CN115252902A - Preparation method of cartilage extracellular matrix, decellularization composition liquid used by same and prepared cartilage extracellular matrix - Google Patents
Preparation method of cartilage extracellular matrix, decellularization composition liquid used by same and prepared cartilage extracellular matrix Download PDFInfo
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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
- A61L27/3612—Cartilage, synovial fluid
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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
- A61L27/3687—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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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- 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
- A61L27/3695—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 function or physical properties of the final product, where no specific conditions are defined to achieve this
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Abstract
The invention discloses a preparation method of a cartilage extracellular matrix, a decellularization combined solution used by the preparation method and the prepared cartilage extracellular matrix. The preparation method of the cartilage extracellular matrix comprises the following steps: s1, grinding animal source articular cartilage to obtain bone powder; s2, washing the cartilage powder by using a buffer solution containing a protease inhibitor, and centrifuging to obtain a centrifugal precipitate; s3, washing the centrifugal precipitate by using a buffer solution, filtering, and freeze-drying to obtain cartilage powder; s4, adopting the cell removal liquid A, the cell removal liquid B and the cell removal liquid C to sequentially perform cell removal treatment on the cartilage powder, flushing and eluting the cartilage tissue subjected to cell treatment by using a buffer solution containing a protease inhibitor and performing centrifugal treatment to obtain the cell-removed cartilage tissue; and S5, treating the decellularized cartilage tissue with nuclease and centrifuging, washing the centrifuged precipitate with buffer solution, and freeze-drying to obtain the cartilage extracellular matrix. The cartilage extracellular matrix prepared by the method has high quality.
Description
Technical Field
The invention belongs to the technical field of tissue engineering, and particularly relates to a preparation method of a cartilage extracellular matrix, a decellularization combined solution used in the preparation method and the prepared cartilage extracellular matrix.
Background
Articular cartilage is a hyaline cartilage rich in type II collagen and glycosaminoglycan, and plays a role in bearing mechanical load and lubricating joints during joint movement. Unlike other tissues, cartilage tissue has no blood vessels and nerves, and has extremely limited ability of autogenous repair after degeneration and injury, and the cartilage tissue is difficult to repair after the injury reaches 3mm, and the cartilage tissue can cause injury to the lower bone after the injury reaches 6 mm. At present, the surgical methods for treating articular cartilage injury mainly include microfracture, autologous Chondrocyte Implantation (ACI), mosaic plasty, allogeneic cartilage transplantation and the like. However, these methods have certain limitations, cannot completely restore the original cartilage, and the prognosis has uncertainty, so based on this, the implantation of a suitable tissue engineering scaffold provides a better solution to the problem.
The cartilage tissue engineering is to culture and expand cartilage seed cells in vitro under the participation of some factors favorable to cell differentiation and proliferation, culture a certain amount of seed cells on a biomaterial scaffold, then transplant the cells to cartilage defect parts to form new cartilage tissues, reshape the cartilage tissues and integrate the new cartilage tissues with body tissues to achieve the regeneration and repair of damaged cartilage. The content of cartilage tissue engineering is mainly three aspects: the cartilage tissue engineering scaffold comprises seed cells, a biomaterial scaffold and growth factors, wherein the biomaterial scaffold is used as a temporary substitute of extracellular matrix, provides a three-dimensional geometric shape for the engineered cartilage tissue, provides a living microenvironment for the seed cells, and is a key for the adhesion, proliferation and differentiation of the cells and finally forming a new cartilage tissue basic framework and metabolic site, thereby being the successful of the cartilage tissue engineering.
The existing biological scaffold materials mainly comprise natural materials and synthetic materials, wherein artificially synthesized polymers comprise polyglycolic acid, polylactic acid, polyglycolic acid compound, polyvinyl alcohol and the like, and natural polymers comprise cartilage extracellular matrix, agarose, gelatin, hyaluronic acid and the like. Among them, the chondrocyte extracellular matrix is widely used for a scaffold material for tissue engineering due to its excellent biosafety and biocompatibility, and sites for cell recognition and adhesion.
The cartilage extracellular matrix material is prepared by subjecting cartilage tissue to decellularization treatment to remove all cells, antigens, lipids, soluble proteins and other substances in the tissue, and retaining extracellular matrix with intact morphology, histology and ultrastructure. The development of extracellular matrix preparation technology is also directed to complete decellularization while retaining the bioactive components in the maximum amount and reducing various complications caused by materials, and patent 101496913B discloses a method for preparing articular cartilage extracellular matrix, which comprises pulverizing cartilage, and then removing the cellular components by continuous shaking using 0.5-2% triton X-100 Tris-HCl buffer solution. In patent CN107823710B, the cell components are removed by treating with sodium dodecyl sulfate at a concentration of 0.005-0.1% or Triton X-100% at a concentration of 0.01-1%. However, the extracellular matrix obtained by the above method still has a part of impurities such as cells, lipids and the like which are not removed, and the impurities greatly affect the usability of the extracellular matrix, so that it is extremely important to provide a reasonable preparation method of the extracellular matrix.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a decellularization solution used in preparation of a cartilage extracellular matrix, a preparation method of the cartilage extracellular matrix and the prepared cartilage extracellular matrix. The cartilage extracellular matrix prepared by the preparation method of the cartilage extracellular matrix has the characteristics of thorough decellularization, clean antigen removal, complete collagen preservation and the like.
The invention provides a preparation method of cartilage extracellular matrix, which comprises the following steps:
1. cleaning and disinfecting the joints of four limbs of animal sources, wherein the joints of the animal sources comprise the joints of pigs, cows, sheep and horses;
2. opening the joint cavity under the aseptic condition, and cutting cartilage which does not contain lower bone, wherein the operation is carried out under the aseptic condition;
3. grinding the cartilage by adopting a high-flux tissue grinder, wherein the grinding process comprises the following steps: a. putting cartilage into grinding jar, and freezing in liquid nitrogen for 10-15min; b. quickly transferring into a grinding instrument, and starting grinding with the grinding frequency of 50-60Hz and the grinding time of 30-60s;
4. washing cartilage powder by using a buffer solution, wherein the buffer solution is one of Phosphate Buffer Solution (PBS) and 10mM Tris-HCl; the buffer solution also comprises a protease inhibitor, the protease inhibitor is one or two of dimethyl sulfoxide solution of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), phenylmethylsulfonyl fluoride (PMSF) and Diisopropyl Fluorophosphate (DFP), and the concentration of the protease inhibitor is 0.1-0.35mM; preferably, the buffer solution is 10mM Tris-HCl, the protease inhibitor is AEBSF, and the concentration is 0.25-0.3mM; the volume ratio of the buffer solution to the cartilage powder is 10-20:1;
5. putting cartilage powder containing buffer solution into an ultrasonic cleaning machine, and performing ultrasonic treatment for 30-60min at an ultrasonic frequency of 20-25KHZ and an ultrasonic temperature of 30-40 deg.C;
6. centrifuging cartilage powder containing buffer solution by using a centrifuge, wherein the rotating speed of the centrifuge is 7000-10000r/min, and the centrifuging temperature is 4 ℃;
7. washing the centrifuged precipitate with a buffer solution, wherein the buffer solution is one of Phosphate Buffered Saline (PBS) and 10mM Tris-HCl; filtering, and freeze-drying the cartilage powder by using a freeze dryer, wherein the freezing temperature of the freeze dryer is-80 ℃, and the vacuum pressure of the freeze dryer is 10-15Pa, and the method comprises the following specific steps: spreading cartilage powder on a watch glass, freezing in-20 deg.C refrigerator for 1-2 hr, transferring to-80 deg.C refrigerator for freezing for 30-60min, quickly transferring to freeze dryer, and vacuum freeze drying for 12-24 hr to obtain dried cartilage powder;
8. weighing 1 part by weight of cartilage powder, adding 5-10 parts by weight of cell removal liquid A, and oscillating for 2 hours in a table type constant temperature shaking table at the temperature of 4 ℃ and the speed of the shaking table of 100-180rpm; b. stopping the shaking table, sucking the cell removal solution A out, adding the cell removal solution B again, and continuing to start the shaking table for 4h at the temperature of 4 ℃ and the speed of 100-180rpm; c. stopping the shaking table, sucking the cell removal solution B out, adding the cell removal solution C again, and continuously starting the shaking table for 16h at the temperature of 4 ℃;
the cell removal liquid A comprises the following raw materials in parts by weight:
the cell removal solution B comprises the following raw materials in parts by weight:
the cell removal liquid C comprises the following raw materials in parts by weight:
9. washing away the cartilage tissue of the cells by using a buffer solution, wherein the volume ratio of the buffer solution to the cartilage powder is 10-20:1, the buffer solution is one of Phosphate Buffer Solution (PBS) and 10mM Tris-HCl, the buffer solution also comprises protease inhibitors, the protease inhibitors are dimethyl sulfoxide solutions of one or two of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), phenylmethylsulfonyl fluoride (PMSF) and Diisopropyl Fluorophosphate (DFP), and the concentration of the protease inhibitors is 0.1-0.35mM; preferably, the buffer solution is 10mM Tris-HCl, the protease inhibitor is AEBSF, and the concentration is 0.25-0.3mM; after washing, centrifuging at the centrifuge rotation speed of 7000-10000r/min and the centrifugation temperature of 4 ℃;
10. treating the decellularized cartilage tissue by adopting a digestive enzyme mixed solution, wherein the digestive enzyme mixed solution comprises 50-100U/ml DNase and 0.5-2U/ml RNase, and is placed in a constant temperature shaking table to be digested for 12-24 hours at 37 ℃, the oscillation frequency is 100-180rpm, the centrifugation is carried out, the rotation speed of a centrifuge is 7000-10000r/min, and the centrifugation temperature is 4 ℃;
11. washing the centrifugal precipitate by using a buffer solution, wherein the buffer solution is any one of Phosphate Buffer Solution (PBS) and 10mM Tris-HCl, then filtering, and freeze-drying the decellularized cartilage powder by using a freeze dryer, wherein the freezing temperature of the freeze dryer is-80 ℃, and the vacuum pressure is 10-15Pa, and the specific steps are as follows: spreading cartilage powder on a watch glass, freezing in a-20 deg.C refrigerator for 1-2 hr, transferring to a-80 deg.C refrigerator for 30-60min, quickly transferring to a freeze dryer, and vacuum lyophilizing for 12-24 hr to obtain dried cartilage extracellular matrix.
The invention has the beneficial effects that: in the prior art, a single SDS or Triton X-100 is often adopted to treat cartilage tissues for a long time, and in the methods, lipid and cell impurities cannot be well removed when the dosage of the surfactant is too small, the structure of the extracellular matrix is damaged when the dosage is too large, and the overall structure of the extracellular matrix cannot be well maintained. In the invention, a plurality of surfactants are compounded, a plurality of types of surfactants act together, composite acellular fluids with different concentration gradients are adopted, the acellular fluids with higher concentration are adopted for strengthening treatment to remove most of lipids, open cell membranes and remove most of cells, then the acellular fluids with low concentration are adopted for long-time soaking treatment to remove a small amount of residual lipids, cells, soluble proteins and other impurities, and meanwhile, the complete morphology, the histology and the ultrastructure of the extracellular matrix can be maintained.
Drawings
Fig. 1 is a graph of cartilaginous tissue HE staining (× 40);
fig. 2 is a graph of HE staining (× 40) of the extracellular matrix after treatment in example 1;
fig. 3 is a sirius red staining pattern (x 40) of cartilage tissue;
fig. 4 is an extracellular matrix sirius red staining pattern (× 40).
Detailed Description
In order to make the present invention more clear and intuitive for those skilled in the art, the present invention will be further described with reference to the accompanying drawings.
The acellular combined liquid used by the preparation method of the cartilage extracellular matrix comprises acellular liquid A, acellular liquid B and acellular liquid C.
The cell removal solution A comprises the following raw materials in parts by weight:
0.5-0.7 part by weight of sodium laurate glutamate, 0.2-0.4 part by weight of cocamidobetaine, 0.7-1 part by weight of sodium lauryl sulfate, 1-2 parts by weight of AEBSF/dimethyl sulfoxide solution, 0.1-0.3 part by weight of EDTA-2 sodium, and 94.9-97.5 parts by weight of pure water;
the cell removal liquid B comprises the following raw materials in parts by weight:
0.2-0.4 part by weight of sodium laurate glutamate, 0.2-0.4 part by weight of cocamidobetaine, 0.4-0.6 part by weight of sodium lauryl sulfate, 1-2 parts by weight of AEBSF/dimethyl sulfoxide solution, 0.1-0.3 part by weight of EDTA-2 sodium, and 96-97.8 parts by weight of pure water;
the cell removal liquid C comprises the following raw materials in parts by weight:
0.05-0.1 part of sodium laurate glutamate, 0.05-0.1 part of cocoamido betaine, 0.1-0.3 part of sodium lauryl sulfate, 1-2 parts of AEBSF/dimethyl sulfoxide solution, 0.1-0.3 part of EDTA-2 sodium and 97.2-98.7 parts of pure water.
PREFERRED EMBODIMENTS
A preparation method of cartilage extracellular matrix comprises the following steps:
1. cleaning and disinfecting four limb joints of animal sources, wherein the animal source joints are achyranthes bidentata Blume cartilage;
2. opening the joint cavity under aseptic conditions, and cutting cartilage without lower bone, wherein the operations are carried out under aseptic conditions;
3. grinding the cartilage by adopting a high-flux tissue grinder, wherein the grinding process comprises the following steps: a. putting cartilage into grinding jar, and freezing in liquid nitrogen for 10min; b. quickly transferring the mixture into a grinding instrument, and starting grinding at a grinding frequency of 50Hz for 60s;
4. washing the cartilage powder with a buffer solution, wherein the buffer solution is Phosphate Buffered Saline (PBS); the buffer solution also contains a protease inhibitor, the protease inhibitor is a phenylmethylsulfonyl fluoride (PMSF) dimethyl sulfoxide solution, and the concentration of the protease inhibitor is 0.25mM; the dosage ratio of the buffer solution to the cartilage powder is 10:1;
5. putting the cartilage powder containing the buffer solution into an ultrasonic cleaning machine, and carrying out ultrasonic treatment for 30min, wherein the ultrasonic frequency of the ultrasonic cleaning machine is 2KHZ, and the ultrasonic temperature is 30 ℃;
6. centrifuging the cartilage powder containing the buffer solution by adopting a centrifuge, wherein the rotating speed of the centrifuge is 7000r/min, and the centrifuging temperature is 4 ℃;
7. washing the centrifugal precipitate with a buffer solution, filtering, and freeze-drying the cartilage powder by using a freeze dryer, wherein the freezing temperature of the freeze dryer is-80 ℃, and the vacuum pressure is 10Pa, and the method comprises the following specific steps: spreading cartilage powder on a watch glass, freezing in a-20 deg.C refrigerator for 2 hr, transferring to a-80 deg.C refrigerator for freezing for 60min, quickly transferring to a freeze dryer, and vacuum lyophilizing for 24 hr to obtain dry cartilage powder;
8. weighing 1 part by weight of cartilage powder, adding 10 parts by weight of cell removal liquid A, and shaking for 2 hours in a table type constant temperature shaking table at the temperature of 4 ℃ and the speed of the shaking table of 150rpm; b. stopping the shaking table, sucking the cell removal solution A out, adding the cell removal solution B again, and continuing to start the shaking table for 4 hours at the temperature of 4 ℃ and the speed of 150rpm; c. stopping the shaking table, sucking the cell removal solution B out, adding the cell removal solution C again, and continuing to start the shaking table for 18h, wherein the speed of the shaking table is 120rpm, and the temperature of the shaking table is 4 ℃;
the cell removal liquid A component consists of the following raw materials in parts by weight:
the cell removal liquid B component consists of the following raw materials in parts by weight:
the cell removal liquid C comprises the following raw materials in parts by weight:
9. washing away cartilage tissues of cells by using a buffer solution, wherein the buffer solution is Phosphate Buffered Saline (PBS), the buffer solution also comprises a protease inhibitor, the protease inhibitor is a solution of phenylmethylsulfonyl fluoride (PMSF) and dimethyl sulfoxide, and the concentration of the protease inhibitor is 0.25mM; after washing, centrifuging at the rotating speed of 7000r/min and the centrifuging temperature of 4 ℃;
10. treating the decellularized cartilage tissue by adopting a digestive enzyme mixed solution, wherein the digestive enzyme mixed solution contains 50U/ml DNase and 0.5U/ml RNase, and is placed in a constant temperature shaking table for digestion at 37 ℃ for 12 hours, and the oscillation frequency is 150rpm;
11. washing the centrifugal precipitate with buffer solution, filtering, and freeze-drying the cell-removed cartilage powder with a freeze dryer at-80 deg.C under vacuum pressure of 10Pa, which comprises the following steps: spreading cartilage powder on a watch glass, freezing for 2h in a-20 ℃ refrigerator, then transferring to a-80 ℃ refrigerator for freezing for 60min, quickly transferring to a freeze dryer for vacuum freeze-drying for 24h, and finally obtaining dry cartilage extracellular matrix;
and (4) performance testing:
1, HE staining tissue staining observation, wherein hematoxylin staining solution is alkaline, and is mainly used for making chromatin in cell nuclei and ribosome in cytoplasm bluish; eosin is an acid dye that primarily reddens components of the cytoplasm and extracellular matrix; before decellularization, the cartilage tissue has complete cell morphology, the cell nucleus edge is clearly visible and is in obvious cartilage tissue morphology (figure 1), after the cartilage tissue is treated by the method, the cartilage tissue is basically cytoplasm and extracellular matrix (the image observed in the experiment is red), no cell structure exists, and no cell nucleus exists (the cell nucleus in the experiment is purple blue), as shown in figure 2.
2. The observation of the dyeing of the scarlet tissues shows that the staining solution of the scarlet can dye collagen into red, the cell morphology of the cartilage tissue is complete before the cartilage tissue is decellularized (figure 3), the original cell morphology completely disappears after the treatment of the method of the invention, and the content of the residual collagen is rich and is not obviously reduced (figure 4).
3. And (3) quantitatively testing the collagen content, namely testing the collagen content before and after reaction of the cartilage tissue by using a collagen quantitative kit, and respectively testing five groups, wherein the test results are shown in table 1, and the retention amount of the collagen in the ECM is 86.2% by calculation.
TABLE 1
| Collagen content (ug/mg) | 1 | 2 | 3 | 4 | 5 | Average out |
| Cartilage tissue | 332.6 | 327.4 | 334.6 | 341.5 | 322.7 | 331.8 |
| ECM | 286.3 | 283.2 | 290.1 | 288.7 | 281.6 | 285.9 |
Glycosaminoglycan GAGs quantitative test using GAGs quantitative test kits to test the collagen content before and after cartilage reaction, the test results are shown in table 2, and the retention amount of GAGs in ECM is 93.3% by calculation.
TABLE 2
| Content of GAGs (ug/mg) | 1 | 2 | 3 | 4 | 5 | Average out |
| Cartilage tissue | 239 | 244 | 242 | 243 | 244 | 242.4 |
| ECM | 225 | 228 | 226 | 227 | 225 | 226.2 |
And (2) DNA residue quantitative testing, wherein a DNA quantitative kit is adopted to test the DNA content before and after reaction, and the DNA residue in the extracellular matrix (ECM) is tested for three times respectively, as shown in Table 3, the average residual quantity of the DNA in the ECM is 29.98ng/mg, the DNA removal rate reaches 96.5%, and the DNA removal effect is good by combining a dyeing result.
TABLE 3
| Cartilage tissue | ECM 1 | ECM 2 | ECM 3 | |
| DNA content (ng/mg) | 850.32 | 32.67 | 26.35 | 30.92 |
The above medicines are purchased from outsourcing, wherein sodium laurate glutamate, cocoamidobetaine, sodium lauryl sulfate, and EDTA-2 sodium are purchased from Allantin, and PBS, phenylmethylsulfonyl fluoride, DNase, RNase, and dimethyl sulfoxide are purchased from sigma.
The embodiments described above are presented to facilitate one of ordinary skill in the art to understand and practice the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art should make modifications and alterations to the present invention in light of the present disclosure.
Claims (10)
1. A method for preparing cartilage extracellular matrix is characterized by comprising the following steps:
s1, grinding animal source articular cartilage to obtain bone powder;
s2, washing the cartilage powder by using a buffer solution containing a protease inhibitor, and centrifuging to obtain a centrifugal precipitate;
s3, washing the centrifugal precipitate by using a buffer solution, filtering, and freeze-drying to obtain cartilage powder;
s4, carrying out decellularization treatment on the cartilage powder by adopting the decellularized solution A, the decellularized solution B and the decellularized solution C in sequence, flushing and eluting the cartilage tissue subjected to the cell treatment by using a buffer solution containing a protease inhibitor, and carrying out centrifugal treatment to obtain a decellularized cartilage tissue;
s5, treating the decellularized cartilage tissue with nuclease and centrifuging, washing the centrifugal precipitate with buffer solution, and freeze-drying to obtain cartilage extracellular matrix;
wherein the cell removal solution A comprises the following raw materials in parts by weight:
0.5-0.7 part by weight of sodium laurate glutamate, 0.2-0.4 part by weight of cocamidobetaine, 0.7-1 part by weight of sodium lauryl sulfate, 1-2 parts by weight of AEBSF/dimethyl sulfoxide solution, 0.1-0.3 part by weight of EDTA-2 sodium, and 94.9-97.5 parts by weight of pure water;
the cell removal solution B comprises the following raw materials in parts by weight:
0.2-0.4 part by weight of sodium laurate glutamate, 0.2-0.4 part by weight of cocamidobetaine, 0.4-0.6 part by weight of sodium lauryl sulfate, 1-2 parts by weight of AEBSF/dimethyl sulfoxide solution, 0.1-0.3 part by weight of EDTA-2 sodium, and 96-97.8 parts by weight of pure water;
the cell removal liquid C comprises the following raw materials in parts by weight:
0.05-0.1 part of sodium laurate glutamate, 0.05-0.1 part of cocamidobetaine, 0.1-0.3 part of sodium dodecyl sulfate, 1-2 parts of AEBSF/dimethyl sulfoxide solution, 0.1-0.3 part of EDTA-2 sodium and 97.2-98.7 parts of pure water.
2. The production method according to claim 1, characterized in that: in step S2, the cartilage powder is washed in an ultrasonic cleaning machine, preferably, the ultrasonic time is 30-60min, the ultrasonic frequency of the ultrasonic cleaning machine is 20-25KHZ, and the ultrasonic temperature is 30-40 ℃.
3. The method of claim 1, wherein: in the step S1, the animal source articular cartilage is articular cartilage of pigs and/or cows and/or sheep and/or horses; the grinding process of the animal source articular cartilage comprises the following steps: a. putting cartilage into grinding jar, and freezing in liquid nitrogen for 10-15min; b. quickly transferring into a grinding instrument, and starting grinding with a grinding frequency of 50-60Hz and a grinding time of 30-60s.
4. The method of claim 1, wherein: in the steps S2-S5, the buffer solution is one of phosphate buffer solution and 10mM Tris-HCl, preferably the buffer solution is 10mM Tris-HCl;
in the steps S2 and S4, the protease inhibitor is one or two of dimethyl sulfoxide solution of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate, and the concentration of the protease inhibitor is 0.1-0.35mM;
preferably, the protease inhibitor is 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride at a concentration of 0.25-0.3mM.
5. The production method according to claim 1, characterized in that: in the steps S2, S4 and S5, the rotating speed of the centrifugal machine is 7000-10000r/min, and the centrifugal temperature is 4 ℃.
6. The production method according to claim 1, characterized in that: in steps S3 and S5, the step of freeze-drying is: spreading cartilage powder on a watch glass, freezing in-20 deg.C refrigerator for 1-2 hr, transferring to-80 deg.C refrigerator for freezing for 30-60min, quickly transferring to freeze dryer, and vacuum freeze drying for 12-24 hr to obtain dried cartilage powder; preferably, the freeze-drying machine has a freezing temperature of-80 deg.C and a vacuum pressure of 10-15Pa.
7. The method of claim 1, wherein: in step S4, the process of removing cells from the cartilage powder is:
weighing 1 part by weight of cartilage powder, adding 5-10 parts by weight of cell removal liquid A, and oscillating for 2h in a table type constant temperature shaking table at the temperature of 4 ℃ and the speed of the shaking table of 100-180rpm;
stopping the shaking table, sucking out the cell removal solution A, adding the cell removal solution B again, and continuously starting the shaking table for 4h at the temperature of 4 ℃ and the speed of 100-180rpm;
stopping the shaking table, sucking the cell removal solution B out, adding the cell removal solution C again, and continuously starting the shaking table for 16h at the temperature of 4 ℃.
8. The method of claim 1, wherein: in step S5, the digestive enzyme mixed solution contains 50-100U/ml DNase and 0.5-2U/ml RNase, and is placed in a constant temperature shaking table for digestion at 37 ℃ for 12-24 hours, and the oscillation frequency is 100-180rpm.
9. A decellularized composition liquid for use in the preparation of a chondrocyte extracellular matrix, comprising: comprises a cell removal solution A, a cell removal solution B and a cell removal solution C, wherein:
the cell removal solution A comprises the following raw materials in parts by weight:
0.5-0.7 part of sodium laurate glutamate, 0.2-0.4 part of cocoamido betaine, 0.7-1 part of sodium lauryl sulfate, 1-2 parts of AEBSF/dimethyl sulfoxide solution, 0.1-0.3 part of EDTA-2 sodium and 94.9-97.5 parts of pure water;
the cell removal liquid B comprises the following raw materials in parts by weight:
0.2-0.4 part by weight of sodium laurate glutamate, 0.2-0.4 part by weight of cocamidobetaine, 0.4-0.6 part by weight of sodium lauryl sulfate, 1-2 parts by weight of AEBSF/dimethyl sulfoxide solution, 0.1-0.3 part by weight of EDTA-2 sodium, and 96-97.8 parts by weight of pure water;
the cell removal liquid C comprises the following raw materials in parts by weight:
0.05-0.1 part of sodium laurate glutamate, 0.05-0.1 part of cocamidobetaine, 0.1-0.3 part of sodium dodecyl sulfate, 1-2 parts of AEBSF/dimethyl sulfoxide solution, 0.1-0.3 part of EDTA-2 sodium and 97.2-98.7 parts of pure water.
10. A chondrocyte extracellular matrix produced by the method for producing a chondrocyte extracellular matrix according to any one of claims 1 to 8.
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| CN113577391A (en) * | 2021-08-20 | 2021-11-02 | 浙江大学医学院附属邵逸夫医院 | Preparation method of epiphyseal cartilage combined bone acellular material from natural tissue source |
| WO2021235352A1 (en) * | 2020-05-20 | 2021-11-25 | 株式会社ダイセル | Emulsifiable preparation, aqueous cosmetic, food or beverage and pharmaceutical composition |
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| CN105664255A (en) * | 2016-01-20 | 2016-06-15 | 林贤丰 | Method for preparing synchondrosis bone acellular materials from natural tissue origins |
| WO2021235352A1 (en) * | 2020-05-20 | 2021-11-25 | 株式会社ダイセル | Emulsifiable preparation, aqueous cosmetic, food or beverage and pharmaceutical composition |
| CN113577391A (en) * | 2021-08-20 | 2021-11-02 | 浙江大学医学院附属邵逸夫医院 | Preparation method of epiphyseal cartilage combined bone acellular material from natural tissue source |
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