-10% when the glycerol concentration is 10-50% of the glycerol solution by volume percent; optionally, the concentration of ethanol in the solvent is 5% by volume; the ethanol solution with the concentration is helpful for removing the epidermis and assisting the glycerin to remove the epidermis.
Optionally, when the solvent of the glycerol solution is ethanol water solution, the ethanol concentration of the solvent is 50-85% by volume when the glycerol concentration is 50-85% by volume; optionally, the concentration of ethanol in the solvent is 75% by volume; the ethanol solution with the concentration is helpful for removing viruses and killing bacteria.
Optionally, when the solvent of the glycerol solution is ethanol water solution, the ethanol concentration of the solvent is 95-99% when the glycerol concentration is 85-98% of the glycerol solution by volume. Optionally, the concentration of ethanol in the solvent is 99% by volume; the ethanol solution with the concentration can help dehydration, and can assist glycerol dehydration and cell removal.
Optionally, the low osmotic pressure solution is water or hypotonic brine. Optionally, the hypotonic brine is sodium chloride solution with mass fraction less than 0.9%.
In the preparation method of the acellular dermis, the condition of soaking the raw dermis in the low osmotic pressure solution is that the raw dermis is soaked for 7-12 hours at room temperature;
optionally, the hide is washed with a low osmotic pressure solution prior to soaking in the low osmotic pressure solution. The cleaning function is to clean the reagent used in the previous step and the cells and epidermis which are removed after the previous step, and the residual cells can be burst and removed due to the change of osmotic pressure while cleaning.
The preparation method of the acellular dermis further comprises the step of soaking the raw dermis processed in the step S3 in a surfactant, PBS buffer solution, DNA hydrolase and/or amino acid solution. In the steps, the surfactant plays a role in further decellularizing, the PBS buffer solution plays a role in cleaning, the DNA hydrolase plays a role in further removing broken cell residual DNA, and the amino acid solution plays a role in supplementing nutrition to tissue materials.
Optionally, the surfactant comprises 0.05-0.4% SDS solution, polyethylene glycol solution, polysorbate 80 solution, triton X-100 solution, tributyl phosphate solution, triton X-200 solution or deoxycholate sodium solution;
Optionally, the concentration of the surfactant is an aqueous solution containing 0.2-0.5% of Triton X-100 by volume percent and/or 0.05-0.4% of SDS by mass percent;
optionally, when the surfactant is adopted for soaking treatment, oscillating for 1-2 hours at room temperature, wherein the oscillating speed is 80-100rpm, and liquid is changed every 10-30 min;
optionally, the amino acid solution comprises at least one of glutamic acid, glycine, alanine, or aspartic acid;
optionally, the concentration of the amino acid solution is 0.2-1% by mass; alternatively, 0.5%;
optionally, soaking for 4 hours at 4 ℃ when adopting amino acid solution for soaking treatment;
optionally, the concentration of the DNA hydrolase is 3-7g/L, and the pH is 7.0;
optionally, soaking with DNA hydrolase at 30deg.C for 3-7 hr;
optionally, packaging and sterilizing the acellular dermis.
In the preparation method of the acellular dermis, the raw hide treated in the step S3 is soaked in a surfactant, a PBS buffer solution, DNA hydrolase and an amino acid solution in sequence;
in the preparation method of the acellular dermis, the raw skin is allogenic skin.
The invention provides the acellular dermis prepared by the preparation method;
Optionally, the acellular dermal DNA residue is less than or equal to 1.0ng/mg, and no cell residue exists;
more optionally, the decellularized dermis has a tensile strength of 10.25-11.56MPa and a suture strength of 17.1-18.1N. The acellular dermis prepared by the invention does not damage the structure of collagen tissues, so that the structure is more compact, the elasticity is higher, and the advantages of the acellular dermis for repairing soft tissues and burning wounds are more outstanding.
The technical scheme of the invention has the following advantages:
1. the preparation method of the acellular dermis provided by the invention comprises the following steps: s1, soaking raw leather by adopting a glycerol solution; s2, soaking the raw hide in the step S1 by adopting a low osmotic pressure solution; s3, repeating the steps S1-S2 for more than or equal to 0 times; the method can effectively remove the epidermis and the virus and ensure the structural integrity of the basement membrane and the acellular dermis, the method can effectively remove the epidermis, the acellular and the virus and the bacteria, simultaneously, the dermis can obtain an integral collagen structure and the basement membrane, the method does not use a cross-linking agent, does not have the slow toxicity caused by the residual cross-linking agent, does not use strong reagents such as strong alkali and the like, does not damage the collagen structure of the acellular dermis, has mild acellular process, completely retains the collagen structure of the basement membrane and the dermis, thoroughly removes the cells, has low DNA residual quantity, has no immunogenicity, can not obtain a certain soft tissue with good adhesion strength, has good pathogen-free and has good tissue-repairing capability, and is easy to repair.
2. The preparation method of the acellular dermis comprises the following steps of (1) sequentially soaking raw dermis by adopting glycerol solution with gradually increased concentration gradient; the gradient concentration glycerol solution has the function of a dehydrating agent and the function of fixing free water in tissue cells, so that the cells can be separated from in-situ tissues, the hypotonic solution is matched as a rehydrating agent, the skin tissues are repeatedly treated by using the osmotic pressure principle, residual cells are dehydrated and then are absorbed and burst, the effects of the glycerol solution with different concentrations on the decellularized dermis are different, the gradient concentration treatment is more beneficial to the dehydration separation and the subsequent rehydration burst of the cells, the decellularized epidermis removing effect is better and more thorough, the structures of collagen and basement membrane are not damaged, and the glycerol with higher concentration has the effect of killing bacteria and viruses and can fix the tissues.
3. The invention provides a preparation method of decellularized dermis, which adopts a gradient concentration glycerol solution ultrasonic oscillation and low osmotic pressure solution repeated soaking osmotic pressure method to perform decellularized epidermis removal virus removal fixed tissue treatment, and combines a surfactant, PBS buffer solution, DNA hydrolase, amino acid and other reagents to further treat the decellularized dermis, so as to finally achieve the effect of thoroughly removing epidermis decellularized fixed dermis tissue.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a view of a tissue section of the decellularized dermis of example 1 of the present invention;
FIG. 2 is a tissue section of the decellularized dermis of example 2 of the invention;
FIG. 3 is a view of a tissue section of the decellularized dermis of comparative example 1 of the invention;
FIG. 4 is a view of a tissue section of the decellularized dermis of comparative example 2 of the invention;
FIG. 5 is a porous structure (magnification ×10000) of the decellularized dermis treated in example 1 of the present invention under a scanning electron microscope;
FIG. 6 is a verification result of the effect of virus removal of the decellularized dermis after the treatment of example 1 of the present invention;
FIG. 7 is a photograph of MHC antigens stained before and after the decellularized dermis treatment in example 1 of the present invention under an optical microscope (magnification ×100);
FIG. 8 is a tissue slice of the epidermal mastoid of normal skin;
FIG. 9 is a tissue section of the epidermal mastoid at the site of repair of the decellularized dermis of example 1 of the present invention.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
The glycerol ethanol solution in the following examples is prepared by dissolving glycerol in ethanol water solution.
The glycerol physiological saline solution is prepared by dissolving glycerol in saline, and the concentration of sodium chloride in the obtained glycerol physiological saline solution is 0.9%.
The room temperature in the examples below was in the range of 10-30 ℃.
The raw skin material in the present invention may be human-derived skin or animal-derived skin, such as pig-derived skin, sheep-derived skin, etc., and the raw skin material used in the following examples is human-derived skin.
Example 1
The embodiment provides a preparation method of acellular dermis, comprising the following steps:
(1) Placing the raw skin in a glycerol solution with the glycerol concentration of 10-50% by volume (the ethanol concentration of the solvent used is 0.2-10% by volume), fully reacting, selecting the glycerol solution in the embodiment (the solvent used is 5% ethanol water solution by volume, and the glycerol volume percentage is 30%), and soaking for 3h at room temperature;
(2) Taking out the raw skin in the step (1), placing the raw skin in a glycerol solution with the concentration of 50-85% by volume (the concentration of ethanol in the solvent used is 50-85% by volume), fully reacting, selecting the glycerol solution in the embodiment (the solvent used is 75% ethanol water solution by volume and 85% by volume of glycerol), and soaking for 4 hours at room temperature;
(3) Taking out the raw skin in the step (2), placing the raw skin in a glycerol solution with the concentration of 85-98% by volume, fully reacting, selecting a glycerol solution (the solvent used is a 99% ethanol water solution with the volume percentage, and the glycerol volume percentage is 98%) in the embodiment, soaking the raw skin at 35 ℃ for 12 hours, carrying out ultrasonic oscillation for 20 minutes every 1 hour, and then changing the raw skin into the new glycerol ethanol solution under the ultrasonic condition: 50KHz,220W;
(4) Taking out the raw material skin in the step (3), rinsing the raw material skin in a low-osmotic-pressure solution for 5 times, soaking the raw material skin in the low-osmotic-pressure solution, wherein the low-osmotic-pressure solution can be water or low-osmotic-pressure brine (sodium chloride solution with the concentration of less than 0.9% by mass), fully reacting, taking out the raw material skin in purified water for 5 times (each 25KHz ultrasonic treatment for 5 minutes) in the embodiment, and soaking the raw material skin in the purified water for 7 hours at room temperature;
(5) Placing the raw skin in the step (4) into a glycerol solution with the glycerol concentration of 10-50% by volume percent, fully reacting, selecting a glycerol solution with the glycerol concentration of 30% by volume percent (the used solvent is physiological saline) in the embodiment, and soaking for 10 hours at room temperature;
(6) Taking out the raw skin in the step (5), placing the raw skin in a glycerol solution (the used solvent is normal saline) with the concentration of 50-85% of glycerol by volume, fully reacting, selecting the glycerol solution (the used solvent is normal saline) with the concentration of 85% of glycerol by volume in the embodiment, and treating for 4 hours at room temperature;
(7) Taking out the raw skin in the step (6), placing the raw skin in a glycerol solution (the used solvent is normal saline) with the concentration of 85-98% by volume, fully reacting, selecting the glycerol solution (the used solvent is normal saline) with the concentration of 98% by volume in the embodiment, treating for 3 hours at 35 ℃, and changing the glycerol solution into the new glycerol solution after soaking for 1 hour for 20 minutes by ultrasonic oscillation, wherein the ultrasonic conditions are as follows: 50KHz,220W;
(8) Taking out the raw hide in the step (7), placing the raw hide in low-osmotic-pressure solution, rinsing the raw hide in the low-osmotic-pressure solution, placing the raw hide in normal saline for soaking, wherein the low-osmotic-pressure solution can be water or low-osmotic-pressure saline (sodium chloride solution with the concentration of less than 0.9% by mass), taking out the raw hide in purified water for rinsing for 5 times in the embodiment, placing the raw hide in the normal saline, and soaking the raw hide in the normal saline for 12 hours at room temperature;
(9) And (3) surfactant treatment: taking out the raw hide in the step (8), soaking in a surfactant, and in the embodiment, selecting a 0.5% TritonX-100 solution by volume percent, oscillating for 2 hours at room temperature, wherein the oscillating speed is 100rpm, and changing the liquid once every 10 minutes of soaking;
(10) DNA hydrolase treatment: taking out the raw skin in the step (9), rinsing the raw skin with purified water for 5 times, and then soaking the raw skin in DNA hydrolase (DNase) with the concentration of 6g/L and the pH of 7.0 for reaction for 5 hours at the temperature of 30 ℃;
(11) Amino acid solution treatment: taking out the raw hide in the step (10), rinsing with purified water, placing the rinsed raw hide in an amino acid solution for soaking treatment, and selecting a glutamic acid solution with the concentration of 0.5% by mass in the embodiment, and soaking for 2 hours at room temperature;
(12) PBS buffer treatment: taking out the raw hide in the step (11), rinsing with purified water for 5 times (each time is treated by 25KHz ultrasonic for 2 minutes), and soaking in PBS buffer solution (pH=7.2-7.4) at room temperature for 2 hours;
(13) Packaging and sterilizing: washing with physiological saline for two times, bagging, sealing, and sterilizing the packaged product with cobalt-60 or electron beam radiation at a dose of 25-40kGy.
Example 2
The embodiment provides a preparation method of acellular dermis, comprising the following steps:
(1) Placing the raw skin in a glycerol solution with the glycerol concentration of 10-50% by volume (the solvent used is ethanol with the ethanol concentration of 0.2-10% by volume) for full reaction, selecting the glycerol solution with the glycerol concentration of 10% by volume (the solvent used is water solution with the ethanol concentration of 0.2% by volume) in the embodiment, and soaking for 6 hours at room temperature;
(2) Taking out the raw skin in the step (1), placing the raw skin in a glycerol solution with the glycerol concentration of 50-85% by volume, fully reacting, selecting a glycerol solution with the glycerol concentration of 50% by volume (the solvent used is an aqueous solution with the ethanol concentration of 50% by volume) in the embodiment, and soaking for 6 hours at room temperature;
(3) Taking out the raw skin in the step (2), placing the raw skin in a glycerol solution with the glycerol concentration of 85-98% by volume, fully reacting, selecting a glycerol solution with the glycerol concentration of 85% by volume (the solvent used is an aqueous solution with ethanol concentration of 95% by volume) in the embodiment, and soaking at 33 ℃ for 10 hours; after each soaking time is 0.5h and ultrasonic oscillation is carried out for 10min, liquid is changed, and ultrasonic conditions are as follows: 50KHz,220W;
(4) Taking out the raw hide in the step (3), placing the raw hide in a low-osmotic-pressure solution, rinsing the raw hide in the low-osmotic-pressure solution, placing the raw hide in the low-osmotic-pressure solution for soaking, wherein the low-osmotic-pressure solution can be water or low-osmotic-salt water (the mass fraction of the low-osmotic-pressure solution is less than 0.9% of sodium chloride solution), fully reacting, taking out the raw hide in purified water for rinsing 5 times (each time of 25KHz ultrasonic treatment for 5 minutes) in the purified water, and soaking the raw hide in the purified water for 12 hours at room temperature;
(5) And (3) surfactant treatment: taking out the raw hide in the step (4), soaking in a surfactant solution, and selecting a TritonX-100 (volume percent) in the embodiment to be soaked for 1h in a shaking table at room temperature, wherein the shaking speed is 80rpm after the soaking is carried out once every 30 minutes;
(6) DNA hydrolase treatment: taking out the raw skin in the step (5), rinsing the raw skin with purified water for 5 times, and then reacting the raw skin in DNA hydrolase (DNase) with the concentration of 5g/L and the pH of 7.0 at the temperature of 30 ℃ for 7h;
(7) Amino acid solution treatment: taking out the raw hide in the step (6), rinsing with purified water, placing the rinsed raw hide in an amino acid solution for soaking treatment, and selecting a glutamic acid solution with the concentration of 0.5% by mass in the embodiment, and soaking for 2 hours at room temperature;
(8) PBS buffer treatment: taking out the raw hide in the step (7), rinsing with purified water for 5 times (each time is treated by 25KHz ultrasonic for 2 minutes), and then soaking in PBS buffer solution (pH 7.2-7.4) for 2 hours at room temperature;
(9) Packaging and sterilizing: washing with physiological saline for two times, bagging, sealing, and sterilizing the packaged product with cobalt-60 or electron beam radiation at a dose of 25-40kGy.
Example 3
The embodiment provides a preparation method of acellular dermis, comprising the following steps:
(1) Placing the raw skin in a glycerol solution with the glycerol concentration of 10-50% by volume (the ethanol concentration of 0.2-10%) for full reaction, selecting a glycerol solution with the glycerol concentration of 50% by volume (the ethanol concentration of 10% by volume) in the embodiment, and soaking for 10 hours at room temperature;
(2) Taking out the raw skin in the step (1), placing the raw skin in a glycerol solution with the glycerol concentration of 50-85% by volume, fully reacting, selecting a glycerol solution with the glycerol concentration of 75% by volume (the solvent used is an aqueous solution with the ethanol concentration of 85% by volume) in the embodiment, and soaking at room temperature for 8 hours;
(3) Taking out the raw skin in the step (2), placing the raw skin in a glycerol solution with the concentration of 85% -98%, fully reacting, selecting the glycerol solution (the solvent is an ethanol water solution with the volume percentage of 97% and the glycerol concentration is 85%) in the embodiment, and soaking the raw skin at 37 ℃ for 3 hours; after each soaking time of 1h, the liquid is changed after ultrasonic oscillation is carried out for 20min, and the ultrasonic conditions are as follows: 50KHz,220W;
(4) Taking out the raw hide in the step (3), placing the raw hide in a low-osmotic-pressure solution, rinsing the raw hide in the low-osmotic-pressure solution, placing the raw hide in the low-osmotic-pressure solution for soaking, wherein the low-osmotic-pressure solution can be water or low-osmotic-salt water (the mass fraction is less than 0.9% of sodium chloride solution), fully reacting, taking out the raw hide in purified water for rinsing 5 times (each time of 25KHz ultrasonic treatment is 5 minutes), placing the raw hide in the sodium chloride solution with the mass fraction of 0.7%, and soaking for 10 hours at room temperature;
(5) Taking out the raw skin in the step (4), placing the raw skin in a glycerol solution (the used solvent is normal saline) with the concentration of 85-98% by volume, fully reacting, selecting a glycerol solution (the used solvent is normal saline) with the concentration of 90% by volume in the embodiment, and treating for 10h at 37 ℃; after each soaking time of 1.5h, ultrasonic oscillation is carried out for 30min, liquid is changed, and ultrasonic conditions are as follows: 20KHz,220W;
(6) Taking out the raw skin in the step (5), placing the raw skin in a glycerol solution (the used solvent is normal saline) with the concentration of 50-85% of the glycerol volume percentage, fully reacting, selecting a glycerol solution (the used solvent is normal saline) with the glycerol concentration of 78% of the glycerol volume percentage in the embodiment, and treating for 8 hours at room temperature;
(7) Placing the raw skin in the step (6) into a glycerol solution with the glycerol concentration of 10-50% by volume percent, fully reacting, selecting a glycerol physiological saline solution with the glycerol concentration of 50% by volume percent in the embodiment, and soaking for 8 hours at room temperature;
(8) Taking out the raw hide in the step (7), placing the raw hide in a low-osmotic-pressure solution, rinsing the raw hide in the low-osmotic-pressure solution, placing the raw hide in normal saline for soaking, wherein the low-osmotic-pressure solution can be water or low-osmotic-pressure saline (sodium chloride solution with the concentration of less than 0.9% by mass), taking out the raw hide in the 0.7% sodium chloride aqueous solution for rinsing for 5 times (each time of 25KHz ultrasonic treatment for 5 minutes), placing the raw hide in the normal saline, and soaking for 10 hours at room temperature;
(9) And (3) surfactant treatment: taking out the raw hide in the step (8), placing the raw hide in a surfactant solution for soaking treatment, selecting a mixed solution containing 0.5% of Triton X-100 by volume percent and 0.4% of SDS by mass percent in the embodiment, oscillating and soaking the mixed solution in a shaking table at room temperature for 1h, and changing the liquid once every 20 minutes of soaking at the oscillating speed of 90 rpm;
(10) DNA hydrolase treatment: taking out the raw skin in the step (9), rinsing the raw skin with purified water for 5 times, and then reacting the raw skin in DNA hydrolase (DNase) with the concentration of 6g/L and the pH of 7.0 at the temperature of 30 ℃ for 3 hours;
(11) Amino acid solution treatment: taking out the raw hide in the step (10), rinsing with purified water, placing the rinsed raw hide in an amino acid solution for soaking treatment, and selecting a mixed solution with the concentration of 0.5 mass percent of glycine and 0.5 mass percent of alanine in the embodiment, and soaking for 2 hours at room temperature;
(12) PBS buffer treatment: taking out the raw hide in the step (11), rinsing with purified water for 5 times (each time is treated by 25KHz ultrasonic for 2 minutes), and then soaking in PBS buffer solution (pH 7.2-7.4) for 2 hours at room temperature;
(13) Packaging and sterilizing: washing with physiological saline for two times, bagging, sealing, and sterilizing the packaged product with cobalt-60 or electron beam radiation at a dose of 25-40kGy.
Example 4
(1) The raw hide is taken out and placed into a glycerin aqueous solution with the concentration of 85-98% by volume, the glycerin aqueous solution (the glycerin volume percentage is 98%) is selected in the embodiment, the raw hide is soaked for 12 hours at 35 ℃, the raw hide is replaced by the new glycerin aqueous solution after being soaked for 1 hour and vibrated for 20 minutes by ultrasound, and the ultrasound conditions are as follows: 50KHz,220W;
(2) Taking out the raw material skin in the step (1), rinsing the raw material skin in a low-osmotic-pressure solution for 5 times, soaking the raw material skin in the low-osmotic-pressure solution, wherein the low-osmotic-pressure solution can be water or low-osmotic-pressure brine (sodium chloride solution with the concentration of less than 0.9% by mass), fully reacting, taking out the raw material skin in purified water for 5 times (each 25KHz ultrasonic treatment for 5 minutes) in the embodiment, and soaking the raw material skin in the purified water for 7 hours at room temperature;
(3) Taking out the raw skin in the step (2), placing the raw skin in a glycerin water solution with the concentration of 50-85% by volume, fully reacting, selecting the glycerin water solution (the glycerin volume percentage is 85%) in the embodiment, and soaking at room temperature for 4h;
(4) Taking out the raw material skin in the step (3), rinsing the raw material skin in a low-osmotic-pressure solution for 5 times, soaking the raw material skin in the low-osmotic-pressure solution, wherein the low-osmotic-pressure solution can be water or low-osmotic-pressure brine (sodium chloride solution with the concentration of less than 0.9% by mass), fully reacting, taking out the raw material skin in purified water for 5 times (each 25KHz ultrasonic treatment for 5 minutes) in the embodiment, and soaking the raw material skin in the purified water for 7 hours at room temperature;
(5) Placing the raw skin in the step (4) in a glycerol aqueous solution with the glycerol concentration of 10-50% by volume percent for full reaction, and selecting the glycerol aqueous solution (30% by volume of glycerol) in the embodiment, and soaking at room temperature for 3h;
(6) Taking out the raw hide in the step (5), placing the raw hide in a glycerin water solution with the concentration of 85-98% by volume, fully reacting, selecting the glycerin water solution (the glycerin volume percentage is 98%) in the embodiment, soaking for 12h at 35 ℃, carrying out ultrasonic oscillation for 20min every 1h, and replacing the raw hide with the new glycerin water solution, wherein the ultrasonic conditions are as follows: 50KHz,220W;
(7) Taking out the raw material skin in the step (6), rinsing the raw material skin in a low-osmotic-pressure solution for 5 times, soaking the raw material skin in the low-osmotic-pressure solution, wherein the low-osmotic-pressure solution can be water or low-osmotic-pressure brine (sodium chloride solution with the concentration of less than 0.9% by mass), fully reacting, taking out the raw material skin in purified water for 5 times (each 25KHz ultrasonic treatment for 5 minutes) in the embodiment, and soaking the raw material skin in the purified water for 7 hours at room temperature;
(8) Taking out the raw hide in the step (7), placing the raw hide in low-osmotic-pressure solution, rinsing the raw hide in the low-osmotic-pressure solution, placing the raw hide in normal saline for soaking, wherein the low-osmotic-pressure solution can be water or low-osmotic-pressure saline (sodium chloride solution with the concentration of less than 0.9% by mass), taking out the raw hide in purified water for rinsing for 5 times in the embodiment, placing the raw hide in the normal saline, and soaking the raw hide in the normal saline for 12 hours at room temperature;
(9) And (3) surfactant treatment: taking out the raw hide in the step (8), soaking in a surfactant, and in the embodiment, selecting a 0.5% TritonX-100 solution by volume percent, oscillating for 2 hours at room temperature, wherein the oscillating speed is 100rpm, and changing the liquid once every 10 minutes of soaking;
(10) DNA hydrolase treatment: taking out the raw skin in the step (9), rinsing the raw skin with purified water for 5 times, and then reacting the raw skin in DNA hydrolase (DNase) with the concentration of 6g/L and the pH of 7.0 for 5 hours at the temperature of 30 ℃;
(11) Amino acid solution treatment: taking out the raw hide in the step (10), rinsing with purified water, placing the rinsed raw hide in an amino acid solution for soaking treatment, and selecting a glutamic acid solution with the concentration of 0.5% by mass in the embodiment, and soaking for 2 hours at room temperature;
(12) PBS buffer treatment: taking out the raw hide in the step (11), rinsing with purified water for 5 times (each time is treated by 25KHz ultrasonic for 2 minutes), and then soaking in PBS buffer solution (pH 7.2-7.4) for 2 hours at room temperature;
(13) Packaging and sterilizing: washing with physiological saline for two times, bagging, sealing, and sterilizing the packaged product with cobalt-60 or electron beam radiation at a dose of 25-40kGy.
Example 5
(1) Taking out the raw hide, placing the raw hide in a glycerol solution with the volume percentage concentration of 85% -98%, fully reacting, selecting the glycerol solution (the solvent used is an ethanol water solution with the volume percentage of 97%, and the glycerol concentration is 85%) in the embodiment, and soaking the raw hide for 3 hours at 37 ℃; after each soaking time of 1h, the liquid is changed after ultrasonic oscillation is carried out for 20min, and the ultrasonic conditions are as follows: 50KHz,220W;
(2) Taking out the raw skin in the step (1), placing the raw skin in a glycerol solution with the glycerol concentration of 50-85% by volume, fully reacting, selecting a glycerol solution with the glycerol concentration of 75% by volume (the solvent used is an aqueous solution with the ethanol concentration of 85% by volume) in the embodiment, and soaking at room temperature for 8 hours;
(3) Placing the raw skin in the step (2) in a glycerol solution with the glycerol concentration of 10-50% by volume (the solvent used is an aqueous solution with the ethanol concentration of 0.2-10% by volume), fully reacting, selecting a glycerol solution with the glycerol concentration of 50% by volume (the solvent used is an aqueous solution with the ethanol concentration of 10% by volume), and soaking for 10 hours at room temperature;
(4) Taking out the raw skin in the step (3), placing the raw skin in a glycerol solution with the concentration of 85% -98%, fully reacting, selecting the glycerol solution (the solvent is an ethanol water solution with the volume percentage of 97% and the glycerol concentration is 85%) in the embodiment, and soaking the raw skin at 37 ℃ for 3 hours; after each soaking time of 1h, the liquid is changed after ultrasonic oscillation is carried out for 20min, and the ultrasonic conditions are as follows: 50KHz,220W;
(5) Taking out the raw hide in the step (4), placing the raw hide in a low-osmotic-pressure solution, rinsing the raw hide in the low-osmotic-pressure solution, placing the raw hide in the low-osmotic-pressure solution for soaking, wherein the low-osmotic-pressure solution can be water or low-osmotic-salt water (the mass fraction is less than 0.9% of sodium chloride solution), fully reacting, taking out the raw hide in purified water for rinsing 5 times (each time of 25KHz ultrasonic treatment is 5 minutes), placing the raw hide in the sodium chloride solution with the mass fraction of 0.7%, and soaking for 10 hours at room temperature;
(6) Taking out the raw skin in the step (5), placing the raw skin in a glycerol solution (the used solvent is normal saline) with the concentration of 85-98% by volume, fully reacting, selecting a glycerol solution (the used solvent is normal saline) with the concentration of 90% by volume in the embodiment, and treating for 10h at 37 ℃; after each soaking time of 1.5h, ultrasonic oscillation is carried out for 30min, liquid is changed, and ultrasonic conditions are as follows: 20KHz,220W;
(7) Taking out the raw skin in the step (6), placing the raw skin in a glycerol solution (the used solvent is normal saline) with the concentration of 50-85% of the glycerol volume percentage, fully reacting, selecting a glycerol solution (the used solvent is normal saline) with the glycerol concentration of 78% of the glycerol volume percentage in the embodiment, and treating for 8 hours at room temperature;
(8) Placing the raw skin in the step (7) into a glycerol solution (the used solvent is normal saline) with the glycerol concentration of 10-50% by volume for full reaction, selecting the glycerol solution (the used solvent is normal saline) with the glycerol concentration of 50% by volume in the embodiment, and soaking for 8 hours at room temperature;
(9) Taking out the raw hide in the step (8), placing the raw hide in a low-osmotic-pressure solution, rinsing the raw hide in the low-osmotic-pressure solution, placing the raw hide in normal saline for soaking, wherein the low-osmotic-pressure solution can be water or low-osmotic-pressure saline (sodium chloride solution with the concentration of less than 0.9% by mass), taking out the raw hide in the 0.2% sodium chloride aqueous solution for rinsing for 5 times (each time of 25KHz ultrasonic treatment for 5 minutes), placing the raw hide in the normal saline, and soaking for 10 hours at room temperature;
(10) And (3) surfactant treatment: taking out the raw hide in the step (9), placing the raw hide in a surfactant solution for soaking treatment, selecting a mixed solution containing 0.5% of Triton X-100 by volume percent and 0.4% of SDS by mass percent in the embodiment, oscillating and soaking the mixed solution in a shaking table at room temperature for 1h, and changing the liquid once every 20 minutes of soaking at the oscillating speed of 90 rpm;
(11) DNA hydrolase treatment: taking out the raw skin in the step (10), rinsing the raw skin with purified water for 5 times, and then reacting the raw skin in DNA hydrolase (DNase) with the concentration of 6g/l and the pH of 7.0 at the temperature of 30 ℃ for 3 hours;
(12) Amino acid solution treatment: taking out the raw hide in the step (11), rinsing with purified water, placing the rinsed raw hide in an amino acid solution for soaking treatment, and selecting a mixed solution with the concentration of 0.5 mass percent of glycine and 0.5 mass percent of alanine in the embodiment, and soaking for 2 hours at room temperature;
(13) PBS buffer treatment: taking out the raw hide in the step (12), rinsing with purified water for 5 times (each time is treated by 25KHz ultrasonic for 2 minutes), and then soaking in PBS buffer solution (pH 7.2-7.4) for 2 hours at room temperature;
(14) Packaging and sterilizing: washing with physiological saline for two times, bagging, sealing, and sterilizing the packaged product with cobalt-60 or electron beam radiation at a dose of 25-40kGy.
Example 6
(1) Placing the raw skin in a glycerol solution with the glycerol concentration of 10-50% by volume (the solvent used is an aqueous solution with the ethanol concentration of 0.2-10% by volume), fully reacting, selecting a glycerol solution with the glycerol concentration of 10% by volume (the solvent used is an aqueous solution with the ethanol concentration of 0.2% by volume) in the embodiment, and soaking at room temperature for 6 hours;
(2) Taking out the raw material skin in the step (1), rinsing the raw material skin in a low-osmotic-pressure solution for 5 times, soaking the raw material skin in the low-osmotic-pressure solution, wherein the low-osmotic-pressure solution can be water or low-osmotic-pressure brine (sodium chloride solution with the concentration of less than 0.9% by mass), fully reacting, taking out the raw material skin in purified water for 5 times (each 25KHz ultrasonic treatment for 5 minutes) in the embodiment, and soaking the raw material skin in the purified water for 7 hours at room temperature;
(3) Taking out the raw skin in the step (2), placing the raw skin in a glycerol solution with the glycerol concentration of 50-85% by volume, fully reacting, selecting a glycerol solution with the glycerol concentration of 50% by volume (the solvent used is an aqueous solution with the ethanol concentration of 50% by volume) in the embodiment, and soaking for 6 hours at room temperature;
(4) Taking out the raw material skin in the step (3), rinsing the raw material skin in a low-osmotic-pressure solution for 5 times, soaking the raw material skin in the low-osmotic-pressure solution, wherein the low-osmotic-pressure solution can be water or low-osmotic-pressure brine (sodium chloride solution with the concentration of less than 0.9% by mass), fully reacting, taking out the raw material skin in purified water for 5 times (each 25KHz ultrasonic treatment for 5 minutes) in the embodiment, and soaking the raw material skin in the purified water for 7 hours at room temperature;
(5) Taking out the raw skin in the step (4), placing the raw skin in a glycerol solution with the glycerol concentration of 85-98% by volume, fully reacting, selecting a glycerol solution with the glycerol concentration of 85% by volume (the used solvent is an ethanol water solution with the glycerol concentration of 95% by volume) in the embodiment, and soaking at 33 ℃ for 10 hours; after each soaking time is 0.5h and ultrasonic oscillation is carried out for 10min, liquid is changed, and ultrasonic conditions are as follows: 50KHz,220W;
(6) Taking out the raw hide in the step (5), placing the raw hide in a low-osmotic-pressure solution, rinsing the raw hide in the low-osmotic-pressure solution, placing the raw hide in the low-osmotic-pressure solution for soaking, wherein the low-osmotic-pressure solution can be water or low-osmotic-salt water (the mass fraction of the low-osmotic-pressure solution is less than 0.9% of sodium chloride solution), fully reacting, taking out the raw hide in purified water for rinsing 5 times (each time of 25KHz ultrasonic treatment for 5 minutes) in the purified water, and soaking the raw hide in the purified water for 12 hours at room temperature;
(7) And (3) surfactant treatment: taking out the raw hide in the step (6), soaking in a surfactant solution, and selecting a TritonX-100 (volume percent) in the embodiment to be soaked for 1h in a shaking table at room temperature, wherein the shaking speed is 80rpm after the soaking is carried out once every 30 minutes;
(8) DNA hydrolase treatment: taking out the raw skin in the step (7), rinsing the raw skin with purified water for 5 times, and then reacting the raw skin in DNA hydrolase (DNase) with the concentration of 5g/L and the pH of 7.0 at the temperature of 30 ℃ for 7h;
(9) Amino acid solution treatment: taking out the raw hide in the step (8), rinsing with purified water, placing the rinsed raw hide in an amino acid solution for soaking treatment, and selecting a glutamic acid solution with the concentration of 0.5% by mass in the embodiment, and soaking for 2 hours at room temperature;
(10) PBS buffer treatment: taking out the raw hide in the step (9), rinsing with purified water for 5 times (each time is treated by 25KHz ultrasonic for 2 minutes), and then soaking in PBS buffer solution (pH 7.2-7.4) for 2 hours at room temperature;
(11) Packaging and sterilizing: washing with physiological saline for two times, bagging, sealing, and sterilizing the packaged product with cobalt-60 or electron beam radiation at a dose of 25-40kGy.
Comparative example 1
The comparative example provides a method for preparing acellular dermis, comprising the following steps:
(1) Placing the raw hide in a glycerol solution of alkali (sodium hydroxide with the concentration of 0.5% by mass), dissolving sodium hydroxide in glycerol (glycerol with the commercial purity of 95%) to prepare a glycerol solution with the concentration of 0.5% by mass), and soaking at room temperature for 1h;
(2) And (3) surfactant treatment: taking out the raw hide in the step (1), rinsing with purified water, soaking in a surfactant, and in the embodiment, selecting a 0.5% TritonX-100 solution by volume percent, oscillating for 2 hours at room temperature, wherein the oscillating speed is 100rpm, and changing the liquid once every 10 minutes of soaking;
(3) DNA hydrolase treatment: taking out the raw skin in the step (2), rinsing the raw skin with purified water for 5 times, and then reacting the raw skin in DNA hydrolase (DNase) with the concentration of 6g/L and the pH of 7.0 for 5 hours at the temperature of 30 ℃;
(4) Amino acid solution treatment: taking out the raw hide in the step (3), rinsing with purified water, placing the rinsed raw hide in an amino acid solution for soaking treatment, and selecting a glutamic acid solution with the concentration of 0.5% by mass in the embodiment, and soaking for 2 hours at room temperature;
(5) PBS buffer treatment: taking out the raw hide in the step (4), rinsing with purified water for 5 times (each time is treated by 25KHz ultrasonic for 2 minutes), and then soaking in PBS buffer solution for 2 hours at room temperature;
(6) Packaging and sterilizing: washing with physiological saline for two times, bagging, sealing, and sterilizing the packaged product with cobalt-60 or electron beam radiation at a dose of 25-40kGy.
Comparative example 2
This comparative example is substantially similar to comparative example 1 except that in step (1), the raw hide is placed in glycerin with a purity of 95%, and is immersed at room temperature for 1 hour.
Effect example 1
The decellularized dermis obtained in example 1, example 2, comparative example 1, comparative example 2 was tested for the following performance index.
(1) Cell, epidermal residue:
the detection method comprises the following steps: HE staining, microscopic observation
The results are shown in FIGS. 1 to 5, and it can be seen from FIG. 1 that the acellular dermis of example 1 has no cell residue, the appearance and section observation show that the epidermis is completely removed, and the collagen structure and the basement membrane structure are completely preserved (FIGS. 1 and 5), and that the porous structure of the extracellular matrix is completely preserved and ordered in the arrangement as can be seen from FIG. 5. As can be seen from fig. 2, the acellular dermis of example 2 has no cell residue, and the appearance and section observation show that the epidermis is completely removed, and the collagen structure and the basement membrane structure are completely maintained. As can be seen from FIG. 3, the acellular dermis of comparative example 1 has no cell residue, and the appearance and section observation show that the epidermis is completely removed, but the collagen structure is loose and the basal membrane structure is destroyed. As can be seen from FIG. 4, the acellular dermis of comparative example 2 was not obtained, and the epidermis was still free from signs of exfoliation, the skin was of a more rigid, wrinkled texture, and a large amount of cells remained.
(2) DNA residue:
the detection method comprises the following steps: according to YY/T0606.25-2014 method for measuring animal-derived biological material DNA residues: the detection method defined in the fluorescent staining method is used for detection.
The test results are shown in Table 1 below:
TABLE 1 DNA residues
| Example 1 | Example 2 | Comparative example 1 | Comparative example 2 | |
| DNA residue | 0.2ng/mg | 0.3ng/mg | 0.7ng/mg | 78ng/mg |
Effect example 2
The decellularized dermis obtained in example 1 was subjected to the following performance index investigation.
(1) Effect of removing inactivated virus
The detection method comprises the following steps: and verifying the virus removal effect according to the method of the verification technology examination guiding principle of the virus inactivation technology of the allograft implantable medical device.
Detection result: the process of decellularized dermis of example 1 can achieve an effect of inactivating viruses with an acceptable level of virus reduction index. In this experimental example, only the dermis treated in step (2) of example 1 was subjected to a virus inactivation index test of one virus (the indicator virus in this experimental example is human parvovirus B19), and the test results are shown in fig. 6.
(2) Immunogenicity of
The detection method comprises the following steps: taking the raw leather before the treatment of the example 1 and the decellularized dermis after the treatment of the example 1, respectively performing MHC (tissue compatibility complex) antigen staining and optical microscopy;
detection result: as shown in FIG. 7, the left panel shows the results of MHC antigen staining of the untreated raw dermis of example 1 as positive, and the right panel shows the results of MHC antigen staining of the decellularized dermis obtained after the treatment of example 1 as negative, and it can be seen that the decellularized dermis treated by example 1 has no immunogenicity.
(3) Tissue repair effect
The detection method comprises the following steps: the site after repair of normal skin and the decellularized dermal tissue of implant example 1 was removed and observed by HE staining.
As shown in fig. 8 to 9, fig. 8 is a view of the epidermis mastoid tissue of the normal skin, and fig. 9 is a view of the epidermis mastoid tissue of the example 1 after the decellularized dermis repair site, and as can be seen from comparison of the figures, the decellularized dermis repair site of the example 1 has the same structure as the epidermis mastoid tissue of the normal skin, thus indicating that the decellularized dermis of the example 1 has a good tissue repair effect.
(4) Mechanical properties
The detection method comprises the following steps: determination of tensile Properties of plastics according to GBT 1040.3-2006 part 3: test conditions of the films and sheets the tensile strength of the decellularized dermis product was measured, and the suture strength of the decellularized dermis product was measured according to the method of measuring the tensile strength in the vascular prosthesis of the YY 0500-2004 cardiovascular implant, the measurement results are shown in table 2 below.
TABLE 2 mechanical Properties
(5) After irradiation, sterility was detected.
(6) Biocompatibility of
The detection method comprises the following steps: the decellularized dermis after treatment of example 1-example 6 was tested according to 16886 series standard.
The detection results are as follows:
1. in vitro cytotoxicity: according to the leaching proportion required by the product, the survival rate of the test sample cells is 96%.
2. Skin sensitization: the sample did not elicit a sensitization reaction.
3. Intradermal reaction: without any signs of irritation that would result in the animal.
4. Acute systemic toxicity: has no acute systemic toxicity.
5. Pyrogen test: no thermal reaction.
6. Sub-chronic toxicity: the sample does not show clinically significant abnormal indicators or toxic target organs.
7. Genotoxicity: the results were negative and non-genotoxic.
8. And (3) implantation: no difference from the control (marketed product).
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (9)
1. A method of preparing decellularized dermis comprising:
s1, soaking raw leather by adopting a glycerol solution; the method sequentially comprises the following steps: the condition of soaking the raw skin by adopting a glycerol solution with the glycerol concentration of 10-50% by volume is that the raw skin is soaked for 3-10 hours at room temperature; the condition of soaking the raw skin by adopting a glycerol solution with the glycerol concentration of 50-85% by volume is that the raw skin is soaked for 4-8 hours at room temperature; the condition of soaking the raw skin by adopting a glycerol solution with the glycerol concentration of 85-98% by volume is that the raw skin is soaked for 3-12h at the temperature of 33-37 ℃; carrying out liquid exchange after carrying out ultrasonic oscillation for 10-30min every 0.5-1.5h, wherein the ultrasonic condition is 20KHz-50KHz,220W; the solvent of the glycerol solution is ethanol water solution, and when the glycerol concentration is 10-50% of the glycerol solution, the ethanol concentration in the solvent is 0.2-10% of the glycerol solution; when the concentration of the glycerol is 50-85% of the glycerol solution by volume, the concentration of ethanol in the solvent is 50-85% by volume; when the glycerol concentration is 85-98% of the glycerol solution, the ethanol concentration in the solvent is 95-99% of the glycerol solution;
S2, soaking the raw hide in the step S1 by adopting a low osmotic pressure solution; the condition of soaking the raw hide in the low osmotic pressure solution is that the raw hide is soaked for 7-12 hours at room temperature;
s3, repeating the steps S1-S2 for more than or equal to 0 times;
the raw skin is human skin or animal skin.
2. The method of claim 1, wherein the low osmotic pressure solution is water or hypotonic saline.
3. The method for preparing acellular dermis according to claim 2,
the hypotonic brine is sodium chloride solution with mass fraction less than 0.9%.
4. The method for preparing acellular dermis according to claim 1,
the skin is rinsed with a low osmotic pressure solution prior to soaking in the low osmotic pressure solution.
5. The method for preparing decellularized dermis according to claim 1-4, further comprising the step of immersing the raw dermis treated in step S3 in surfactant, PBS buffer, DNA hydrolase and/or amino acid solution.
6. The method for preparing acellular dermis according to claim 5, wherein,
the surfactant comprises 0.05-0.4% SDS solution, polyethylene glycol solution, polysorbate 80 solution, tritonX-100 solution, tributyl phosphate solution, tritonX-200 solution or deoxycholate sodium solution;
And/or, when the surfactant is adopted for soaking treatment, oscillating for 1-2 hours at room temperature, wherein the oscillating speed is 80-100rpm, and liquid is changed every 10-30 min;
and/or the amino acid solution comprises at least one of glutamic acid, glycine, alanine or aspartic acid;
and/or the concentration of the amino acid solution is 0.2-1% by mass;
and/or soaking for 2-4h at 4 ℃ when adopting amino acid solution for soaking treatment;
and/or, the concentration of the DNA hydrolase is 3-7g/L, and the pH is 7.0;
and/or soaking with DNA hydrolase at 30deg.C for 3-7 hr;
and/or, further comprising packaging and sterilizing the prepared acellular dermis.
7. The method for preparing acellular dermis according to claim 6,
the surfactant is an aqueous solution containing 0.2-0.5% of TritonX-100 by volume percent and/or 0.05-0.4% of SDS by mass percent;
and/or the concentration of the amino acid solution is 0.5% by mass.
8. An acellular dermis prepared by the method of preparing an acellular dermis according to any of claims 1-7.
9. The decellularized dermis according to claim 8, wherein said decellularized dermis has no cell residue and no DNA residue of less than 0.3 ng/mg;
And/or
The tensile strength of the acellular dermis is 10.58-11.56MPa, and the suture strength is 17.5-17.9N.
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| CN103908700A (en) * | 2013-01-06 | 2014-07-09 | 陕西佰傲再生医学有限公司 | Decellularization cornea preparation method |
| CN106730008A (en) * | 2016-12-29 | 2017-05-31 | 北京桀亚莱福生物技术有限责任公司 | A kind of anal fistula sticking patch or bolt, preparation method and applications |
| CN108888804A (en) * | 2018-07-27 | 2018-11-27 | 山东省眼科研究所 | A kind of soft tissue repair material and preparation method thereof |
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