CN115252902B - Preparation method of cartilage extracellular matrix, decellularized combined solution used by preparation method and prepared cartilage extracellular matrix - Google Patents
Preparation method of cartilage extracellular matrix, decellularized combined solution used by preparation method and prepared cartilage extracellular matrix Download PDFInfo
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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
- A61L27/3612—Cartilage, synovial fluid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A—HUMAN NECESSITIES
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- 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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- 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
- A61L27/3695—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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
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Abstract
The invention discloses a preparation method of a cartilage extracellular matrix, a decellularized 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-derived articular cartilage to obtain bone meal; s2, flushing cartilage powder with a buffer solution containing a protease inhibitor, and centrifuging to obtain centrifugal precipitate; s3, cleaning the centrifugal precipitate by using a buffer solution, filtering and freeze-drying to obtain cartilage powder; s4, sequentially performing cell removal treatment on the cartilage powder by using a cell removal liquid A, a cell removal liquid B and a cell removal liquid C, flushing the cell-removed cartilage tissue by using a buffer solution containing a protease inhibitor, and performing centrifugal treatment to obtain the cell-removed cartilage tissue; s5, treating the decellularized cartilage tissue with nuclease, centrifuging, washing the centrifugal precipitate with buffer solution, and freeze-drying to obtain the cartilage extracellular matrix. The cartilage extracellular matrix prepared by the invention 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 decellularized combined solution used by the preparation method and the prepared cartilage extracellular matrix.
Background
The articular cartilage is hyaline cartilage rich in type II collagen and glycosaminoglycan, and plays roles in bearing mechanical load and lubricating joints in joint movement. Unlike other tissues, cartilage tissue has no blood vessel or nerve, and has extremely limited self-repairing capability after degeneration and injury, and is difficult to repair after injury reaches 3mm, and injury to lower bone is caused after injury reaches 6 mm. At present, the operation methods for treating the articular cartilage injury mainly comprise micro fracture, autologous Chondrocyte Implantation (ACI), mosaic forming, allogenic cartilage transplantation and the like. However, these methods have certain limitations, such as the inability to fully restore autologous original cartilage and uncertainty in prognosis, and implantation of appropriate scaffolds for tissue engineering provides a better solution to this 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 certain amount of seed cells on a biological material bracket, then transplant the seed cells to cartilage defect parts to form new cartilage tissues, and integrate the new cartilage tissues with body tissues after reshaping to achieve the regeneration and repair of damaged cartilage. The content of cartilage tissue engineering is mainly three aspects: seed cells, biomaterial scaffolds and growth factors, wherein the biomaterial scaffolds serve as temporary substitutes of extracellular matrixes, provide three-dimensional geometric shapes for engineering cartilage tissues, provide living microenvironments for the seed cells, adhere, proliferate and differentiate the cells and finally form new cartilage tissue basic frameworks and metabolic sites, and are key to success of cartilage tissue engineering.
The current biological scaffold materials mainly comprise natural and synthetic polymers, wherein the synthetic polymers comprise polyglycolic acid, polylactic acid, polyglycolic acid compound, polyvinyl alcohol and the like, and the natural polymers comprise cartilage extracellular matrix, agarose, gelatin, hyaluronic acid and the like. Among them, the chondrocyte extracellular matrix is widely used for tissue engineering scaffold materials due to its good biosafety and biocompatibility and sites for cell recognition and adhesion.
The cartilage extracellular matrix material is prepared by removing all cells, antigens, lipids, soluble proteins and other substances from cartilage tissue by decellularizing, and retaining extracellular matrix with complete morphology, histology and ultrastructure. The development of the extracellular matrix preparation technology is also to completely decellularize, simultaneously maximally limit the retention of bioactive components and reduce various complications caused by materials, and in patent 101496913B, a preparation method of articular cartilage extracellular matrix is disclosed, cartilage is firstly crushed, and then 0.5-2% Triton X-100 Tris-HCl buffer solution is used for continuously shaking to remove the cellular components. In patent CN107823710B, the cell components are removed by treatment 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 method still has a part of unremoved impurities such as cells and lipids in the system, and the impurities influence the usability of the extracellular matrix to a great extent, 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 decellularized liquid used in the 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 cell removal, clean antigen removal, complete collagen preservation and the like.
The invention provides a preparation method of a cartilage extracellular matrix, which comprises the following steps:
1. cleaning and sterilizing animal-derived joints of limbs including joints of pigs, cattle, sheep and horses;
2. opening the joint cavity under aseptic condition, and cutting cartilage which does not contain lower bone, wherein the operation is required to be carried out under aseptic condition;
3. the cartilage is ground by a high-flux tissue grinder, and the grinding process is as follows: a. placing cartilage into grinding tank, and freezing in liquid nitrogen for 10-15min; b. quickly transferring into a grinding instrument, starting grinding with the grinding frequency of 50-60Hz and the grinding time of 30-60s;
4. flushing cartilage powder by using a buffer solution, wherein the buffer solution is one of Phosphate Buffer (PBS) and 10mM Tris-HCl; the buffer solution also contains a protease inhibitor, wherein the protease inhibitor is one or two dimethyl sulfoxide solutions 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 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, a step of;
5. placing the cartilage powder containing the buffer solution into an ultrasonic cleaner, and performing ultrasonic treatment for 30-60min, wherein the ultrasonic frequency of the ultrasonic cleaner is 20-25KHZ, and the ultrasonic temperature is 30-40 ℃;
6. centrifuging cartilage powder containing buffer solution by adopting a centrifuge, wherein the rotation speed of the centrifuge is 7000-10000r/min, and the centrifuging temperature is 4 ℃;
7. washing the centrifugal precipitate by using a buffer solution, wherein the buffer solution is one of Phosphate Buffer (PBS) and 10mM Tris-HCl; filtering, freeze drying cartilage powder with freeze drier at-80 deg.c and vacuum pressure of 10-15Pa, and the process includes the following steps: spreading cartilage powder on a surface dish, freezing in a refrigerator at-20deg.C for 1-2 hr, transferring to a refrigerator at-80deg.C, freezing for 30-60min, and rapidly transferring to a freeze dryer for vacuum freeze drying for 12-24 hr to obtain dry cartilage powder;
8. the process of removing cells of cartilage powder comprises a. Weighing 1 part by weight of cartilage powder, adding 5-10 parts of cell removing liquid A, oscillating at constant temperature in a table type constant temperature shaking table for 2h, wherein the shaking table temperature is 4 ℃, and the shaking table speed is 100-180rpm; b. stopping the shaking table, sucking out the decellularized liquid A, adding the decellularized liquid B again, continuously starting the shaking table for 4 hours, wherein the temperature of the shaking table is 4 ℃, and the shaking table speed is 100-180rpm; c. stopping the shaking table, sucking out the cell removing liquid B, adding the cell removing liquid C again, and continuously starting the shaking table for 16 hours at the temperature of 4 ℃;
the decellularized liquid A comprises the following raw materials in parts by weight:
the decellularized liquid B comprises the following raw materials in parts by weight:
the decellularized liquid C comprises the following raw materials in parts by weight:
9. rinsing the cell-free cartilage tissue with a buffer solution, wherein the volume ratio of the buffer solution to the cartilage powder is 10-20: 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 dimethyl sulfoxide solutions 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 is 10mM Tris-HCl, the protease inhibitor is AEBSF, and the concentration is 0.25-0.3mM; after washing, centrifuging, wherein the rotation speed of the centrifuge is 7000-10000r/min, and the centrifuging temperature is 4 ℃;
10. treating the cell-removed 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 for digestion for 12-24 hours at 37 ℃, the oscillating frequency is 100-180rpm, the centrifugal machine rotating speed is 7000-10000r/min, and the centrifugal 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, filtering, and freeze-drying the cell-free cartilage powder by adopting 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 surface dish, freezing in a refrigerator at-20deg.C for 1-2 hr, transferring to a refrigerator at-80deg.C, freezing for 30-60min, and rapidly transferring to a freeze dryer for vacuum freeze drying for 12-24 hr to obtain dry cartilage extracellular matrix.
The invention has the beneficial effects that: in the prior art, single SDS or Triton X-100 is often adopted to treat cartilage tissues for a long time, the surfactant is excessively small in the methods, lipid and cell impurities cannot be removed well, the structure of extracellular matrix can be destroyed due to the excessive amount of surfactant, and the integral structure of extracellular matrix cannot be maintained well. The invention adopts the combination of a plurality of surfactants and the combined action of a plurality of types of surfactants, adopts the compound decellularized liquid with different concentration gradients, starts to adopt the reinforced treatment of the decellularized liquid with higher concentration, removes most of lipid, opens cell membranes, removes most of cells, adopts the long-time soaking treatment of the decellularized liquid with low concentration to remove the remaining few lipid, cell, soluble protein and other impurities, and simultaneously can also maintain the complete morphology, histology and ultrastructure of the extracellular matrix.
Drawings
Fig. 1 is a graph of HE staining of cartilage tissue (x 40);
fig. 2 is a HE staining pattern of the extracellular matrix after treatment in example 1 (x 40);
fig. 3 is a graph of staining of the cartilage tissue with sirius scarlet (x 40);
fig. 4 is a graph of staining of the extracellular matrix sirius scarlet (x 40).
Detailed Description
The present invention will be further described with reference to the accompanying drawings for a clear and intuitive understanding to those skilled in the art.
The acellular composite solution used in the preparation method of the cartilage extracellular matrix comprises acellular solution A, acellular solution B and acellular solution C.
The decellularized liquid A comprises the following raw materials in parts by weight:
0.5 to 0.7 weight part of lauric acid sodium glutamate, 0.2 to 0.4 weight part of cocamidobetaine, 0.7 to 1 weight part of sodium dodecyl sulfate, 1 to 2 weight parts of AEBSF/dimethyl sulfoxide solution, 0.1 to 0.3 weight part of EDTA-2 sodium and 94.9 to 97.5 weight parts of pure water;
the decellularized liquid B comprises the following raw materials in parts by weight:
0.2-0.4 part by weight of lauric acid sodium glutamate, 0.2-0.4 part by weight of cocamidobetaine, 0.4-0.6 part by weight of sodium dodecyl 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 decellularized liquid C comprises the following raw materials in parts by weight:
0.05-0.1 part of lauric acid sodium 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.
PREFERRED EMBODIMENTS
A method for preparing a chondrocyte extracellular matrix, comprising the steps of:
1. cleaning and sterilizing the joints of the limbs of animal sources, wherein the joints of animal sources are achyranthes bidentata cover cartilages;
2. opening the joint cavity under aseptic condition, and cutting cartilage which does not contain lower bone, wherein the operation is required to be carried out under aseptic condition;
3. the cartilage is ground by a high-flux tissue grinder, and the grinding process is as follows: a. placing cartilage into a grinding tank, and freezing in liquid nitrogen for 10min; b. quickly transferring into a grinding instrument, starting grinding with the grinding frequency of 50Hz and the grinding time of 60s;
4. rinsing the cartilage powder with a buffer, wherein the buffer is Phosphate Buffer (PBS); the buffer solution also comprises a protease inhibitor, wherein the concentration of the protease inhibitor is 0.25mM, and the protease inhibitor is a phenylmethylsulfonyl fluoride (PMSF) dimethyl sulfoxide solution; the dosage ratio of the buffer solution to the cartilage powder is 10:1, a step of;
5. placing the cartilage powder containing the buffer solution into an ultrasonic cleaner, and performing ultrasonic treatment for 30min, wherein the ultrasonic frequency of the ultrasonic cleaner is 2KHZ, and the ultrasonic temperature is 30 ℃;
6. centrifuging cartilage powder containing buffer solution by adopting a centrifuge, wherein the rotation speed of the centrifuge is 7000r/min, and the centrifugation temperature is 4 ℃;
7. washing the centrifugal precipitate by using a buffer solution, filtering, and freeze-drying the cartilage powder by adopting a freeze dryer, wherein the freezing temperature of the freeze dryer is-80 ℃ and the vacuum pressure is 10Pa, and the specific steps are as follows: spreading cartilage powder on a surface dish, freezing in a refrigerator at-20deg.C for 2 hr, transferring to a refrigerator at-80deg.C for 60min, rapidly transferring to a freeze dryer, and vacuum freeze-drying for 24 hr to obtain dry cartilage powder;
8. the process of removing cells of cartilage powder comprises a. Weighing 1 part by weight of cartilage powder, adding 10 parts of cell removing liquid A, shaking at constant temperature in a table type constant temperature shaking table for 2h, wherein the shaking table temperature is 4 ℃, and the shaking table speed is 150rpm; b. stopping the shaking table, sucking out the decellularized liquid A, adding the decellularized liquid B again, continuously starting the shaking table for 4 hours, wherein the temperature of the shaking table is 4 ℃, and the shaking table speed is 150rpm; c. stopping the shaking table, sucking out the cell removing liquid B, adding the cell removing liquid C again, continuously starting the shaking table for 18 hours, wherein the shaking table speed is 120rpm, and the shaking table temperature is 4 ℃;
the acellular fluid A consists of the following raw materials in parts by weight:
the acellular fluid B comprises the following raw materials in parts by weight:
the acellular fluid C comprises the following raw materials in parts by weight:
9. rinsing the decellularized cartilage tissue with a buffer solution, wherein the buffer solution is Phosphate Buffer (PBS), and the buffer solution further comprises a protease inhibitor, wherein the protease inhibitor is a phenylmethylsulfonyl fluoride (PMSF) dimethyl sulfoxide solution, and the concentration of the protease inhibitor is 0.25mM; after washing, centrifuging, wherein the rotation speed of the centrifuge is 7000r/min, and the centrifuging temperature is 4 ℃;
10. treating the cell-removed cartilage tissue by adopting a digestive enzyme mixed solution, wherein the digestive enzyme mixed solution comprises 50U/ml DNase and 0.5U/ml RNase, and the digestive enzyme mixed solution is placed in a constant-temperature shaking table for digestion for 12 hours at 37 ℃ and has a shaking frequency of 150rpm;
11. washing the centrifugal precipitate by using a buffer solution, filtering, and freeze-drying the cell-free cartilage powder by adopting a freeze dryer, wherein the freezing temperature of the freeze dryer is-80 ℃ and the vacuum pressure is 10Pa, and the specific steps are as follows: spreading cartilage powder on a surface dish, freezing in a refrigerator at-20deg.C for 2 hr, transferring to a refrigerator at-80deg.C for 60min, rapidly transferring to a freeze dryer, and vacuum freeze-drying for 24 hr to obtain dry cartilage extracellular matrix;
performance test:
HE staining tissue staining observation, wherein hematoxylin staining solution is alkaline, and mainly causes chromatin in cell nuclei and ribosomes in cytoplasm to be purple blue; eosin is an acid dye that primarily reds the cytoplasmic and extracellular matrix components; the cartilage tissue was intact before decellularization, the cell morphology was clearly visible at the nucleus border, and it was in the form of distinct cartilage tissue (fig. 1), and after treatment by the method of the invention, it was essentially cytoplasmic and extracellular matrix (red in the experimentally observed image), no cellular structure was present, and no nucleus was present (purple blue in the experimentally observed nucleus), as shown in fig. 2.
2. The staining observation of the sirius red tissue shows that the sirius red staining solution can stain 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 by the method of the invention, and the residual collagen content is rich and does not obviously decrease (figure 4).
3. And (3) quantitatively testing the collagen content, wherein five groups of the collagen content are tested respectively before and after the cartilage tissue reaction by adopting a collagen quantitative kit, and the test results are shown in table 1, and the retention of the collagen in the ECM is 86.2% by calculation.
TABLE 1
| Collagen content (ug/mg) | 1 | 2 | 3 | 4 | 5 | Average of |
| 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 |
Glycosaminoglycans quantitative test the collagen content of cartilage before and after reaction was tested using a GAGs quantitative test kit, and the test results are shown in table 2, and the retention of GAGs in ECM was 93.3% by calculation.
TABLE 2
| GAGs content (ug/mg) | 1 | 2 | 3 | 4 | 5 | Average of |
| Cartilage tissue | 239 | 244 | 242 | 243 | 244 | 242.4 |
| ECM | 225 | 228 | 226 | 227 | 225 | 226.2 |
DNA residue quantitative test, the DNA content before and after the reaction is tested by using a DNA quantitative kit, and the DNA residues in extracellular matrix (ECM) are respectively tested for three times, as shown in Table 3, the average residual quantity of the DNA in the ECM is 29.98ng/mg through calculation, the DNA removal rate reaches 96.5 percent, and the DNA removal effect can be obtained by combining the dyeing result.
TABLE 3 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 Allatin, PBS, phenylmethylsulfonyl fluoride, DNase, RNase, and dimethyl sulfoxide.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments 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, based on the present disclosure, should make improvements and modifications within the scope of the present invention.
Claims (12)
1. A method for preparing a chondrocyte extracellular matrix, comprising the steps of:
s1, grinding animal-derived articular cartilage to obtain bone meal;
s2, flushing cartilage powder with a buffer solution containing a protease inhibitor, and centrifuging to obtain centrifugal precipitate;
s3, cleaning the centrifugal precipitate by using a buffer solution, filtering and freeze-drying to obtain cartilage powder;
s4, sequentially performing cell removal treatment on the cartilage powder by using a cell removal liquid A, a cell removal liquid B and a cell removal liquid C, flushing the cell-removed cartilage tissue by using a buffer solution containing a protease inhibitor, and performing centrifugal treatment to obtain the cell-removed cartilage tissue;
s5, treating the decellularized cartilage tissue with nuclease and centrifuging, cleaning the centrifugal precipitate with buffer solution, and freeze-drying to obtain a cartilage extracellular matrix;
wherein, the decellularized liquid A comprises the following raw materials in parts by weight:
0.5 to 0.7 weight part of lauric acid sodium glutamate, 0.2 to 0.4 weight part of cocamidobetaine, 0.7 to 1 weight part of sodium dodecyl sulfate, 1 to 2 weight parts of AEBSF/dimethyl sulfoxide solution, 0.1 to 0.3 weight part of EDTA-2 sodium and 94.9 to 97.5 weight parts of pure water;
the decellularized liquid B comprises the following raw materials in parts by weight:
0.2-0.4 part by weight of lauric acid sodium glutamate, 0.2-0.4 part by weight of cocamidobetaine, 0.4-0.6 part by weight of sodium dodecyl 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 decellularized liquid C comprises the following raw materials in parts by weight:
0.05-0.1 part of lauric acid sodium 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 method of manufacturing according to claim 1, characterized in that: in the step S2, the cartilage powder is washed in an ultrasonic cleaner for 30-60min, the ultrasonic frequency of the ultrasonic cleaner is 20-25KHZ, and the ultrasonic temperature is 30-40 ℃.
3. The method of manufacturing according to claim 1, characterized in that: in the step S1, the animal-derived articular cartilage is the articular cartilage of pigs and/or cattle and/or sheep and/or horses; the grinding process of the animal-derived articular cartilage is as follows: a. placing cartilage into grinding tank, and freezing in liquid nitrogen for 10-15min; b. quickly transferring into a grinder, starting grinding with the grinding frequency of 50-60Hz and the grinding time of 30-60s.
4. The method of manufacturing according to claim 1, characterized in that: in the step S2-S5, the buffer solution is one of phosphate buffer solution and 10mM Tris-HCl;
in the steps S2 and S4, the protease inhibitor is one or two dimethyl sulfoxide solutions of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate, and the concentration of the protease inhibitor is 0.1-0.35mM.
5. The method of manufacturing according to claim 4, wherein: in the steps S2-S5, the buffer solution is 10mM Tris-HCl;
in steps S2 and S4, the protease inhibitor is 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride at a concentration of 0.25-0.3mM.
6. The method of manufacturing according to claim 1, characterized in that: in the steps S2, S4 and S5, the rotation speed of the centrifugal machine is 7000-10000r/min, and the centrifugal temperature is 4 ℃.
7. The method of manufacturing according to claim 1, characterized in that: in the steps S3 and S5, the freeze drying steps are as follows: spreading cartilage powder on a surface dish, freezing in a refrigerator at-20deg.C for 1-2 hr, transferring to a refrigerator at-80deg.C, freezing for 30-60min, and rapidly transferring to a freeze dryer for vacuum freeze drying for 12-24 hr to obtain dry cartilage powder.
8. The method of manufacturing according to claim 7, wherein: in the steps S3 and S5, the freezing temperature of the freeze dryer is-80 ℃ and the vacuum pressure is 10-15Pa.
9. The method of manufacturing according to claim 1, characterized in that: in step S4, the process of decellularizing cartilage powder is:
weighing 1 part by weight of cartilage powder, adding 5-10 parts by weight of decellularized liquid A, oscillating for 2 hours at constant temperature in a table type constant-temperature shaking table, wherein the shaking table temperature is 4 ℃, and the shaking table speed is 100-180rpm;
stopping the shaking table, sucking out the decellularized liquid A, adding the decellularized liquid B again, continuously starting the shaking table for 4 hours, wherein the temperature of the shaking table is 4 ℃, and the shaking table speed is 100-180rpm;
stopping shaking table, sucking out the cell removing liquid B, adding the cell removing liquid C again, and continuously starting the shaking table for 16h at the temperature of 4 ℃.
10. The method of manufacturing according to claim 1, characterized in that: in the step S5, the nuclease comprises 50-100U/ml DNase and 0.5-2U/ml RNase, and is put into a constant temperature shaking table for digestion for 12-24 hours at 37 ℃ and the shaking frequency is 100-180rpm.
11. A decellularized composite fluid for use in preparing a chondrocyte extracellular matrix, comprising: comprises a decellularized liquid A, a decellularized liquid B and a decellularized liquid C which are respectively and independently used in preparation of a cartilage extracellular matrix, wherein:
the decellularized liquid A comprises the following raw materials in parts by weight:
0.5 to 0.7 weight part of lauric acid sodium glutamate, 0.2 to 0.4 weight part of cocamidobetaine, 0.7 to 1 weight part of sodium dodecyl sulfate, 1 to 2 weight parts of AEBSF/dimethyl sulfoxide solution, 0.1 to 0.3 weight part of EDTA-2 sodium and 94.9 to 97.5 weight parts of pure water;
the decellularized liquid B comprises the following raw materials in parts by weight:
0.2-0.4 part by weight of lauric acid sodium glutamate, 0.2-0.4 part by weight of cocamidobetaine, 0.4-0.6 part by weight of sodium dodecyl 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 decellularized liquid C comprises the following raw materials in parts by weight:
0.05-0.1 part of lauric acid sodium 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.
12. A chondrocyte extracellular matrix prepared by the method for preparing a chondrocyte extracellular matrix according to any one of claims 1 to 10.
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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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