CN113425906A - Cartilage material and preparation method and application thereof - Google Patents
Cartilage material and preparation method and application thereof Download PDFInfo
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- CN113425906A CN113425906A CN202010208243.XA CN202010208243A CN113425906A CN 113425906 A CN113425906 A CN 113425906A CN 202010208243 A CN202010208243 A CN 202010208243A CN 113425906 A CN113425906 A CN 113425906A
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- 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
- A61L27/3612—Cartilage, synovial fluid
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- A61L27/3641—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 site of application in the body
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- A61L27/3654—Cartilage, e.g. meniscus
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- 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
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- 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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- 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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- 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
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- 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
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Abstract
The invention relates to the field of biological materials, in particular to a cartilage material and a preparation method and application thereof. The immunogenicity of the cartilage material processed and prepared by the method is reduced from the source of raw materials by a gene editing technology, and the immunogenicity is further reduced by using an optimized acellular processing technology and a cross-linking technology. The cartilage material of the invention has low immunogenicity and good biological activity, and is used for the repair and regeneration of clinical cartilage tissues.
Description
Technical Field
The invention relates to the field of biological materials, in particular to a cartilage material and a preparation method and application thereof.
Background
Animal-derived cartilage materials have been widely used in cartilage tissue engineering and cartilage regeneration research, and show great potential in clinical applications. The immunogenicity problem presented by xenogenic cartilage material is generally reduced by the removal of xenogenic cells from the cartilage tissue. The existing methods for reducing the immunogenicity of cartilage materials mainly comprise physical methods, chemical methods and biological methods (enzyme methods). Only to the extent of decellularization, products with higher degrees of decellularization can be prepared by various methods. However, the biological activity of cartilage materials and the like are still unsatisfactory from the viewpoint of clinical application effects. The main reasons are that: the existing acellular scheme inevitably leads to the loss of extracellular matrix components, particularly soluble proteoglycan and glycoprotein components, and the loss rate can reach 80 percent. In order to improve the product performance and satisfy the clinical treatment effect, a technical scheme for further optimizing and reducing the immunogenicity of the cartilage material is needed.
Disclosure of Invention
In view of the above, the present invention provides a cartilage material, a preparation method thereof and uses thereof. The immunogenicity of the cartilage material processed and prepared by the method is reduced from the source of raw materials by a gene editing technology, and the immunogenicity is further reduced by using an optimized acellular processing technology and a cross-linking technology. The cartilage material is used for repairing and regenerating clinical cartilage tissues, and active molecules and/or living cells can be combined in the using process.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a cartilage material, which comprises the following steps:
step 1, breeding a gene editing animal;
step 2, collecting cartilage of the animal in the step 1 as a raw material;
step 3, taking the raw material prepared in the step 2 to remove cells;
step 4, crosslinking the material prepared in the step 3;
wherein, the step 3 is specifically as follows:
immersing in 0.01-4 wt.% SDS solution for 3-24 h, with oscillation frequency of 100-1000 rpm and temperature of 0-40 ℃;
ultrasonic cleaning, processing for 2-12 h at 40-120 KHz, with the power of 5-10 KW and the temperature of 25-40 ℃;
placing the mixture at a temperature of between 40 ℃ below zero and 80 ℃ below zero for 2 to 6 hours, placing the mixture at a temperature of between 4 ℃ below zero and 40 ℃ for 1 to 3 hours, placing the mixture at a temperature of between 20 ℃ below zero and 80 ℃ below zero for 3 to 6 hours, and placing the mixture at a temperature of between 10 ℃ below zero and 40 ℃ for 1 to 3 hours;
ultrasonic cleaning, treating for 2-6 h at 60-120 KHz with the power of 0.5-5 KW and the temperature of 25-40 ℃;
immersing the substrate in 0.001-1 wt.% SDS solution for 3-12 h, wherein the oscillation frequency is 300-1000 rpm, and the temperature is 20-40 ℃;
ultrasonic cleaning, processing for 2-6 h at 40-120 KHz, with the power of 1-5 KW and the temperature of 25-40 ℃;
the step 4 specifically comprises the following steps:
immersing in 0.005-5 wt.% glutaraldehyde solution for 1-12 h at 25-40 ℃ for 1-5 times;
immersing the substrate in 0.00001-1 wt.% genipin solution for 1-12 h at 25-40 ℃, and repeating the immersion for 1-10 times.
In some embodiments of the invention, step 3 is specifically:
submersed in a 0.5 wt.% SDS solution for 3h with an oscillation frequency of 200rpm at a temperature of 10 ℃;
ultrasonic cleaning, treating with 100KHz for 60min, with power of 6KW and temperature of 25 deg.C;
placing at-40 deg.C for 6h, 20 deg.C for 2h, at-80 deg.C for 3h, and at 30 deg.C for 1 h;
ultrasonic cleaning, treating with 30KHz for 120min, with power of 3KW and temperature of 20 deg.C;
submersed in a 0.001 wt.% SDS solution for 12h with an oscillation frequency of 600rpm at a temperature of 20 ℃;
ultrasonic cleaning, processing for 4h at 40KHz, with the power of 1KW and the temperature of 25 ℃;
the step 4 specifically comprises the following steps:
immersing in 0.005 wt.% glutaraldehyde solution for 1h at 25 deg.C for 3 times;
immersing in 0.0001 wt.% genipin solution for 4h at 30 deg.C, and repeating the immersion for 6 times.
In some embodiments of the invention, step 3 is specifically:
submersed in a 0.07 wt.% SDS solution for 10h with an oscillation frequency of 1000rpm at a temperature of 25 ℃;
ultrasonic cleaning, treating for 8h at 60KHz, with the power of 5KW and the temperature of 30 ℃;
placing at-80 deg.C for 2h, 10 deg.C for 3h, at-20 deg.C for 5h, and at 40 deg.C for 3 h;
ultrasonic cleaning, processing for 2h at 120KHz, with the power of 3KW and the temperature of 40 ℃;
submersed in a 0.001 wt.% SDS solution for 10h with an oscillation frequency of 300rpm at a temperature of 40 ℃;
ultrasonic cleaning, processing for 2h at 40KHz, with the power of 1KW and the temperature of 25 ℃;
the step 4 specifically comprises the following steps:
immersing in 0.006 wt.% glutaraldehyde solution for 3h at 25 deg.C for 2 times;
immersing in 0.0001 wt.% genipin solution for 12h at 25 deg.C, and repeating the immersion for 10 times.
In some embodiments of the invention, step 3 is specifically:
submersed in 0.1 wt.% SDS solution for 7h with an oscillation frequency of 600rpm at a temperature of 20 ℃;
ultrasonic cleaning, processing for 6h at 80KHz, with the power of 6KW and the temperature of 30 ℃;
placing at-60 deg.C for 4h, at 30 deg.C for 2h, at-60 deg.C for 4h, and at 37 deg.C for 2 h;
ultrasonic cleaning, processing for 5h at 80KHz, with the power of 3KW and the temperature of 30 ℃;
submersed in a 0.001 wt.% SDS solution for 9h with an oscillation frequency of 700rpm at a temperature of 25 ℃;
ultrasonic cleaning, treating for 3h at 60KHz, with power of 4KW and temperature of 25 deg.C;
the step 4 specifically comprises the following steps:
immersing in 0.005 wt.% glutaraldehyde solution for 6h at 25 deg.C for 5 times;
immersing in 0.0005 wt.% genipin solution for 8h at 40 deg.C, and repeating the immersing 10 times.
In some embodiments of the invention, the gene editing comprises at least one of meganucleases (Meganuclease), Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR/Cas) systems.
In some embodiments of the invention, the gene editing comprises gene knock-out and/or gene transfer;
the gene knocked out in the gene knock-out comprises at least one of GGTA1, CMAH, β 4GalNT2, or PERV;
the gene transferred by the gene transfer comprises at least one of hCD46, hCD55, hCD47, LEA29Y, hTBM, hTFPI or EPCR.
In some embodiments of the invention, the animal comprises at least one of a pig, a cow, a sheep, a horse, a monkey, a dog, a rabbit, a chicken, a mouse, a silkworm, a fish, a marine organism, a donkey; the animals are stably passaged for more than 3 generations after gene editing. In other embodiments, animals are selected for stable passage 4 after gene editing.
The fish comprises freshwater fish.
The marine organisms include marine animals and marine plants; the marine animal comprises one or more of a marine mammal, a marine reptile, a marine fish, a marine arthropod, a barnacle, a marine mollusk, a marine echinoderm, or a marine coelenterate. The marine mammal comprises one or more of blue whale, sperm whale, tiger whale, tooth whale, dolphin, seal, sea lion, and dugent. The marine reptile comprises one or more of sea snake and sea turtle. The marine fish comprises one or more of manta ray, ray asteroides, ray hurricane, ray gordonii, starfish, eel, Kargy eel, catfish, takifugu obscurus, bream , sea horse, brachymystax, red snout fish, shark, butterfly fish, caper, nojirimus fish, grouper, rough skin, lame fish, bat fish, clown fish, hairtail, lobster fish, candlelia fish, candlelike fish and car fish. The marine arthropod comprises one or more of horseshoe crab, shrimp, and crab. The marine mollusk comprises one or more of Amyda sinensis, Sinonovacula Constricta and Carnis Leporis. The marine echinoderm comprises one or more of starfish, sea urchin and sea cucumber. The marine coelenterate comprises one or more of jellyfish, Obelia, jellyfish, coral or sunflower. The marine plants comprise planktonic algae and benthic algae; the benthic algae include green algae, brown algae and red algae.
On the basis of the research, the invention also provides the animal-derived cartilage material prepared by the method.
The invention also provides the application of the animal derived cartilage material in the preparation of tissue engineering materials, regenerative medical materials and transformation medical materials.
On the basis of the research, the invention also provides a composition, which comprises the animal-derived cartilage material and medically or pharmaceutically acceptable active molecules or living cells, wherein the active molecules comprise growth factors; the living cells include stem cells.
The invention also provides the application of the composition in preparing tissue engineering materials, regenerative medical materials and transformation medical materials.
The invention provides a preparation method of a cartilage material, the cartilage material prepared by the method and application thereof. (1) The gene editing technology is used for obtaining animals with low immunogenicity through the gene editing technology, and cartilage raw materials are collected, namely the immunogenicity of animal-derived cartilage materials is reduced from material sources through the gene editing technology; (2) the gradual cell removal technology is different from the conventional method (such as one-time cell removal by a high-concentration reagent), and the cell removal treatment is carried out by adopting the principle of a small amount of times, for example, the steps of soaking by using a Sodium Dodecyl Sulfate (SDS) solution for many times, repeatedly freezing and thawing, ultrasonic treatment and the like are adopted to remove active ingredients such as cells, polysaccharides and the like in the cartilage raw material, so that the immunogenicity is reduced, and the influence on the activity of the cartilage material in the treatment process is reduced as much as possible; (3) "gradual crosslinking technology", unlike conventional methods (such as one-time crosslinking with high concentration of crosslinking agent), the immunogenicity of the antigen active site in the material is reduced by multiple crosslinking with low concentration of crosslinking agent. The cartilage material processed and prepared by the method does not contain living cells, the immunogenicity of the cartilage material is reduced from a raw material source by a gene editing technology, and the immunogenicity is further reduced by using an optimized acellular processing technology and a crosslinking technology. The cartilage material is used for clinical cartilage repair and regeneration, and active molecules and/or living cells can be combined in the using process.
The invention relates to a preparation method of a cartilage material, which relates to a gene editing technology, is different from a conventional method (treating the existing animal tissues/organs by physical, chemical and biological methods), and the gene editing technology is used for carrying out fixed-point 'editing' on a target gene, realizing the modification of a specific DNA fragment and reducing the immunogenicity of the cartilage material from the source (collecting animals). In the process, according to the characteristics of the cartilage material, an editing system, a target gene, an animal species and the like are determined through screening, wherein the target gene range comprises knockout genes such as GGTA1, CMAH, beta 4GalNT2, PERV and the like, and transgenes such as hCD46, hCD55, hCD47, LEA29Y, hTBM, hTFPI, EPCR and the like. Taking alpha-Gal as an example, when xenoantigen remained in cartilage material of animal origin enters into human body, hyperacute immune rejection reaction is initiated, and the main target antigen of the immune rejection reaction is considered to be caused by alpha-Gal antigen existing in animal tissue, and the alpha-Gal antigen exists in most mammals except human and higher primates; the immunogenicity of the cartilage material of animal origin can be obviously reduced by developing a-Gal antigen knockout (GTKO) through a gene editing technology.
On the other hand, the preparation method of the animal-derived cartilage material relates to a decellularization technology and a crosslinking technology, and is different from the conventional method (such as single decellularization of a high-concentration SDS solution or single crosslinking of high-concentration glutaraldehyde), and optimizes decellularization treatment and crosslinking treatment according to the effect of reducing the immunogenicity of the animal-derived cartilage material by a gene editing technology, such as multiple decellularization of a low-concentration SDS solution or multiple crosslinking of low-concentration glutaraldehyde, and minimizes the influence on the biological activity of the material in the treatment process by a mild treatment mode.
Therefore, the cartilage material provided by the invention and the preparation method and application thereof have important practical significance.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the results of immunohistochemical assays of example 1 and comparative example 1;
FIG. 2 shows the results of the bioactivity tests of example 1 and comparative example 1;
FIG. 3 shows the results of immunohistochemical assays of example 2 and comparative example 2;
FIG. 4 shows the results of the bioactivity test of example 2 and comparative example 2;
FIG. 5 shows the results of immunohistochemical assays of example 3 and comparative example 3;
fig. 6 shows the results of the bioactivity tests of example 3 and comparative example 3.
Detailed Description
The invention discloses a cartilage material and a preparation method and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides a preparation method of a cartilage material, which comprises the following steps:
a. breeding the gene editing animals;
b. collecting cartilage of the animal as a raw material;
c. performing multiple circulation treatments on the raw material, wherein the treatment modes comprise repeated freeze thawing, ultrasound and Sodium Dodecyl Sulfate (SDS) solution soaking;
d. the treated material was immersed in the crosslinker solution multiple times.
In some embodiments, the gene editing techniques include at least one of meganucleases (Meganuclease), Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR/Cas) systems.
In some embodiments, the gene editing techniques include both gene knock-out and gene transfer, wherein the knocked-out gene comprises at least one of GGTA1, CMAH, β 4GalNT2, and PERV, and the transferred gene comprises at least one of hCD46, hCD55, hCD47, LEA29Y, hTBM, hTFPI, and EPCR.
In some embodiments, the animal comprises at least one of a pig, a cow, a sheep, a horse, a monkey, a dog, a rabbit, a chicken, a mouse, a silkworm, a fish, a marine organism, a donkey; the animal is an animal which is subjected to stable passage for more than 3 generations after gene editing, and further an animal which is subjected to stable passage for 4 generations after gene editing is selected.
In some embodiments, the freezing temperature range in the repeated freeze-thaw treatment is-20 to-80 ℃, the freezing time is 30min to 12h, the thawing temperature range is 4 to 40 ℃, and the thawing time is 0.5 to 6 h; the low-frequency range of the ultrasonic treatment is 10-40 KHz, the low-frequency treatment time is 5 min-24 h, the high-frequency range is 60-120 KHz, the high-frequency treatment time is 5 min-24 h, the ultrasonic power is 100W-10 KW, and the ultrasonic temperature is 0-40 ℃; in the SDS treatment, the concentration range of SDS is 0.001-5 wt.%, the oscillation frequency is 100-3000 rpm, the treatment temperature is 0-40 ℃, and the treatment time is 0.5-24 h; the multiple-cycle treatment is to combine the treatment methods for use, and the use frequency of each treatment method is 0-10 times.
In some embodiments, the crosslinking agent comprises at least one of a chemical crosslinking agent and a biological crosslinking agent; further, the chemical crosslinking agent includes at least one of aldehydes, imido esters, N-hydroxysuccinimide esters (NHS esters), maleimides, haloacetyl groups, dithiodipyridine, hydrazides, and carbodiimides; further, the aldehyde includes at least one of glutaraldehyde and paraformaldehyde; the biological cross-linking agent comprises at least one of procyanidine and genipin; the concentration range of the cross-linking agent is 0.00001-5 wt.%, the immersion temperature is 0-40 ℃, the single immersion time is 0.5-12 h, and the immersion times are 1-10.
The invention also provides the animal origin cartilage material prepared by the method.
The invention also provides the application of the cartilage material in the preparation of tissue engineering materials, regenerative medical materials and transformation medical materials, active molecules and/or living cells can be combined in the using process, and the active molecules comprise growth factors; living cells include stem cells.
The cartilage material provided by the invention, the preparation method and the reagent used in the application can be purchased from the market.
The invention is further illustrated by the following examples:
example 1
A pig cartilage material, its preparation method and cartilage regeneration application are provided.
The preparation method comprises the following steps:
a. GGTA1 and CMAH are knocked out by gene editing of a ZFNs system, the pig is transferred into hCD47, and the pig is edited by using genes with stable passage of 3 generations;
b. collecting cartilage of a gene editing pig as a raw material;
c. the cartilage starting material is treated as follows:
c1. submersed in a 0.5 wt.% SDS solution for 3h with an oscillation frequency of 200rpm at a temperature of 10 ℃;
c2. immersing in an ultrasonic cleaner, treating for 60min at 100KHz with power of 6KW and temperature of 25 deg.C;
c3. placing in-40 deg.C refrigerator for 6h, 20 deg.C refrigerator for 2h, at-80 deg.C refrigerator for 3h, and 30 deg.C refrigerator for 1 h;
c4. immersing in an ultrasonic cleaner, treating for 120min at 30KHz with power of 3KW and temperature of 20 deg.C;
c5. submersed in a 0.001 wt.% SDS solution for 12h with an oscillation frequency of 600rpm at a temperature of 20 ℃;
c6. ultrasonic cleaning, processing for 4h at 40KHz, with the power of 1KW and the temperature of 25 ℃;
d. immersing in 0.005 wt.% glutaraldehyde solution for 1h at 25 deg.C for 3 times;
immersing in 0.0001 wt.% genipin solution for 4h at 30 deg.C, and repeating the immersion for 6 times.
The pig-derived cartilage material prepared by the steps is used for cartilage regeneration, and bone marrow mesenchymal stem cells are added into the material.
Comparative example 1
Collecting cartilage materials of common pigs. The cell removing treatment comprises the following steps: submersed in a 5 wt.% SDS solution for 24h with a frequency of 200rpm shaking at a temperature of 25 ℃. The crosslinking treatment comprises the following steps: immersed in a2 wt.% glutaraldehyde solution for 3h at 25 ℃.
Effect example 1 immunogenicity test
1.1Gal content detection: and quantitatively detecting the Gal content in the sample by using a human alpha galactosidase (alpha GAL) ELISA kit.
The results are as follows:
refer to industry Standard tissue engineering medical appliance product animalThe method of the detection of residual alpha Gal antigen of original scaffold material (YY/T1561-2017) is carried out according to the detection standard of residual alpha-Gal antigen content and Gal antigen clearance rate in animal source medical maple of quality evaluation room of the institute of medical and hospitalization facilities (NIFDC-SOP-F-T-3001), and the Gal antigen content of the cartilage sample of the pig (GTKO pig, pig used in example 1) with GGTA1 knocked out is 2.17 +/-0.39 multiplied by 1012The Gal antigen content of wild pig (pig used in comparative example 1) in control group is 5.36 + -1.08 × 10 per mg (wet weight)14One/mg (wet weight) with a minimum detection limit of 0.03125 (relative to Gal-BSA) μ g/mL (corresponding to 8.25X 10)11Individual epitopes/each reaction). Each sample is tested by at least 5 different samples, Gal antigen content is calculated, and statistical analysis of data through t-test software shows that p<0.05 had significant differences. Indicating that the sample of example 1 is less immunogenic.
1.2 immunohistochemical detection: the samples were implanted subcutaneously in rats and after a specified period of time, the experimental rats were sacrificed and the implant material and surrounding skin tissue were removed, fixed with 4 wt.% paraformaldehyde fixative and immunohistochemically stained with CD68 monoclonal antibody.
The results are shown in FIG. 1:
the CD68 mab was subjected to immunohistochemical staining, primarily to identify macrophages present in the test sample (dark brown) due to immune rejection. The results show that the example 1 sample found fewer macrophages than the comparative example 1 after 1 month of subcutaneous implantation in rats, indicating that the example 1 sample was less immunogenic than the comparative example 1.
Effect example 2 biological Activity test
2.1 histocompatibility assay: the samples were implanted into rat muscles and after a specific time the experimental rats were sacrificed and the implant material and surrounding muscle tissue were removed and fixed with 4 wt.% paraformaldehyde fixing solution and HE stained.
The results are shown in FIG. 2:
HE staining showed that after 2 months of rat muscle implantation, the sample of example 1 was completely fused with the surrounding muscle tissue and cells were grown into the sample, whereas the sample of comparative example 1 and the surrounding muscle tissue had distinct boundaries, indicating that the sample of example 1 was more bioactive.
Example 2
Bovine cartilage material, its preparation method and cartilage regeneration application are provided.
The preparation method comprises the following steps:
a. preparing a knockout GGTA1 through CRISPR/Cas system gene editing, transferring the knockout GGTA1 into a cattle with hCD46, and editing the cattle by using a gene with stable passage of 5 generations;
b. collecting cartilage of a gene-edited cow as a raw material;
c. the cartilage starting material is treated as follows:
submersed in a 0.07 wt.% SDS solution for 10h with an oscillation frequency of 1000rpm at a temperature of 25 ℃;
ultrasonic cleaning, treating for 8h at 60KHz, with the power of 5KW and the temperature of 30 ℃;
standing at-80 deg.C for 2 hr, at 10 deg.C for 3 hr, at-20 deg.C for 5 hr, and at 40 deg.C for 3 hr;
ultrasonic cleaning, processing for 2h at 120KHz, with the power of 3KW and the temperature of 40 ℃;
submersed in a 0.001 wt.% SDS solution for 10h with an oscillation frequency of 300rpm at a temperature of 40 ℃;
ultrasonic cleaning, processing for 2h at 40KHz, with the power of 1KW and the temperature of 25 ℃;
d. immersing in 0.006 wt.% glutaraldehyde solution for 3h at 25 deg.C for 2 times;
immersing in 0.0001 wt.% genipin solution for 12h at 25 deg.C, and repeating the immersion for 10 times.
The bovine-derived cartilage material prepared by the steps is used for cartilage regeneration.
Comparative example 2
Collecting the common cattle cartilage material. The cell removing treatment comprises the following steps: immersed in a 10 wt.% SDS solution for 12h with an oscillation frequency of 600rpm and a temperature of 25 ℃. The crosslinking treatment comprises the following steps: immersed in a 5 wt.% glutaraldehyde solution for 6h at 25 ℃.
Effect example 3 immunogenicity test
3.1 immunohistochemical detection: the samples were implanted subcutaneously in rats and after a specified period of time, the experimental rats were sacrificed and the implant material and surrounding skin tissue were removed, fixed with 4 wt.% paraformaldehyde fixative and immunohistochemically stained with CD3 monoclonal antibody.
The results are shown in FIG. 3:
CD3 mab was subjected to immunohistochemical staining, primarily to identify T cells (dark brown) present in the test sample due to immune rejection. The results show that the sample of example 2 found fewer T cells than comparative example 2 after 1 month of subcutaneous implantation in rats, indicating that the sample of example 2 was less immunogenic than comparative example 2.
Effect example 4 biological Activity test
4.1 histocompatibility assay: the samples were implanted into rat muscles and after a specific time the experimental rats were sacrificed and the implant material and surrounding muscle tissue were removed and fixed with 4 wt.% paraformaldehyde fixing solution and HE stained.
The results are shown in FIG. 4:
HE staining showed that after 1 month of rat muscle implantation, the sample of example 2 was completely fused with the surrounding muscle tissue and cells were grown into the sample, whereas the sample of comparative example 2 and the surrounding muscle tissue had distinct boundaries, indicating that the sample of example 2 was more bioactive.
Example 3
A rabbit cartilage material and its preparation method and cartilage regeneration application are provided.
The preparation method comprises the following steps:
a. preparing a rabbit with GGTA1 knocked out by TALEN system gene editing, and editing the rabbit by using a gene with stable passage of 3 generations;
b. collecting cartilage of a gene editing rabbit as a raw material;
c. the cartilage starting material is treated as follows:
submersed in 0.1 wt.% SDS solution for 7h with an oscillation frequency of 600rpm at a temperature of 20 ℃;
ultrasonic cleaning, processing for 6h at 80KHz, with the power of 6KW and the temperature of 30 ℃;
placing at-60 deg.C for 4h, at 30 deg.C for 2h, at-60 deg.C for 4h, and at 37 deg.C for 2 h;
ultrasonic cleaning, processing for 5h at 80KHz, with the power of 3KW and the temperature of 30 ℃;
submersed in a 0.001 wt.% SDS solution for 9h with an oscillation frequency of 700rpm at a temperature of 25 ℃;
ultrasonic cleaning, treating for 3h at 60KHz, with power of 4KW and temperature of 25 deg.C;
d. immersing in 0.005 wt.% glutaraldehyde solution for 6h at 25 deg.C for 5 times;
immersing in 0.0005 wt.% genipin solution for 8h at 40 deg.C, and repeating the immersing 10 times.
The rabbit-derived cartilage material prepared by the steps is used for articular cartilage regeneration.
Comparative example 3
Collecting cartilage materials of common rabbits. The cell removing treatment comprises the following steps: immersed in a 5 wt.% SDS solution for 20h with an oscillation frequency of 400rpm and a temperature of 25 ℃. The crosslinking treatment comprises the following steps: submersed in a2 wt.% glutaraldehyde solution for 12h at a submersion temperature of 25 ℃.
Effect example 5 immunogenicity test
5.1 immunohistochemical detection: the samples were implanted subcutaneously in rats and after a specified period of time, the experimental rats were sacrificed and the implant material and surrounding skin tissue were removed, fixed with 4 wt.% paraformaldehyde fixative and immunohistochemically stained with CD3 monoclonal antibody.
The results are shown in FIG. 5:
CD3 mab was subjected to immunohistochemical staining, primarily to identify T cells (dark brown) present in the test sample due to immune rejection. The results show that the sample of example 3 found fewer T cells than comparative example 3 after 1 month of subcutaneous implantation in rats, indicating that the sample of example 3 was less immunogenic than comparative example 3.
Effect example 6 biological Activity test
6.1 histocompatibility assay: the samples were implanted into rat muscles and after a specific time the experimental rats were sacrificed and the implant material and surrounding muscle tissue were removed and fixed with 4 wt.% paraformaldehyde fixing solution and HE stained.
The results are shown in FIG. 6:
HE staining showed that after 1 month of rat muscle implantation, the example 3 sample was completely fused with the surrounding muscle tissue and cells were grown into the sample, whereas the comparative example 3 sample and the surrounding muscle tissue had distinct boundaries, indicating that the example 3 sample was more bioactive.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A preparation method of a cartilage material is characterized by comprising the following steps:
step 1, breeding a gene editing animal;
step 2, collecting cartilage of the animal in the step 1 as a raw material;
step 3, taking the raw material prepared in the step 2 to remove cells;
step 4, crosslinking the material prepared in the step 3;
wherein, the step 3 is specifically as follows:
immersing in 0.01-4 wt.% SDS solution for 3-24 h, with oscillation frequency of 100-1000 rpm and temperature of 0-40 ℃;
ultrasonic cleaning, processing for 2-12 h at 40-120 KHz, with the power of 5-10 KW and the temperature of 25-40 ℃;
placing the mixture at a temperature of between 40 ℃ below zero and 80 ℃ below zero for 2 to 6 hours, placing the mixture at a temperature of between 4 ℃ below zero and 40 ℃ for 1 to 3 hours, placing the mixture at a temperature of between 20 ℃ below zero and 80 ℃ below zero for 3 to 6 hours, and placing the mixture at a temperature of between 10 ℃ below zero and 40 ℃ for 1 to 3 hours;
ultrasonic cleaning, treating for 2-6 h at 60-120 KHz with the power of 0.5-5 KW and the temperature of 25-40 ℃;
immersing the substrate in 0.001-1 wt.% SDS solution for 3-12 h, wherein the oscillation frequency is 300-1000 rpm, and the temperature is 20-40 ℃;
ultrasonic cleaning, processing for 2-6 h at 40-120 KHz, with the power of 1-5 KW and the temperature of 25-40 ℃;
the step 4 specifically comprises the following steps:
immersing in 0.005-5 wt.% glutaraldehyde solution for 1-12 h at 25-40 ℃ for 1-5 times;
immersing the substrate in 0.00001-1 wt.% genipin solution for 1-12 h at 25-40 ℃, and repeating the immersion for 1-10 times.
2. The method of claim 1, wherein the gene editing comprises at least one of meganucleases (Meganuclease), Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR/Cas) systems.
3. The method of claim 1 or 2, wherein said gene editing comprises gene knock-out and/or gene transfer;
the gene knocked out in the gene knock-out comprises at least one of GGTA1, CMAH, β 4GalNT2, or PERV;
the gene transferred by the gene transfer comprises at least one of hCD46, hCD55, hCD47, LEA29Y, hTBM, hTFPI or EPCR.
4. The method of any one of claims 1 to 3, wherein the animal comprises at least one of a pig, a cow, a sheep, a horse, a monkey, a dog, a rabbit, a chicken, a mouse, a silkworm, a fish, a marine organism, a donkey; the animals are stably passaged for more than 3 generations after gene editing; preferably, animals are selected for stable passage 4 after gene editing.
5. Cartilage material of animal origin obtainable by a process according to any one of claims 1 to 4.
6. The use of the cartilage material of animal origin according to claim 5 for the preparation of tissue engineering materials, regenerative medical materials, transformation medical materials.
7. A composition comprising the cartilage material of animal origin of claim 5 and a pharmaceutically acceptable excipient.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030108531A1 (en) * | 2001-11-30 | 2003-06-12 | Hideshige Moriya | Genes for transfection into bony tissues |
| US20140115728A1 (en) * | 2012-10-24 | 2014-04-24 | A. Joseph Tector | Double knockout (gt/cmah-ko) pigs, organs and tissues |
| CN103785065A (en) * | 2014-01-24 | 2014-05-14 | 北京大清生物技术有限公司 | Preparation method of cartilage regeneration scaffold material |
| CN105078889A (en) * | 2015-07-15 | 2015-11-25 | 魏垒 | Liposome delivery system for treating cartilage diseases and preparation method of liposome delivery system |
| WO2016065046A1 (en) * | 2014-10-22 | 2016-04-28 | Indiana University Research & Technology Corporation | Triple transgenic pigs suitable for xenograft |
| CN108642054A (en) * | 2018-05-17 | 2018-10-12 | 武汉博杰生物医学科技有限公司 | Reduce immunogenicity sgRNA, low immunogenicity dressing and preparation method thereof |
| CN109621010A (en) * | 2018-12-17 | 2019-04-16 | 浙江华臻医疗器械有限公司 | A kind of Acellular cartilaginous matrix and preparation method thereof |
| WO2019214591A1 (en) * | 2018-05-07 | 2019-11-14 | 创观（苏州）生物科技有限公司 | Blood product derived from gene knockout pig and use thereof |
-
2020
- 2020-03-23 CN CN202010208243.XA patent/CN113425906A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030108531A1 (en) * | 2001-11-30 | 2003-06-12 | Hideshige Moriya | Genes for transfection into bony tissues |
| US20140115728A1 (en) * | 2012-10-24 | 2014-04-24 | A. Joseph Tector | Double knockout (gt/cmah-ko) pigs, organs and tissues |
| CN103785065A (en) * | 2014-01-24 | 2014-05-14 | 北京大清生物技术有限公司 | Preparation method of cartilage regeneration scaffold material |
| WO2016065046A1 (en) * | 2014-10-22 | 2016-04-28 | Indiana University Research & Technology Corporation | Triple transgenic pigs suitable for xenograft |
| CN105078889A (en) * | 2015-07-15 | 2015-11-25 | 魏垒 | Liposome delivery system for treating cartilage diseases and preparation method of liposome delivery system |
| WO2019214591A1 (en) * | 2018-05-07 | 2019-11-14 | 创观（苏州）生物科技有限公司 | Blood product derived from gene knockout pig and use thereof |
| CN108642054A (en) * | 2018-05-17 | 2018-10-12 | 武汉博杰生物医学科技有限公司 | Reduce immunogenicity sgRNA, low immunogenicity dressing and preparation method thereof |
| CN109621010A (en) * | 2018-12-17 | 2019-04-16 | 浙江华臻医疗器械有限公司 | A kind of Acellular cartilaginous matrix and preparation method thereof |
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Application publication date: 20210924 |