CN111110919A

CN111110919A - Preparation method of omentum majus acellular matrix material and construction method of cartilage tissue

Info

Publication number: CN111110919A
Application number: CN201911397490.2A
Authority: CN
Inventors: 万绵水; 林创鑫
Original assignee: Guangdong Hongzhi Biotechnology Co ltd
Current assignee: Guangdong Hongzhi Biotechnology Co ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2020-05-08
Also published as: WO2021135564A1

Abstract

The invention provides a preparation method of a large omentum decellularized matrix material and a construction method of cartilage tissue. The preparation method comprises the following steps: cleaning the omentum majus, performing freeze-thaw cycle treatment to obtain a first intermediate, adding the first intermediate into a solution containing trypsin and phenylmethylsulfonyl fluoride and having a pH value of 7.8-8.2, continuing for a first preset time to obtain a second intermediate, performing degreasing treatment on the second intermediate, then cleaning to obtain a third intermediate, adding the third intermediate into a solution containing trypsin and phenylmethylsulfonyl fluoride and having a pH value of 7.8-8.2, continuing for a second preset time to obtain a fourth intermediate, performing cell removal treatment on the fourth intermediate, then cleaning to obtain a fifth intermediate, and drying and sterilizing the fifth intermediate to obtain the omentum majus acellular matrix material. The omentum majus acellular matrix material prepared by the preparation method provided by the application is particularly suitable for regeneration culture of cartilage tissues.

Description

Preparation method of omentum majus acellular matrix material and construction method of cartilage tissue

Technical Field

The invention relates to the technical field of tissue engineering, in particular to a preparation method of a large omentum acellular matrix material and a construction method of cartilage tissue.

Background

Trauma, osteoarthritis and the like are common orthopedic diseases in clinic, the disease has high incidence rate and relatively more inducements, most patients are accompanied by subchondral bone defects after injury, and if the defects cannot be timely and effectively repaired, other diseases can be induced, and even serious patients can cause the loss of joint functions. At present, an ideal repair method for bone joint cartilage defects is still lacking clinically, a common repair method has certain limitations, and the traditional cartilage injury treatment methods comprise joint grinding and shaping, drilling, microfracture and arthroscopic lavage, which cannot repair damaged cartilage and subchondral bone into the original normal tissue structures.

The greater omentum is one of the peritoneum. The peritoneum is a layer of mucous membrane existing in the abdominal cavity of higher vertebrates, which is mainly composed of mesothelial cells, and is a membranous tissue formed by the support of connective tissue. The peritoneum coats most organs in the abdominal cavity, and can secrete mucus to wet the surfaces of the viscera and reduce friction among the viscera. The peritoneum migrates from the parietal layer to the visceral layer, or from one organ to another, constituting a bilayer structure. Blood vessels, nerves and lymphatic vessels are usually running between the two layers of the peritoneal. These forms are named ligament, omentum and mesentery, respectively, in terms of their own structural features and the association of specific organs. The greater omentum is the peritoneum that connects the greater curvature of the stomach to the transverse colon, for a total of four layers: the peritoneum including the anterior and posterior walls of the stomach heals at the greater curvature of the stomach to form the first two layers of the greater omentum, extends downward to slightly below the umbilical plane, then turns back upward to coat the transverse colon to form the second two layers of the greater omentum. The greater omentum is a very thin tissue with great elasticity and flexibility and a highly vascularized structure, which makes it an excellent natural biomaterial in the fields of tissue engineering and regenerative medicine.

At present, in the field of tissue engineering research, omentum majus can be used as a 'natural bioreactor' for in vitro transplantation of a construct, promotes vascularization of the construct, enhances the function of the transplantation construct, and has been successfully applied to the fields of myocardial tissue engineering, nervous tissue engineering and the like. However, no research report on the application of the large omentum acellular matrix to cartilage tissue engineering is found at present.

Disclosure of Invention

In view of the above-mentioned situation, the main object of the present invention is to provide a method for preparing a large omentum acellular matrix material and a method for constructing a cartilage tissue, which solve the above-mentioned problems of the conventional large omentum acellular matrix material.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the first aspect of the present invention provides a preparation method of a large omentum acellular matrix material, comprising the steps of:

s100, cleaning a omentum majus;

s200, carrying out freeze-thaw cycle treatment on the cleaned omentum majus to obtain a first intermediate;

s300, adding the first intermediate into a solution containing trypsin and phenylmethylsulfonyl fluoride and having a pH value of 7.8 to 8.2, and keeping for a first preset time to obtain a second intermediate, wherein the pH value can be 7.8, 7.9, 8, 8.1, 8.2 and is further preferably 8;

s400, degreasing the second intermediate, and cleaning to obtain a third intermediate;

s500, adding the third intermediate into a solution containing trypsin and phenylmethylsulfonyl fluoride and having a pH value of 7.8 to 8.2, and keeping for a second preset time to obtain a fourth intermediate, wherein the pH value can be 7.8, 7.9, 8, 8.1, 8.2 and is further preferably 8;

s600, carrying out tissue cell removal treatment on the fourth intermediate, and then cleaning to obtain a fifth intermediate;

s700, drying and sterilizing the fifth intermediate to obtain the omentum majus acellular matrix material.

The omentum majus is subjected to freeze-thaw cycle treatment, freezing is formed in the process of the freeze-thaw cycle, and the concentration change of a salt solution for the freeze-thaw cycle (the solubility of salt in unfrozen water changes along with the temperature reduction in the freezing process of the freeze-thaw cycle, the salt concentration gradually rises, and the concentration difference between the inside and the outside of a cell membrane is generated) causes the cell to be ruptured, so that the cell lysis effect is improved, and a larger total surface area is obtained to be suitable for tissue regeneration of cartilage tissues.

Before the defatting treatment and the decellularization treatment, a pretreatment process of cutting off carboxyl groups in lysine and arginine residues by trypsin in a liquid environment at a pH of 7.8 to 8.2 to break up cells for the subsequent decellularization treatment and, in addition, since many proteases are released from the inside of cells into the treatment solution during the subsequent decellularization process and damage other protein structures (structures constituting the matrix material) of the omentum majus tissue, the benzyl sulfonyl fluoride is added into the solution in the pretreatment process and is used as a protease inhibitor, so that the damage process can be prevented, therefore, the generated three-dimensional space of the matrix material is more stable, and in addition, the three-dimensional space of the matrix material generated by the method can promote the proliferation and migration of chondrocytes and improve the stability of the chondrocytes expressed according to the original phenotype.

The pretreatment is carried out firstly, and then the decellularization treatment is carried out, so that the surface roughness of the matrix particles can be improved, and the cartilage cells can be more favorably adhered, and the regeneration of cartilage tissues can be favorably carried out.

The omentum majus can be selected from mammalians of pig, cattle, sheep, etc., and can be purchased from slaughterhouse, etc.

Preferably, in the step S100, the omentum majus is washed with physiological saline.

The normal saline has a certain sterilization effect, can cause bacteria to generate intracellular and extracellular concentration difference, further easily causes the bacteria to generate the effect of plasmolysis in cells, inhibits the reproduction of the bacteria, and has no harm to the normal saline so as to ensure the integrity of the omentum majus. The number of washing times may be 2-3.

Preferably, in the step S200, the washed omentum majus is added to a buffer solution containing tris and phenylmethylsulfonyl fluoride and having a PH of 7.8 to 8.2 to perform the freeze-thaw cycle.

By adopting the buffer solution, on one hand, the cell lysis effect can be improved, on the other hand, the addition of the benzyl sulfonyl fluoride can provide a stable environment for tissues, and the damage of protease released in cells to other protein structures (structures forming matrix materials) of the omentum majus tissues is avoided, so that the three-dimensional space of the generated matrix materials is ensured to be more stable.

Preferably, the molar concentration of the tris (hydroxymethyl) aminomethane in the buffer is 1mmol/L to 20mmol/L, such as 1mmol/L, 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L, 10mmol/L, 11mmol/L, 12mmol/L, 13mmol/L, 14mmol/L, 15mmol/L, 16mmol/L, 17mmol/L, 18mmol/L, 19mmol/L, 20mmol/L, more preferably 8mmol/L to 12mmol/L, still more preferably 10mmol/L, and the mass percentage of the phenylmethylsulfonyl fluoride is 0.1% to 1%, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, more preferably 0.8% to 1%, still more preferably 1%.

Preferably, the buffer solution further contains ethylene diamine tetraacetic acid, and the molar concentration of the ethylene diamine tetraacetic acid is 1mmol/L to 10mmol/L, such as 1mmol/L, 2mmol/L, 3mmol/L, 4mmol/L, 5mmol/L, 6mmol/L, 7mmol/L, 8mmol/L, 9mmol/L, 10mmol/L, more preferably 8mmol/L to 10mmol/L, and still more preferably 10 mmol/L.

Utilize ethylenediamine tetraacetic acid to adjust the pH value of buffer solution, make its pH value control in 7.8 to 8.2 within ranges, in addition, because ethylenediamine tetraacetic acid has the inhibitory action, adopt it to carry out pH value and adjust and can avoid leading into new material and cause the destruction to other protein structure (the structure that constitutes matrix material) of macroreticular lamina tissue to further guarantee that the three-dimensional space of matrix material that generates is more firm.

Preferably, one of the freeze-thaw cycles comprises: firstly, freezing treatment is carried out for 1 to 8 hours under the temperature condition of-80 ℃ to-20 ℃, the temperature of the freezing treatment can be-80 ℃, 75 ℃, 70 ℃, 65 ℃, 60 ℃, 55 ℃, 50 ℃, 45 ℃, 40 ℃, 35 ℃, 30 ℃, 25 ℃ and 20 ℃, and the duration of the freezing treatment can be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours and 8 hours; then, the melting treatment is carried out at 30 ℃ to 40 ℃ for 0.5 to 1 hour, and the temperature of the melting treatment may be, for example, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃ and 40 ℃, and the duration of the melting treatment may be, for example, 0.5 hour, 0.6 hour, 0.7 hour, 0.8 hour, 0.9 hour and 1 hour.

Preferably, the number of freeze-thaw cycles may be 2-3, more preferably 3.

Although the freeze-thaw cycle is an existing treatment technology, the requirement of the macroreticular membrane acellular matrix material is very strict, the protein denaturation can be caused by overhigh melting temperature, the incomplete melting can be caused by overlow melting temperature, and the enzymolysis of the extracellular matrix by intracellular protease after the cell lysis can be caused if the half-life of the benzylsulfonyl fluoride protease inhibitor is too short, for example, if the melting treatment time is overlong, so that the technology is not adopted in the preparation process of the existing macroreticular membrane acellular matrix material.

Preferably, in the step S300, the mass percentage of the trypsin is 0.1% to 0.5%, such as 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, and more preferably 0.25%, and the mass percentage of the phenylmethylsulfonyl fluoride is 0.1% to 1%, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, and more preferably 0.8% to 1%, and still more preferably 1%;

in step S500, the trypsin is 0.1% to 0.5% by mass, for example, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, and more preferably 0.25%, and the phenylmethylsulfonyl fluoride is 0.1% to 1% by mass, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1%, and more preferably 0.8% to 1%, and still more preferably 1%.

Preferably, ethylene diamine tetraacetic acid is added to the solution in each of the step S300 and the step S500, and the mass percentage of ethylene diamine tetraacetic acid is 0.01% to 0.1%, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, more preferably 0.8% to 0.1%, and still more preferably 0.1%.

In step S300 and step S500, the PH of the buffer solution is adjusted by using edta to control the PH in the range of 7.8 to 8.2, and in addition, because edta has an inhibitory effect, the PH adjustment using edta can avoid the damage to other protein structures (structures constituting the matrix material) of the macroreticular tissue due to the introduction of new substances, thereby further ensuring that the three-dimensional space of the generated matrix material is more stable.

Preferably, the first predetermined period of time is 6 to 12 hours, such as 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours; further preferably, in step S300, the first intermediate is added to a solution containing trypsin and phenylmethylsulfonyl fluoride at a pH of 7.8 to 8.2, and agitated for a first predetermined period of time.

The second predetermined period of time is 3 to 6 hours, for example 3 hours, 4 hours, 5 hours, 6 hours. Further preferably, in step S500, the third intermediate is added to a solution containing trypsin and phenylmethylsulfonyl fluoride at a pH of 7.8 to 8.2, and shaken for a second predetermined period of time.

Preferably, in the step S400, the second intermediate is degreased using one or a mixture of at least two of isopropyl alcohol, n-butanol, acetonitrile, ethanol, and methanol. Further preferably, the second intermediate is degreased with isopropyl alcohol, which has a high boiling point with respect to methanol, chloroform, and diethyl ether, has no pungent odor, is not easily volatilized, and can reduce environmental pollution. More preferably, in step S400, the second intermediate is placed in one or a mixture of at least two of isopropanol, n-butanol, acetonitrile, ethanol, and methanol, and shaken under constant temperature conditions (30 ℃ to 40 ℃, e.g., 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃) for 40 to 50 hours, e.g., 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 49 hours, 50 hours, and more preferably 48 hours.

Preferably, in step S400, the washing is performed with phosphate buffered saline solution, the number of times of washing is 2-3, and the PH value of phosphate buffered saline solution is preferably 7.8 to 8.2, for example, 7.8, 7.9, 8, 8.1, 8.2, and more preferably 8.

Preferably, in the step S600, the fourth intermediate is subjected to a decellularization treatment using a solution containing magnesium salt, dnase, rnase, and lipase. Further preferably, in step S600, the fourth intermediate is put into a solution containing magnesium salt, DNase, RNase and lipase under constant temperature conditions (30 ℃ to 40 ℃, e.g., 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃) for 10 to 32 hours, e.g., 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, further preferably 14 to 18 hours, further preferably 16 hours with shaking.

In step S600, a solution containing dnase, rnase and lipase is used to perform a degrouping treatment on the fourth intermediate, and the pretreatment process of step S500 is matched to disperse cells, so that all cell components can be effectively removed, and divalent magnesium ions in the magnesium salt have a trypsin inhibiting effect, so that the degrouping treatment can not damage the matrix material, and the integrity of the matrix material is ensured.

Preferably, the magnesium salt is one of magnesium chloride and magnesium sulfate or a mixture of the two.

Preferably, the molar concentration of the magnesium salt is 0.005mmol/L to 0.05mmol/L, such as 0.005mmol/L, 0.01mmol/L, 0.015mmol/L, 0.02mmol/L, 0.025mmol/L, 0.03mmol/L, 0.035mmol/L, 0.04mmol/L, 0.045mmol/L, 0.05mmol/L, the DNase is contained in an amount of 5KU/L to 50KU/L, such as 5KU/L, 10KU/L, 15KU/L, 20KU/L, 25KU/L, 30KU/L, 35KU/L, 40KU/L, 45KU/L, 50KU/L, more preferably 10KU/L to 20KU/L, still more preferably 15KU/L, the DNase is of Type II, Type II-S or Type II-S, the ribonuclease content is 5mg/L to 20mg/L, for example, 5mg/L, 6mg/L, 7mg/L, 8mg/L, 9mg/L, 10mg/L, 10.5mg/L, 11mg/L, 11.5mg/L, 12mg/L, 12.5mg/L, 13mg/L, 13.5mg/L, 14mg/L, 15mg/L, 16mg/L, 17mg/L, 18mg/L, 19mg/L, 20mg/L, further preferably 10mg/L to 14mg/L, further preferably 12.5mg/L, ribonuclease Type of enzyme is Type I-A, Type I-AS, Type II-A, Type III-A, Type X-A, Type XII-A, the content of the lipase is 1KU/L to 10KU/L, such as 1KU/L, 2KU/L, 3KU/L, 4KU/L, 5KU/L, 6KU/L, 7KU/L, 8KU/L, 9KU/L, 10KU/L, more preferably 1KU/L to 4KU/L, still more preferably 2KU/L, and the Type of the lipase is Type II or VI-S.

Preferably, in step S600, washing is performed by using phosphate buffered saline solution, the PH of phosphate buffered saline solution is preferably 7.8 to 8.2, such as 7.8, 7.9, 8, 8.1, 8.2, and more preferably 8, the number of washing times may be 2-3, and then washing is performed by using ethanol, the concentration of ethanol is preferably 50% to 70%, and the number of washing times may be 2-3.

Preferably, in step S700, drying is performed by using a freeze-drying method, and sterilization is performed by using a radiation sterilization method.

Preferably, in the step S500, the fourth intermediate obtained is washed with phosphate buffered saline solution with PH of 7.8 to 8.2, and then enters the step S600.

In summary, the method of the present application can completely retain the three-dimensional space structure of the extracellular matrix and some growth factors (such as transforming growth factor (TGF- β), basic fibroblast growth factor (bFGF)) which play an important role in cell differentiation, and the three-dimensional space formed by the omentum macroreticular matrix material prepared by the method can promote the proliferation and migration of chondrocytes, improve the stability of the chondrocytes expressed according to the original phenotype, promote the secretion of cell matrix so as to increase the firmness of the scaffold.

A second aspect of the present invention provides a method for constructing a cartilage tissue, the method comprising the steps of:

s10, placing the seed cells for cartilage tissue construction in a nutrient solution for a third preset time; the number of chondrocytes can be expanded using this procedure;

s20, taking cell suspension from the culture solution obtained in the step S10, and inoculating on the omentum majus acellular matrix material prepared by the preparation method;

and S30, placing the inoculated omentum majus acellular matrix material into a culture solution for culture, and continuing for a fourth preset time to obtain the constructed cartilage tissue.

The construction method provided by the application adsorbs seed cells in a stent carrier which is formed by a omentum majus acellular matrix material and has good biocompatibility and can be gradually absorbed by a human body, the carrier provides a living three-dimensional space for the cells, and is beneficial to the cells to obtain enough nutrient substances, grow and expand so as to obtain the constructed cartilage tissue, the constructed cartilage tissue and the stent carrier form an integral graft, the graft is implanted into a tissue lesion part in a body, the implanted cells continue to expand and secrete the matrix to form new corresponding cartilage tissue with the special shape and function of in-situ tissue cells, and connective tissue cells around the implanted part grow into a reticular structure of the omentum majus acellular matrix material, so that a bionic structure similar to normal tissue is gradually formed, thereby playing a repairing role, and a patient can repair, The autologous cartilage graft is transplanted to obtain the repair and functional reconstruction of cartilage defect, and complete cure and durable rehabilitation are realized.

In step S10, the seed cells for cartilage tissue construction may be purchased in a cell bank, or cartilage tissue of a patient or a volunteer may be taken, when cartilage tissue of a patient is taken, cartilage tissue rich in cartilage cells in a non-load-bearing area is preferably taken, when cartilage tissue of a patient is taken, cartilage tissue in a non-load-bearing area may be taken under aseptic conditions, and put into a culture bottle containing nutrient solution to be washed for 2 to 3 times, and then, cartilage tissue is cut into small pieces under aseptic conditions and is dried with sterile filter paper, and put into a culture bottle containing nutrient solution.

In the step S10, the nutrient solution is DMEM/F12 medium containing fetal bovine serum, DMEM (dulbecco 'S modified eagle medium) is a medium containing various amino acids and glucose, F12 (Ham' S F12 nutrient medium) is an animal cell medium, and DMEM/F12 medium is obtained by combining DMEM and F12 at a ratio of 1: 1. The third predetermined period is preferably 6-8 weeks.

Preferably, in the step S30, the culture solution includes a DMEM culture solution. Since DMEM contains various amino acids and glucose, the adhesion rate of cells is greatly improved.

Preferably, the DMEM culture solution is added with fetal bovine serum, penicillin, streptomycin, amphotericin, glutamine, vitamin additives and vitamin C. Wherein the fetal calf serum is 5 to 15 percent by mass, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, more preferably 10 percent by mass, the penicillin is 800IU/ml to 1200IU/ml, such as 800IU/ml, 900IU/ml, 1000IU/ml, 1100IU/ml, 1200IU/ml, more preferably 1000IU/ml, the streptomycin is 0.8mg/ml to 1.2mg/ml, such as 0.8mg/ml, 0.9mg/ml, 1mg/ml, 1.1mg/ml, 1.2mg/ml, more preferably 1mg/ml, the amphotericin is 2 to 3 μ g/ml, such as 2 μ g/ml, 2.1 μ g/ml, 2.2 μ g/ml, 2 μ g/ml, or, 2.3. mu.g/ml, 2.4. mu.g/ml, 2.5. mu.g/ml, 2.6. mu.g/ml, 2.7. mu.g/ml, 2.8. mu.g/ml, 2.9. mu.g/ml, 3. mu.g/ml, more preferably 2.5. mu.g/ml, a glutamine content of 1mmol/L to 3mmol/L, e.g. 1mmol/L, 1.5mmol/L, 2mmol/L, 2.5mmol/L, 3mmol/L, more preferably 2mmol/L, a vitamin additive content of 0.5% to 1.5%, e.g. 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, more preferably 1%, a vitamin C content of 30. mu.g/ml to 70. mu.g/ml, e.g/ml, 35. mu.g/ml, 35. mu.5. mu.g/ml, 3 g/, 40. mu.g/ml, 45. mu.g/ml, 50. mu.g/ml, 55. mu.g/ml, 60. mu.g/ml, 65. mu.g/ml, 70. mu.g/ml, and more preferably 50. mu.g/ml.

Wherein, the fetal calf serum provides necessary nutrient substances for cells, performs acid-base balance on a culture medium, detoxifies and inhibits the activity of certain enzyme preparations; penicillin, streptomycin and amphotericin are mainly used for eliminating pollution, and two kinds of pollution need to be considered in the process of culturing chondrocytes: contamination by microorganisms and the incorporation of other cell lines, both very common and the possible contamination and the effect of contamination on the results must be taken into account, so antibiotics and antimycotics are used to prevent contamination; glutamine is important in cell culture, and after amino groups are removed, L-glutamine can be used as an energy source for culturing cells and participate in protein synthesis and nucleic acid metabolism; the vitamin additive provides nutrition; vitamin C has antioxidant and protective effects on cells.

Preferably, the fourth predetermined period of time is preferably 3-5 days.

Preferably, the liquid level of the culture solution is higher than the surface of the omentum majus acellular matrix material, and the height difference is less than 1 mm.

The liquid level of the culture solution is slightly higher than the surface of the large omentum acellular matrix material, so that the large omentum acellular matrix material planted with cells and the culture solution containing about 5 percent of CO can be prepared₂The air contact of the culture medium ensures the air-liquid surface culture, thereby improving the culture efficiency and ensuring the culture effect.

Preferably, the culture temperature of step S30 is between 35 ℃ and 38 ℃, preferably 37 ℃, and the change of the culture solution is preferably performed once per day.

The invention also provides a construction method of the cartilage tissue, which comprises the following steps:

s01, cleaning the omentum majus;

s02, performing freeze-thaw cycle treatment on the cleaned omentum majus to obtain a first intermediate;

s03, adding the first intermediate into a solution containing trypsin and phenylmethylsulfonyl fluoride and having a pH value of 7.8-8.2, and keeping for a first preset time to obtain a second intermediate;

s04, degreasing the second intermediate, and cleaning to obtain a third intermediate;

s05, adding the third intermediate into a solution containing trypsin and phenylmethylsulfonyl fluoride and having a pH value of 7.8-8.2, and continuing for a second predetermined time to obtain a fourth intermediate;

s06, performing histiocyte removal treatment on the fourth intermediate, and then cleaning to obtain a fifth intermediate;

s07, drying and sterilizing the fifth intermediate to obtain the omentum majus acellular matrix material;

s08, placing the seed cells for cartilage tissue construction in a nutrient solution for a third preset time;

s09, taking cell suspension from the culture solution obtained in the step S08, and inoculating the cell suspension on the omentum majus acellular matrix material obtained in the step S07;

and S010, placing the inoculated omentum majus acellular matrix material into a culture solution for culturing for a fourth preset time to obtain the constructed cartilage tissue.

The prepared omentum majus acellular matrix material has larger total surface area and very stable three-dimensional space, is very suitable for subsequent regeneration culture of cartilage tissues, promotes the proliferation and migration of chondrocytes, and improves the stability of the chondrocytes expressed according to the original phenotype.

Preferably, in the step S01, the greater omentum is washed with physiological saline.

Preferably, in the step S02, the washed omentum majus is added to a buffer solution containing tris and phenylmethylsulfonyl fluoride and having a PH of 7.8 to 8.2 to perform the freeze-thaw cycle.

Preferably, in the buffer solution, the molar concentration of the tris is 1mmol/L to 20mmol/L, and the mass percentage of the phenylmethylsulfonyl fluoride is 0.1% to 1%.

Preferably, the buffer solution also contains ethylene diamine tetraacetic acid, and the molar concentration of the ethylene diamine tetraacetic acid is 1mmol/L to 10 mmol/L.

Preferably, one of the freeze-thaw cycles comprises: freezing treatment is firstly carried out for 1 to 8 hours under the temperature condition of-80 ℃ to-20 ℃, and then thawing treatment is carried out for 0.5 to 1 hour under the temperature condition of 30 ℃ to 40 ℃.

Preferably, in the step S03, the trypsin is 0.1 to 0.5% by mass, and the phenylmethylsulfonyl fluoride is 0.1 to 1% by mass;

in the step S05, the trypsin is 0.1 to 0.5% by mass, and the phenylmethylsulfonyl fluoride is 0.1 to 1% by mass.

Preferably, ethylene diamine tetraacetic acid is added to the solutions in step S03 and step S05, and the mass percentage of ethylene diamine tetraacetic acid is 0.01% to 0.1%.

Preferably, in the step S04, the second intermediate is degreased with one or a mixture of at least two of isopropanol, n-butanol, acetonitrile, ethanol, and methanol.

Preferably, in the step S04, washing is performed with phosphate buffered saline.

Preferably, in the step S06, the fourth intermediate is subjected to a decellularization treatment using a solution containing magnesium salt, dnase, rnase, and lipase.

Preferably, the magnesium salt is selected from one or a mixture of two of magnesium chloride and magnesium sulfate.

Preferably, the molar concentration of the magnesium salt is 0.005mmol/L to 0.05mmol/L, the content of the deoxyribonuclease is 5KU/L to 50KU/L, the content of the ribonuclease is 5mg/L to 20mg/L, and the content of the lipase is 1KU/L to 10 KU/L.

Preferably, in the step S06, the washing is performed by using phosphate buffered saline solution first, and then by using ethanol.

Preferably, in the step S08, the nutrient solution is DMEM/F12 culture solution containing fetal bovine serum.

Preferably, in the step S010, the culture solution includes a DMEM culture solution.

Preferably, the DMEM culture solution is added with fetal bovine serum, penicillin, streptomycin, amphotericin, glutamine, vitamin additives and vitamin C.

Drawings

Preferred embodiments of the present invention will be described below with reference to the accompanying drawings. In the figure:

FIG. 1 is a flow chart of a method for preparing a large omentum acellular matrix material according to the present invention;

FIG. 2 is a flow chart of a method for constructing cartilage tissue according to the present invention;

fig. 3 is a flow chart of a method for constructing cartilage tissue according to another embodiment of the present invention.

Detailed Description

The present invention will be described below based on examples, but the present invention is not limited to only these examples. In the following detailed description of the present invention, certain specific details are set forth in order to avoid obscuring the nature of the present invention, well-known methods, procedures, and components have not been described in detail.

Further, those of ordinary skill in the art will appreciate that the drawings provided herein are for illustrative purposes and are not necessarily drawn to scale.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is, what is meant is "including, but not limited to".

In the description of the present invention, it is to be understood that the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance. In addition, in the description of the present invention, "a plurality" means two or more unless otherwise specified.

Because of the characteristics of cartilage tissues, the omentum majus acellular matrix material prepared by the existing method is not suitable for the regeneration culture of the cartilage tissues, and based on the characteristics, the application provides the preparation method of the omentum majus acellular matrix material. As shown in fig. 1, the preparation method comprises the steps of:

s100, cleaning a omentum majus;

s300, adding the first intermediate into a solution which contains trypsin and phenylmethylsulfonyl fluoride and has a pH value of 7.8-8.2, and continuing for a first preset time to obtain a second intermediate;

s500, adding the third intermediate into a solution which contains trypsin and phenylmethylsulfonyl fluoride and has a pH value of 7.8-8.2, and continuing for a second preset time to obtain a fourth intermediate;

Specific examples of the preparation process are given below:

the first embodiment is as follows:

washing fresh pig omentum majus with physiological saline for 3 times;

adding the washed omentum majus into a buffer solution with the pH value of 8.0 and containing 10mmol/L of Tris (hydroxymethyl) aminomethane (Tris), 10mmol/L of Ethylene Diamine Tetraacetic Acid (EDTA) and 1% of phenylmethylsulfonyl fluoride (PMSF) by mass for freezing and thawing for 3 times, wherein the freezing temperature in one freezing and thawing cycle is-80 ℃, the duration time is 1 hour, the melting temperature is 37 ℃, and the duration time is 0.5 hour;

adding the tissue subjected to freeze-thaw cycle treatment into a solution containing 0.25 mass percent of trypsin, 0.1 mass percent of Ethylene Diamine Tetraacetic Acid (EDTA) and 1 mass percent of phenylmethylsulfonyl fluoride (PMSF), and oscillating for 12 hours at 37 ℃;

defatting the tissue obtained by the above steps with isopropanol at 37 deg.C for 48 hr, and adding Phosphate Buffered Saline (PBS) with pH of 8.0 (8g/L NaCl, 200mg/L KCl, 1g/L Na)₂HPO₄、200mg/L KH₂PO₄) Washing for 3 times.

The washed tissue was added to a solution containing 0.25 mass% of trypsin, 0.1 mass% of ethylenediaminetetraacetic acid (EDTA), and 1 mass% of phenylmethylsulfonyl fluoride (PMSF), shaken at 37 ℃ for 6 hours, and washed with PBS having a pH of 8.0 for 3 times.

Adding the tissue obtained in the previous step to a solution containing 55mmol/LNa₂HPO₄、17mmol/LKH₂PO₄、4.9mmol/LMgSO₄*7H₂O, 15000U/L deoxyribonuclease II (bovine pancreas), 12.5mg/L ribonuclease IIIA (bovine pancreas), 2000U/L lipase VI-S (porcine pancreas), was shaken at 37 ℃ for 16 hours, and then washed 3 times with phosphate buffered saline PBS and 70% ethanol one after the other.

And (3) freeze-drying and irradiating for sterilization of the tissue obtained in the step to obtain the omentum majus acellular matrix material.

Example two:

washing fresh pig omentum majus with physiological saline for 3 times;

adding the washed omentum majus into a buffer solution with the pH value of 7.8 and containing 1mmol/L of Tris (hydroxymethyl) aminomethane (Tris), 1mmol/L of Ethylene Diamine Tetraacetic Acid (EDTA) and 0.1% of phenylmethylsulfonyl fluoride (PMSF) by mass for freeze-thaw cycle for 3 times, wherein the freezing temperature in one freeze-thaw cycle is-20 ℃, the duration time is 8 hours, the melting temperature is 30 ℃, and the duration time is 1 hour;

adding the tissue subjected to freeze-thaw cycle treatment into a solution containing 0.1% by mass of trypsin, 0.01% by mass of ethylenediaminetetraacetic acid (EDTA) and 0.1% by mass of phenylmethylsulfonyl fluoride (PMSF), and oscillating for 6 hours at 30 ℃;

defatting the tissue obtained by the above steps with isopropanol at 30 deg.C for 40 hr, and adding Phosphate Buffered Saline (PBS) with pH of 7.8 (8g/L NaCl, 200mg/L KCl, 1g/L Na)₂HPO₄、200mg/L KH₂PO₄) Washing for 3 times.

The washed tissue was added to a solution containing 0.1% by mass of trypsin, 0.01% by mass of ethylenediaminetetraacetic acid (EDTA), and 0.1% by mass of phenylmethylsulfonyl fluoride (PMSF), and the mixture was shaken at 30 ℃ for 3 hours and washed with PBS having a pH of 7.8 for 3 times.

Adding the tissue obtained in the previous step to a solution containing 55mmol/L Na₂HPO₄、17mmol/L KH₂PO₄、1mmol/LMgSO₄*7H₂O, 5000U/L deoxyribonuclease II (bovine pancreas), 5mg/L ribonuclease IIIA (bovine pancreas) and 1000U/L lipase VI-S (porcine pancreas) in a solution, oscillating for 10 hours at 30 ℃, and then washing 3 times with Phosphate Buffered Saline (PBS) and 70% ethanol in sequence.

Example three:

washing fresh pig omentum majus with physiological saline for 3 times;

adding the washed omentum majus into a buffer solution with the pH value of 8.2 and containing 20mmol/L of Tris (hydroxymethyl) aminomethane (Tris), 8mmol/L of Ethylene Diamine Tetraacetic Acid (EDTA) and 0.8 mass percent of phenylmethylsulfonyl fluoride (PMSF), and performing freeze-thaw cycle for 3 times, wherein the freezing temperature in one freeze-thaw cycle is-50 ℃, the duration time is 5 hours, the melting temperature is 40 ℃, and the duration time is 0.8 hour;

adding the tissue subjected to freeze-thaw cycle treatment into a solution containing 0.5% by mass of trypsin, 0.08% by mass of ethylenediaminetetraacetic acid (EDTA) and 0.8% by mass of phenylmethylsulfonyl fluoride (PMSF), and oscillating at 40 ℃ for 12 hours;

adding the tissue obtained in the previous step into isopropanol, and vibrating at 40 deg.CDefatting for 50 hr, and adding Phosphate Buffered Saline (PBS) with pH 8.2 (8g/L NaCl, 200mg/L KCl, 1g/L Na)₂HPO₄、200mg/L KH₂PO₄) Washing for 3 times.

The washed tissue was added to a solution containing 0.5% by mass of trypsin, 0.08% by mass of ethylenediaminetetraacetic acid (EDTA), and 0.8% by mass of phenylmethylsulfonyl fluoride (PMSF), and the mixture was shaken at 40 ℃ for 6 hours and washed with PBS having a pH of 8.2 for 3 times.

Adding the tissue obtained in the previous step to a solution containing 55mmol/L Na₂HPO₄、17mmol/L KH₂PO₄、3mmol/LMgSO₄*7H₂O, 50000U/L deoxyribonuclease II (bovine pancreas), 20mg/L ribonuclease IIIA (bovine pancreas) and 10000U/L lipase VI-S (porcine pancreas) in a solution, oscillating for 32 hours at 40 ℃, and then washing 3 times by Phosphate Buffered Saline (PBS) and 70% ethanol in sequence.

As shown in fig. 2, the present application also provides a method for constructing cartilage tissue, which comprises the following steps:

Specific examples of the construction method are given below:

example four:

seed cells purchased from a cell bank for cartilage tissue construction were cultured in DMEM/F12 culture solution containing fetal bovine serum for 6 weeks;

inoculating the cell suspension on the omentum majus acellular matrix material;

placing the inoculated omentum majus acellular matrix material into a culture solution for culturing for 5 days, replacing the culture solution once a day, wherein the liquid level of the culture solution is higher than the omentum majus acellular matrix material, the distance between the liquid level and the omentum majus acellular matrix material is less than 1mm, and the culture solution is a DMEM culture solution added with 5 mass percent of fetal calf serum, 800IU/ml of penicillin, 0.8 mass percent of streptomycin, 2 mu g/ml of amphotericin, 1mmol/L of glutamine, 0.5 mass percent of vitamin additive and 30 mu g/ml of vitamin C.

Example five:

taking cartilage tissue of a non-bearing area of a patient under aseptic condition, putting the cartilage tissue into a culture bottle containing nutrient solution, washing for 2-3 times, cutting the cartilage tissue into small pieces under aseptic condition, sucking the small pieces by using sterile filter paper, and putting the small pieces into DMEM/F12 culture solution containing fetal calf serum for culturing for 8 weeks;

inoculating the cell suspension on the omentum majus acellular matrix material;

placing the inoculated omentum majus acellular matrix material into a culture solution for culturing for 3 days, replacing the culture solution once a day, wherein the liquid level of the culture solution is higher than the omentum majus acellular matrix material, the distance between the liquid level and the omentum majus acellular matrix material is less than 1mm, and the culture solution is a DMEM culture solution added with 15% by mass of fetal calf serum, 1200IU/ml of penicillin, 1.2mg/ml of streptomycin, 3 mug/ml of amphotericin, 3mmol/L of glutamine, 1.5% by mass of vitamin additive and 70 mug/ml of vitamin C.

Example six:

taking cartilage tissue of a non-bearing area of a patient under aseptic condition, putting the cartilage tissue into a culture bottle containing nutrient solution, washing for 2-3 times, cutting the cartilage tissue into small pieces under aseptic condition, sucking the small pieces by using sterile filter paper, and putting the small pieces into DMEM/F12 culture solution containing fetal calf serum for culturing for 7 weeks;

inoculating the cell suspension on the omentum majus acellular matrix material;

placing the inoculated omentum majus acellular matrix material into a culture solution for culturing for 4 days, replacing the culture solution once a day, wherein the liquid level of the culture solution is higher than that of the omentum majus acellular matrix material, the distance between the liquid level and the omentum majus acellular matrix material is less than 1mm, and the culture solution is a DMEM culture solution added with 10 mass percent of fetal calf serum, 1000IU/ml of penicillin, 1mg/ml of streptomycin, 2.5 mu g/ml of amphotericin, 2mmol/L of glutamine, 1 mass percent of vitamin additive and 50 mu g/ml of vitamin C.

As shown in fig. 3, the present application also provides a method for constructing cartilage tissue, which comprises the following steps:

s01, cleaning the omentum majus;

Specific examples of the construction method are given below:

example seven:

washing fresh pig omentum majus with physiological saline for 3 times;

inoculating the cell suspension on the omentum majus acellular matrix material;

Example eight:

washing fresh pig omentum majus with physiological saline for 3 times;

inoculating the cell suspension on the omentum majus acellular matrix material;

Example nine:

washing fresh pig omentum majus with physiological saline for 3 times;

defatting the tissue obtained by the above steps with isopropanol at 40 deg.C for 50 hr, and adding Phosphate Buffered Saline (PBS) with pH of 8.2 (8g/L NaCl, 200mg/L KCl, 1g/L Na)₂HPO₄、200mg/L KH₂PO₄) Washing for 3 times.

inoculating the cell suspension on the omentum majus acellular matrix material;

It will be appreciated by those skilled in the art that the above-described preferred embodiments may be freely combined, superimposed, without conflict.

It will be understood that the embodiments described above are illustrative only and not restrictive, and that various obvious and equivalent modifications and substitutions for details described herein may be made by those skilled in the art without departing from the basic principles of the invention.

Claims

1. A preparation method of a large omentum decellularized matrix material is characterized by comprising the following steps:

s100, cleaning a omentum majus;

2. The method for preparing a large omentum acellular matrix material according to claim 1, wherein in the step S100, the large omentum is washed with physiological saline.

3. The method of claim 1, wherein the step S200 of adding the washed omentum majus to a buffer solution containing tris and phenylmethylsulfonyl fluoride and having a pH of 7.8 to 8.2 is performed in the freeze-thaw cycle.

4. The method for preparing a large omentum acellular matrix material according to claim 1, wherein in the step S300, the trypsin is 0.1 to 0.5% by mass, and the phenylmethylsulfonyl fluoride is 0.1 to 1% by mass;

in the step S500, the trypsin is 0.1 to 0.5% by mass, and the phenylmethylsulfonyl fluoride is 0.1 to 1% by mass.

5. The method for preparing a large omentum acellular matrix material according to claim 1, wherein in the step S400, the second intermediate is degreased by using one or a mixture of at least two of isopropanol, n-butanol, acetonitrile, ethanol and methanol.

6. The method of claim 1, wherein in step S600, the fourth intermediate is processed by degrouping with a solution containing magnesium salt, DNase, RNAse and lipase.

7. The method for preparing a large omentum acellular matrix material according to claim 1, wherein in the step S600, the washing is performed by phosphate buffered saline solution firstly, and then the washing is performed by ethanol.

8. A method for constructing a cartilage tissue, comprising the steps of:

s10, placing the seed cells for cartilage tissue construction in a nutrient solution for a third preset time;

s20, taking cell suspension from the culture solution obtained in the step S10, and inoculating on the omentum majus acellular matrix material prepared by the preparation method according to any one of claims 1 to 7;

9. The method for constructing a cartilage tissue according to claim 8 wherein in step S10, the nutrient solution is DMEM/F12 culture solution containing fetal bovine serum.

10. The method for constructing cartilage tissue according to claim 8 or 9, wherein the liquid level of the culture solution is higher than the surface of the omentum majus acellular matrix material, and the height difference is less than 1 mm.