CN109602956B - A kind of extracellular matrix modified multi-layer soft tissue repair material and its preparation method and application - Google Patents

Abstract

The invention discloses a multi-layer soft tissue repair material modified by extracellular matrix, which is a double-layer composite material modified by extracellular matrix bonded by polyurethane; the invention also discloses the preparation method and application of the multi-layer soft tissue repair material . In the present invention, the SIS film and the PU/SIS composite material are firmly bonded together through the water-based polyurethane emulsion to form a multi-layer soft tissue repair material, wherein the PU/SIS composite material acts as a muscle layer to support the SIS film layer, and the SIS film As the intima layer, it is more conducive to the adhesion and growth of cells, and can promote the repair of soft tissues. At the same time, the extracellular matrix-modified multilayer soft tissue repair material prepared by the present invention can promote the differentiation of urine-derived stem cells to specific cells, indicating that the repair material prepared by the present invention has a certain activity of inducing cell differentiation. The repair material prepared by the invention is not only expected to be used to construct soft tissue models for various pathological or biological studies, but also to provide new methods and ideas for soft tissue defect repair.

Description

A multilayer soft tissue repair material modified by extracellular matrix and its preparation method and use

technical field

The invention relates to a multilayer soft tissue repair material modified by extracellular matrix, its preparation method and application.

Background technique

Soft tissue is an important tissue of the human body. However, soft tissue damage or defect caused by various reasons, such as disease and trauma, has become one of the common clinical diseases and seriously threatens human health. Traditional methods for treating soft tissue injuries or defects include autologous transplantation and allogeneic transplantation, but these methods have problems such as limited donors, secondary injury, and immune rejection. It is of great significance to find reliable, safe and effective methods for repairing soft tissue injuries and defects. In recent years, with the development of tissue engineering technology, the use of tissue engineering technology to repair and replace damaged soft tissues has brought new ideas for the treatment of soft tissue injuries and defects.

The small intestinal submucosa (SIS) is an extracellular matrix component rich in a variety of growth factors, which can promote cell growth, stimulate angiogenesis, and induce tissue regeneration at specific concentrations and active states. Moreover, SIS has the characteristics of non-immunogenicity, antimicrobial activity, and anisotropy, and its safety and efficacy have been verified by clinical practice. In addition, SIS is abundant and easy to obtain, which can not only avoid the problem of limited donors in traditional organ transplantation, but also reduce costs and benefit more patients. Therefore, SIS has become a widely studied soft tissue engineering scaffold material. Patents CN107233630A, CN107854727A and CN107281552A are soft tissue repair materials obtained after SIS treatment (such as compounding cells or extracellular matrix on SIS). However, like other bio-natural restoration materials, SIS has unsatisfactory mechanical properties, rapid degradation and uncontrollable properties.

Polymer synthetic materials have the advantages of excellent plasticity, controllable degradation performance and mechanical properties. Polyurethane (PU) is a commonly used medical polymer synthetic material. The combination of PU and SIS is expected to improve the mechanical properties and degradation properties of biological natural repair materials, and obtain more ideal soft tissue repair materials. Patent CN104341608A mixed and cross-linked PU and SIS to obtain a soft tissue repair material (PU/SIS) with excellent resilience, mechanical properties and good biocompatibility. However, this PU/SIS repair material is a single-layer repair material, which cannot simulate and repair soft tissues with double-layer or multi-layer structures, such as uterine wall and skin. At the same time, seeding functional cells on scaffold materials is one of the important means to complete tissue repair from both structural and functional perspectives. However, allogeneic cell transplantation is prone to immune rejection, and autologous functional cells have limitations such as limited sources and damage to the donor site.

Therefore, finding a more ideal solution to construct tissue engineered tissue has become a major challenge in the field of regenerative medicine.

Contents of the invention

The object of the present invention is to provide a multilayer soft tissue repair material modified with extracellular matrix, its preparation method and application.

The invention provides a multi-layer soft tissue repair material modified by extracellular matrix, which is a double-layer composite material modified by extracellular matrix bonded by polyurethane; wherein, one layer of the double-layer composite material is small intestinal submucosa membrane, and the other One layer is a polyurethane/small intestinal submucosa composite material cross-linked after polyurethane and small intestinal submucosa powder are mixed; the extracellular matrix modification is extracellular matrix modified small intestinal submucosa membrane and/or extracellular matrix modified polyurethane/small intestinal mucosa Lower layer composite material; the thickness ratio of the small intestinal submucosa membrane to the polyurethane/small intestinal submucosa composite material layer is 1:10～1:1000; the consumption of the polyurethane emulsion is 0.02～1mL/cm ² ; the polyurethane is water-based polyurethane emulsion.

Further, the solid content of the aqueous polyurethane emulsion is 9wt%-40wt%.

Further, the preparation method of the double-layer composite material modified by the extracellular matrix is as follows:

(1) After coating one layer of polyurethane emulsion on the small intestinal submucosa membrane, paste it on the polyurethane/small intestinal submucosa composite material, and dry it at 37° C. to obtain a double-layer composite material; wherein, the consumption of the polyurethane emulsion is 0.02～1mL/cm ² ;

(2) Composite cells on the double-layer composite material; 1×10 ⁴ to 1×10 ⁸ cells per square centimeter of the double-layer composite material;

(3) Take the double-layer composite material compounded with cells, and perform decellularization treatment to obtain.

Further, the decellularization treatment method is:

Add the decellularized solution dropwise on the double-layer composite material compounded with cells, treat for 10-20 minutes, wash, freeze-dry, and you are ready;

Preferably, the decellularization solution is a 0.5% TritonX-100 solution containing 20 mM NH ₄ OH; the treatment time is 15 min; the cleaning solution is PBS; the freeze-drying temperature is -40°C, Freeze-drying time is 24h.

Further, the cell-compounded double-layer composite material is the small intestinal submucosa membrane composited with cells, and/or the polyurethane/small intestinal submucosa composite material layer is composited with cells.

Further, the preparation method of the small intestinal submucosa membrane is as follows:

Take the small intestine, remove the serosal layer and muscular layer, degrease, decellularize, decontaminate, freeze-dry, and sterilize;

Wherein, the degreasing is soaked in a mixed solution of methanol and chloroform with a volume ratio of 1:1 for 10 to 14 hours, and rinsed with deionized water;

And/or, the decellularization is digested with enzymes, soaked overnight in a 0.25% trypsin solution at 4°C, and rinsed with normal saline to remove the trypsin;

And/or, the descaling is soaking in 0.5% sodium lauryl sulfate solution for 4-6 hours, and then cleaning with deionized water;

And/or, the sterilization is by ethylene oxide sterilization.

Further, the preparation method of the polyurethane/small intestinal submucosa composite material is as follows:

The polyurethane emulsion is blended with small intestinal submucosa powder, the suspension solution is introduced into a mold, lyophilized, cross-linked, washed, and lyophilized to obtain the product;

Wherein, the mass ratio of the polyurethane emulsion to the small intestinal submucosa powder is 5:1 to 9:1;

And/or, the freeze-drying is freeze-drying after pre-freezing in a low-temperature refrigerator at -40°C for 24 hours;

And/or, the crosslinking solution is a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide;

And/or, the cross-linking is carried out under the condition of 37° C. and protected from light for 30-40 hours.

Further, the preparation method of the small intestinal submucosa powder is as follows:

Take the small intestine, remove the serosal layer and muscular layer, degrease, decellularize, decontaminate, freeze-dry, crush at low temperature, and sterilize;

Wherein, the degreasing is soaked in a mixed solution of methanol and chloroform with a volume ratio of 1:1 for 10 to 14 hours, and rinsed with deionized water;

And/or, the decellularization is digested with enzymes, soaked overnight in 0.25% trypsin solution at 4°C, and washed with normal saline to remove the trypsin;

And/or, the descaling is soaking in 0.5% sodium lauryl sulfate solution for 4-6 hours, and then cleaning with deionized water;

And/or, the low-temperature pulverization is carried out by using a ball mill after being cooled by liquid nitrogen, and passed through a 200-mesh sieve;

And/or, the sterilization is by ethylene oxide sterilization.

Further, the preparation method of the polyurethane emulsion is as follows:

(1) Prepolymerization: take hydroxyl donor, isocyanate and catalyst in catalytic amount, prepolymerize;

Wherein, the molar ratio of hydroxyl donor to isocyanate is 1:0.9 to 1:2; preferably, the molar ratio of hydroxyl donor to isocyanate is 1:1.05 to 1:1.85;

The hydroxyl donor is any one or more of polytetrahydrofuran ether, 1,2-pentanediol, polyethylene glycol, and glycerin; preferably, the hydroxyl donor is polytetrahydrofuran ether;

The isocyanate is any one or more of isophorone diisocyanate, methyl diisocyanate, and 1,6-hexamethylene diisocyanate; preferably, the isophorone diisocyanate is isophorone diisocyanate;

The catalyst is stannous octoate or dibutyltin dilaurate; preferably, the catalyst is stannous octoate;

(2) Chain extension: adding a chain extender to extend the chain;

Wherein, the molar ratio of the chain extender to the hydroxyl donor in step (1) is 1:8 to 8:1; preferably, the chain extender

The molar ratio of chain agent and hydroxyl donor is 1:1;

The chain extender is any one or more of 2,2-dimethylolpropionic acid, methyldiethanolamine, dihydroxymethylpropionic acid, butylene glycol sulfate, and sodium ethylenediamine ethanesulfonate; Preferably, the chain extender is 2,2-dimethylolpropionic acid;

(3) Neutralization and emulsification: add a neutralizing agent, stir evenly, add the reaction system dropwise to 20% (v/v) acetone aqueous solution, stir at a high speed for 1-2 hours, remove small molecular organic matter residues, and obtain the product;

Wherein, the molar ratio of the neutralizing agent to the chain extender in step (2) is: 0.5:1 to 3:1; preferably, the molar ratio of the neutralizing agent to the chain extender is 1.5:1;

Described neutralizing agent is triethylamine;

Preferably, in step (1), the prepolymerization temperature is 74°C; the prepolymerization time is 2.5h to 3.5h;

And/or, in step (2), the temperature for chain extension is 50-55°C; the time for chain extension is 3-4 hours;

And/or, in step (3), the method for removing small molecule organic residues is rotary evaporation and dialysis;

And/or, in step (3), the rotating speed of high-speed stirring is 1300rpm.

The present invention also provides a method for preparing the aforementioned restoration material, which comprises the steps of:

(1) After coating one layer of polyurethane emulsion on the small intestinal submucosa membrane, paste it on the polyurethane/small intestinal submucosa composite material, and dry it at 37° C. to obtain a double-layer composite material; wherein, the consumption of the polyurethane emulsion is 0.02～1mL/cm ² ;

(2) Composite cells on the double-layer composite material; 1×10 ⁴ to 1×10 ⁸ cells per square centimeter of the double-layer composite material;

(3) Take the double-layer composite material compounded with cells, and perform decellularization treatment to obtain.

Further, the decellularization treatment method is:

Add the decellularized solution dropwise on the double-layer composite material compounded with cells, treat for 10-20 minutes, wash, freeze-dry, and you are ready;

Preferably, the decellularization solution is a 0.5% TritonX-100 solution containing 20 mM NH ₄ OH; the treatment time is 15 min; the cleaning solution is PBS; the freeze-drying temperature is -40°C, Freeze-drying time is 24h.

Further, the cell-compounded double-layer composite material is the small intestinal submucosa membrane composited with cells, and/or the polyurethane/small intestinal submucosa composite material layer is composited with cells.

The present invention also provides the use of the aforementioned extracellular matrix-modified multi-layer soft tissue repair material in the preparation of soft tissue repair materials.

Further, the soft tissue repair material is a soft tissue repair material for the uterine wall.

In the present invention, the SIS film and the PU/SIS composite material are firmly bonded together through the water-based polyurethane emulsion to form a multi-layer soft tissue repair material, wherein the PU/SIS composite material acts as a muscle layer and supports the SIS film layer, and the SIS As the inner membrane layer, the membrane is more conducive to the adhesion and growth of cells, and can promote the repair of soft tissues. According to the reports in the existing literature, the unmodified water-based polyurethane has poor adhesive properties and is usually difficult to use for bonding, while the modified polyurethane material has the problems of strong toxicity and poor biocompatibility. However, the present invention unexpectedly finds that for the specific SIS film and PU/SIS composite material of the present invention, unmodified water-based polyurethane can be used for effective bonding, which not only achieves effective bonding, but also ensures non-toxicity, biological Excellent compatibility.

At the same time, experiments have proved that the extracellular matrix-modified endometrium repair material and the extracellular matrix-modified myometrium repair material prepared by the present invention can promote the differentiation of urine-derived stem cells into epithelial cells and smooth muscle cells, respectively. . It shows that the extracellular matrix-modified multi-layer soft tissue repair material prepared by the present invention can promote the differentiation of urine-derived stem cells to specific cells. It further demonstrates that the repair material prepared by the present invention has a certain activity of inducing cell differentiation. The repair material prepared by the invention is not only expected to be used to construct soft tissue models for various pathological or biological studies, but also to provide new methods and ideas for soft tissue defect repair.

Obviously, according to the above content of the present invention, according to the common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, more other various forms of modification, substitution or change can also be made.

The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Description of drawings

Fig. 1 is a flow chart of the preparation of the extracellular matrix-modified endometrial layer scaffold material.

Fig. 2 is a flow chart of the preparation of the extracellular matrix-modified myometrium scaffold material.

Fig. 3 is the result of immunofluorescence staining of endometrial epithelial cells co-cultured with SIS for 7 days (scale bar: 20 μm).

Fig. 4 is the result of immunofluorescent staining of type IV collagen co-cultured with uterine smooth muscle cells and PU/SIS (scale bar: 50 μm).

Fig. 5 is the result of immunofluorescence staining of uterine smooth muscle cells and PU/SIS co-cultured for 11 days (scale bar: 50 μm).

Fig. 6 is the DAPI staining result after decellularization of the endometrial epithelial cell-SIS composite material (scale bar: 100 μm).

Fig. 7 is the result of immunofluorescent staining of type IV collagen after decellularization of uterine smooth muscle cell-PU/SIS composite material (scale bar: 50 μm).

Figure 8 shows the expression of E-Cadherin in the co-culture of USCs and ECM _EC -SIS for 21 days (scale bar: 20 μm).

Figure 9 shows the expression of CK18 in USCs co-cultured with ECM _EC -SIS for 21 days (scale bar: 20 μm).

Figure 10 shows the α-SMA expression of USCs and ECM _SMC -PU/SIS co-cultured for 21 days (scale bar: 20 μm).

Figure 11 shows the expression of Desmin in USCs and ECM _SMC -PU/SIS co-cultured for 21 days (scale bar: 20 μm).

Figure 12 shows the Myosin expression of USCs cells and ECM _SMC -PU/SIS co-cultured for 21 days (scale bar: 20 μm).

Detailed ways

Acronyms:

SIS: small intestinal submucosa, PU: polyurethane.

Embodiment 1 Preparation of double-layer composite material of the present invention

1. Preparation of SIS membrane

1) Separation of the small intestinal submucosa: first cut the cleaned porcine small intestine into sections with a length of about 15 cm, then scrape off the muscular layer and serosa layer of the small intestine, wash with normal saline and filter dry to obtain the small intestinal submucosa;

2) Degreasing: immerse the prepared small intestinal submucosa in a degreasing solution (CHCl3:CH3OH=1:1, V/V) for 12 hours, and then wash it repeatedly until there is no peculiar smell;

3) Decellularization: at 4°C, immerse the defatted small intestinal submucosa in 0.25% trypsin solution overnight, rinse until there is no foam, then immerse it in 0.5% sodium lauryl sulfate solution 4h, freeze-dried after washing with deionized water;

4) Sterilized with ethylene oxide for later use.

2. Preparation of SIS powder

1) Separating the submucosa of the small intestine: Cut the cleaned porcine small intestine into sections with a length of about 15 cm, scrape off the muscular layer and serosa layer of the small intestine, wash with normal saline and filter dry to obtain the submucosa of the small intestine;

2) Degreasing: immerse the prepared small intestinal submucosa in a degreasing solution (CHCl ₃ :CH ₃ OH=1:1, V/V) for 12 hours, and then wash repeatedly until there is no peculiar smell;

3) Decellularization: at 4°C, immerse the defatted small intestinal submucosa in 0.25% trypsin solution overnight, rinse until there is no foam, then immerse it in 0.5% SDS solution for 4 hours, deionize Freeze-dried after washing with water;

4) Low-temperature pulverization: after the shredded SIS is cooled by liquid nitrogen, it is pulverized by a ball mill at low temperature and passed through a 200-mesh sieve, and then sterilized by ethylene oxide for use.

3. Preparation of PU emulsion

1) Prepolymerization: Add PTMG 1000 (33.33g, 33.33mmol), IPDI (24.18g, 96.67mmol) and stannous octoate (0.02mL) into a three-necked flask, and react in an oil bath at 74°C for 3h;

2) Chain extension: Add DMBA (4.94g, 33.33mmol) and react at 54°C for 3h. In order to make the reaction proceed smoothly, acetone can be used to adjust the viscosity of the system if necessary;

3) Neutralization and emulsification: Add TEA (7mL), stir at room temperature for 20min, slowly add the reaction product dropwise to acetone aqueous solution (acetone: deionized water = 1:4), stir at 1300rpm for 2h to obtain PU emulsion, and use rotary evaporation And dialysis to remove small molecule organic residues, that is.

4. Preparation of PU/SIS composite material

1) Blending: 1g of SIS powder was added to 5g of PU emulsion with a solid content of 15wt%, and stirred at room temperature for 2.5h to obtain a PU/SIS suspension solution;

2) Freeze-drying: Pour the prepared blend system into a mold, pre-freeze in a low-temperature refrigerator at -40°C, and then freeze-dry for 24 hours;

3) Cross-linking: soak the freeze-dried material in EDC/NHS solution, react in the dark at 37°C for 36 hours, wash 5 times with PBS, 30 minutes each time, wash 3 times with deionized water, and freeze-dry to obtain PU /SIS Composite.

5. Preparation of SIS-PU/SIS double-layer composite material

After coating a layer of PU emulsion on the SIS film, paste it on the PU/SIS composite material, and dry it at 37°C to obtain a SIS-PU/SIS (SPS) double-layer composite material. The amount of PU emulsion used is 0.5mL/cm ² .

Example 2 Preparation of multilayer soft tissue repair material modified by extracellular matrix of the present invention

1. Compound culture of endometrial epithelial cells and SIS membrane

1) Sample preparation: The double-layer composite material was cut into a disc with a diameter of 6 mm, sterilized with ethylene oxide, placed in a 24-well plate, and soaked overnight in EpiCM medium containing FBS.

2) Cell inoculation: discard the medium in the 24-well plate, and place the 24-well plate in a cell incubator at 37° C. and 5% CO ₂ for 20 minutes to further volatilize the medium.

3) Combined culture of endometrial epithelial cells and SIS membrane: Digest, centrifuge and count endometrial epithelial cells with a good growth state and a fusion rate of 70-80%, and adjust the cell density to 5×10 ⁶ cells/mL; Take 10 μL of cell suspension from the well and inoculate evenly on the surface of the SIS membrane, and incubate for 2 hours in a 37°C, 5% CO ₂ cell incubator; drop 500 μL of EpiCM medium containing FBS from the edge, and culture in the cell incubator for 7 days , changing the medium every day to obtain the endometrial epithelial cell-SIS composite material.

2. Decellularization of endometrial epithelial cells-SIS composites

Take out the SIS membrane co-cultured with endometrial epithelial cells for 7 days, and wash 3 times with PBS; add the prepared decellularization solution (0.5% Triton X-100 solution containing 20mM NH ₄ OH) dropwise on the endometrium On the epithelial cell-SIS composite material, treated for 20min; washed with PBS for 3 times, freeze-dried at -40°C, and freeze-dried for 24h, to obtain the endometrial layer scaffold material modified by endometrial epithelial extracellular matrix ( ECM _EC -SIS).

The flow chart of the preparation of the extracellular matrix-modified endometrial layer scaffold material is shown in Fig. 1 .

Example 3 Preparation of multi-layer soft tissue repair material modified by extracellular matrix of the present invention

1. Combined culture of uterine smooth muscle cells and PU/SIS composite material

1) Sample preparation: Cut the double-layer composite material into discs with a diameter of 6 mm, place the composite material in a 24-well plate after ethylene oxide sterilization, and soak overnight in SMCM medium containing FBS.

2) Cell inoculation: discard the medium in the 24-well plate, and place the 24-well plate in a cell incubator at 37° C. and 5% CO ₂ for 20 minutes to further volatilize the medium.

3) Combined culture of uterine smooth muscle cells and PU/SIS composite material layer: Digest, centrifuge and count uterine smooth muscle cells with a good growth state and a fusion rate of 70-80%, and adjust the cell density to 1×10 ⁷ cells/mL; 10 μL of cell suspension was evenly inoculated on the surface of the PU/SIS composite material, and cultured in a 37°C, 5% CO ₂ cell incubator for 2 hours; 500 μL of SMCM medium containing FBS was added dropwise from the edge, and put into the cell incubator respectively. After culturing for 3 days, 7 days and 11 days, the medium was changed every day to obtain uterine smooth muscle cell-PU/SIS composite material.

2. Decellularization treatment of uterine smooth muscle cells-PU/SIS composite material

Take out the PU/SIS composite material co-cultured with uterine smooth muscle cells for 11 days, wash 3 times with PBS; add the decellularized solution (containing 20mM NH ₄ OH concentration of 0.5% Triton X-100 solution) dropwise on the uterine smooth muscle On the cell-PU/SIS composite material, treated for 15 minutes; washed with PBS for 3 times, freeze-dried at -40°C, and freeze-dried for 24 hours, the myometrium scaffold material modified by uterine smooth muscle extracellular matrix (ECM _SMC -PU/SIS).

The flow chart of the preparation of the extracellular matrix-modified myometrium scaffold material is shown in FIG. 2 .

Prove the beneficial effect of the present invention below with the mode of test example:

Immunofluorescence staining observation of test example 1 combined culture of cells and materials

(1) Immunofluorescence staining observation of endometrial epithelial cells and SIS membrane co-cultured

1. Experimental method

In the experiment, E-Cadherin immunofluorescence staining and CK18 immunofluorescence staining were used to observe the culture of the endometrial epithelial cell-SIS composite material in Example 1, and E-Cadherin and CK18 were specifically expressed in epithelial cells.

(1) E-Cadherin immunofluorescence staining

a. Sample cleaning and fixation: Take the endometrial epithelial cell-SIS composite material that has been cultured for 7 days, add PBS dropwise from the edge to wash the cells, add 4% paraformaldehyde to fix for 20 minutes, and then wash with PBS for 3 times;

b. Cell punching: add Triton X-100 with a concentration of 0.5% to the fixed composite material, rupture the membrane at room temperature for 15 minutes, and wash with PBS for 3 times;

c. Sealing: add goat serum dropwise, and place in a 37°C incubator to seal for 30 minutes;

d. Add primary antibody: absorb excess blocking solution with absorbent paper, dilute the primary antibody E-Cadherin at 1:200, drop directly on the composite material, put it in a wet box, and incubate overnight at 4°C;

e. Cleaning: Take out the composite material, rewarm at room temperature for 30 minutes, and wash with PBS for 3 times;

f. Add secondary antibody: Absorb excess liquid with absorbent paper, add freshly prepared goat anti-mouse secondary antibody (1:200) working solution dropwise on the composite material, put it in a wet box, incubate at 37°C for 60 minutes, and wash 3 times with PBS ;

g. DAPI staining: DAPI was added dropwise, incubated in the dark for 3 minutes, and then washed with PBS;

h. Sealing: Seal the slides with a mounting solution containing an anti-fluorescence quenching agent, and collect images under a fluorescent microscope.

(2) CK18 immunofluorescence staining

A. Sample cleaning and fixation: Take the endometrial epithelial cell-SIS composite material that has been cultured for 7 days, and add PBS dropwise from the edge to wash the cells. Add 4% paraformaldehyde to fix for 20min, then wash 3 times with PBS;

B. Cell punching: Add Triton X-100 with a concentration of 0.5% to the fixed composite material, rupture the membrane for 15 minutes at room temperature, and wash with PBS for 3 times;

C. Sealing: add goat serum dropwise, and place in a 37°C incubator to seal for 30 minutes;

D. Add primary antibody: absorb excess blocking solution with absorbent paper, dilute the primary antibody CK18 at 1:100, drop directly on the composite material, put it in a wet box, and incubate overnight at 4°C;

E. Cleaning: Take out the composite material, rewarm at room temperature for 30 minutes, and wash with PBS for 3 times;

F. Add secondary antibody: Absorb excess liquid with absorbent paper, add freshly prepared goat anti-mouse secondary antibody (1:200) working solution dropwise on the composite material, put it in a wet box, incubate at 37°C for 60 minutes, and wash with PBS 3 times ;

G. DAPI staining: add DAPI dropwise, incubate in the dark for 3 minutes, and then wash with PBS;

H. Sealing: Seal the slides with a mounting solution containing anti-fluorescence quenching agent and collect images under a fluorescent microscope.

2. Experimental results

The results of E-Cadherin immunofluorescence staining and CK18 immunofluorescence staining of endometrial epithelial cells cultured with SIS membrane for 7 days are shown in Fig. 3 . Endometrial epithelial cells positively expressing E-Cadherin or CK18 are shown in red; DAPI-labeled nuclei are shown in blue. It can be seen from Figure 3 that the cells on the SIS membrane were positive for E-Cadherin and CK18 antibodies, the cytoplasm was red, and the nucleus was blue, and most of the cells were oval, with full cytoplasm and large nuclei. It grows in clusters and sheets, and the cells are closely arranged between cells. The experimental results showed that the endometrial epithelial cells adhered and grew well in the endometrial epithelial cells-SIS composite material.

(2) Immunofluorescence staining observation of co-cultured uterine smooth muscle cells and PU/SIS composite materials

1. Experimental method

Type Ⅳ collagen immunofluorescence staining, α-SMA immunofluorescence staining, Desmin immunofluorescence staining and Myosin immunofluorescence staining were used to observe the co-culture of uterine smooth muscle cells and PU/SIS.

(1) Type Ⅳ collagen immunofluorescence staining

a. Sample cleaning and fixation: Take the uterine smooth muscle cell-PU/SIS composite material cultured for 3, 7 and 11 days, add PBS dropwise from the edge to wash the cells, add 4% paraformaldehyde to fix for 20 minutes, and wash with PBS for 3 times;

b. Cell punching: add Triton X-100 with a concentration of 0.5% to the fixed composite material, rupture the membrane at room temperature for 15 minutes, and wash with PBS for 3 times;

c. Sealing: add goat serum dropwise, and place in a 37°C incubator to seal for 30 minutes;

d. Add the primary antibody: Absorb excess blocking solution with absorbent paper, dilute the primary antibody type IV collagen at 1:200, drop it directly on the composite material, put it in a wet box, and incubate overnight at 4°C;

e. Cleaning: Take out the composite material, rewarm at room temperature for 30 minutes, and wash with PBS for 3 times;

f. Add secondary antibody: Absorb excess liquid with absorbent paper, add freshly prepared goat anti-rabbit secondary antibody (1:200) working solution dropwise on the composite material, put it in a wet box, incubate at 37°C for 60 minutes, and wash with PBS 3 times ;

g. DAPI staining: DAPI was added dropwise, incubated in the dark for 3 minutes, and then washed with PBS;

h. Sealing: Seal the slides with a mounting solution containing an anti-fluorescence quenching agent and collect images under a fluorescence microscope.

(2) α-SMA immunofluorescence staining

A. Sample cleaning and fixation: Take the uterine smooth muscle cell-PU/SIS composite material cultured for 11 days, add PBS dropwise from the edge to wash the cells, add 4% paraformaldehyde to fix for 20 minutes, and wash with PBS for 3 times;

B. Frozen section: The composite material specimens were embedded with OCT frozen section embedding agent, cut into thin slices with a thickness of 6 μm, immediately fixed with 10% neutral formaldehyde at room temperature for 5 minutes, and then cleaned with PBS;

C. Sealing: add goat serum dropwise, and place in a 37°C incubator to seal for 30 minutes;

D. Add the primary antibody: absorb the excess blocking solution with absorbent paper, dilute the primary antibody α-SMA at 1:100, drop it directly on the composite material, put it in a wet box, and incubate overnight at 4°C;

E. Cleaning: Take out the composite material, rewarm at room temperature for 30 minutes, and wash with PBS for 3 times;

F. Add secondary antibody: Absorb excess liquid with absorbent paper, add freshly prepared goat anti-mouse secondary antibody (1:200) working solution dropwise on the composite material, put it in a wet box, incubate at 37°C for 60min, and wash with PBS 3 times ;

G. DAPI staining: add DAPI dropwise, incubate in the dark for 3 minutes, and then wash with PBS;

H. Sealing: Seal the slides with a mounting solution containing anti-fluorescence quenching agent and collect images under a fluorescent microscope.

(3) Desmin immunofluorescence staining

a. Sample cleaning and fixation: Take the uterine smooth muscle cell-PU/SIS composite material cultured for 11 days, add PBS dropwise from the edge to wash the cells, add 4% paraformaldehyde to fix for 20 minutes, and wash with PBS for 3 times;

b. Cell punching: add Triton X-100 with a concentration of 0.5% to the fixed composite material, rupture the membrane at room temperature for 15 minutes, and wash with PBS for 3 times;

c. Sealing: add goat serum dropwise, and place in a 37°C incubator to seal for 30 minutes;

d. Add the primary antibody: Absorb excess blocking solution with absorbent paper, dilute the primary antibody Desmin at 1:200, drop it directly on the composite material, put it in a wet box, and incubate overnight at 4°C;

e. Cleaning: Take out the composite material, rewarm at room temperature for 30 minutes, and wash with PBS for 3 times;

f. Add secondary antibody: Absorb excess liquid with absorbent paper, add freshly prepared goat anti-mouse secondary antibody (1:200) working solution dropwise on the composite material, put it in a wet box, incubate at 37°C for 60 minutes, and wash with PBS 3 times ;

g. DAPI staining: DAPI was added dropwise, incubated in the dark for 3 minutes, and then washed with PBS;

h. Sealing: Seal the slides with a mounting solution containing an anti-fluorescence quenching agent and collect images under a fluorescence microscope.

(4) Myosin immunofluorescence staining

A. Sample cleaning and fixation: Take the uterine smooth muscle cell-PU/SIS composite material cultured for 11 days, add PBS dropwise from the edge to wash the cells, add 4% paraformaldehyde to fix for 20 minutes, and wash with PBS for 3 times;

B. Cell punching: Add Triton X-100 with a concentration of 0.5% to the fixed composite material, rupture the membrane for 15 minutes at room temperature, and wash with PBS for 3 times;

C. Sealing: add goat serum dropwise, and place in a 37°C incubator to seal for 30 minutes;

D. Add the primary antibody: absorb the excess blocking solution with absorbent paper, dilute the primary antibody Myosin at 1:1000, drop it directly on the composite material, put it in a wet box, and incubate overnight at 4°C;

E. Cleaning: Take out the composite material, rewarm at room temperature for 30 minutes, and wash with PBS for 3 times;

F. Add secondary antibody: Absorb excess liquid with absorbent paper, add freshly prepared goat anti-mouse secondary antibody (1:200) working solution dropwise on the composite material, put it in a wet box, incubate at 37°C for 60min, and wash with PBS 3 times ;

G. DAPI staining: add DAPI dropwise, incubate in the dark for 3 minutes, and then wash with PBS;

H. Sealing: Seal the slides with a mounting solution containing anti-fluorescence quenching agent and collect images under a fluorescent microscope.

2. Experimental results

Figure 4 and Figure 5 show the immunofluorescence staining results of type IV collagen, α-SMA, Desmin and Myosin after the uterine smooth muscle cells were co-cultured with PU/SIS composite materials. Cells positively expressing type Ⅳ collagen, α-SMA, Desmin and Myosin showed red in fluorescent microscope, marking uterine smooth muscle cells, DAPI fluorescence showed blue, marking cell nucleus and PU/SIS composite, while the green fluorescence shown in FITC channel was PU Autofluorescence of /SIS composites. It can be seen from Figure 4 and Figure 5 that after 3 days of co-culture of uterine smooth muscle cells and PU/SIS, the cells gradually fused into sheets on the surface of the material and the secretion of type IV collagen could be observed, and a few cells began to migrate to the inside of the material. After 7 days of compound culture, the cells began to aggregate and grow on both sides of the material. After 11 days of compound culture, the cells were spindle-shaped and proliferated on the material in large quantities. Bunches of smooth muscle cells are arranged in parallel at the edge of the material, overlapping and growing into multiple layers, undulating up and down, in the form of striations. The cells were positive for α-SMA, Desmin and Myosin antibodies, the cytoplasm was red, and the nucleus was blue. The experimental results showed that the uterine smooth muscle cells adhered and grew well in the uterine smooth muscle cell-PU/SIS composite material.

Experimental Example 2 Test of decellularization effect after material decellularization treatment

(1) DAPI staining method to test the decellularization effect of endometrial epithelial cells-SIS composite materials

1. Experimental method

The endometrial layer scaffold material prepared in Example 1 was taken, fixed with 4% paraformaldehyde for 30 min, and washed 3 times with PBS. DAPI solution was added dropwise and incubated in the dark for 2 min. Wash twice with PBS and observe under a fluorescent microscope.

2. Experimental results

The results of DAPI staining after decellularization of endometrial epithelial cells-SIS composites are shown in Figure 6. It can be seen from Figure 6 that before the cells were inoculated, the SIS membrane showed blue color under the fluorescence microscope. After 7 days of combined culture of endometrial epithelial cells and SIS membrane, a large number of blue nuclei can be observed by DAPI staining. After decellularization, no obvious nuclei were observed on the endometrial epithelial cells-SIS composite, and the fibrous structure of the SIS membrane showed blue autofluorescence under the fluorescence microscope. The experimental results showed that after the decellularization treatment, the cells on the endometrial epithelial cell-SIS composite material were basically removed.

(2) Type Ⅳ collagen immunofluorescence staining to test the decellularization effect of uterine smooth muscle cells-PU/SIS composite material

1. Experimental method

(1) Cleaning and fixation of the sample: take the myometrium scaffold material prepared in Example 2, wash it with PBS, add 4% paraformaldehyde to fix it for 20 minutes, and then wash it 3 times with PBS;

(2) Sealing: add goat serum dropwise, place in a 37°C incubator and seal for 30 minutes;

(3) Add the primary antibody: Absorb the excess blocking solution with absorbent paper, dilute the primary antibody type IV collagen at 1:200, drop it directly on the composite material and put it in a wet box, and incubate overnight at 4°C;

(4) Cleaning: Take out the composite material, rewarm at room temperature for 30 minutes, and wash with PBS for 3 times;

(5) Add secondary antibody: Absorb the excess liquid with absorbent paper, add the freshly prepared goat anti-rabbit secondary antibody (1:200) working solution dropwise on the composite material and put it into a wet box, incubate at 37°C for 60min, and wash with PBS for 3 Second-rate;

(6) DAPI staining: DAPI was added dropwise and incubated in the dark for 3 minutes, then washed with PBS;

(7) Seal the slide: seal the slide with a mount solution containing an anti-fluorescence quencher and collect images under a fluorescence microscope.

2. Experimental results

The results of immunofluorescent staining of type IV collagen after decellularization of the uterine smooth muscle cell-PU/SIS composite material are shown in Figure 7. The PU/SIS composite showed green and blue autofluorescence under a fluorescence microscope. After the decellularized cells were treated, no obvious blue nuclei were observed in the DAPI channel on the composite material, but the expression of type Ⅳ collagen, an extracellular matrix component secreted by cells, could be observed on the material, especially at the edge of the material. The experimental results show that after the decellularization treatment, the cells on the uterine smooth muscle cell-PU/SIS composite material are basically removed, and the extracellular matrix secreted by the cells remains on the composite material.

Test Example 3 Co-culture of Urine-derived Stem Cells (USCs) and ECM _EC -SIS

1. Experimental method

(1) Primary culture of USCs

Take the midstream urine of healthy adults aged 18-30 and put it in a 250mL sterile glass bottle. The urine was centrifuged at 1500r/min for 10min. Discard the upper layer of urine, add PBS containing 1% penicillin-streptomycin mixed solution, mix well and centrifuge for 5min. Repeat this step two more times. Discard the supernatant, add 5mL USCs medium to resuspend the cells at the bottom of the centrifuge tube. After clones appeared, the culture medium was replaced, and after the clones formed a certain size, they were trypsinized and passaged.

(2) Co-cultivation and immunofluorescence staining of USCs and ECM _EC -SIS

a. Pretreatment of materials: After the ECM _EC -SIS prepared in Example 1 was sterilized by ethylene oxide, it was placed in a 24-well plate and soaked overnight in DMEM/F12 medium containing FBS;

b. Cell inoculation: Discard the medium, place the 24-well plate in the cell incubator for 20 minutes, and further volatilize the medium;

c. Combined culture of USCs and ECM _EC -SIS: USCs in good growth state and with a fusion rate of 70-80% were digested, centrifuged and counted. The cell density was adjusted to 5×10 ⁶ cells/mL, and 10 μL of cell suspension was uniformly inoculated on the surface of ECM _EC -SIS from each well, and cultured in a cell incubator for 2 h. 500 μL of DMEM/F12 medium containing FBS was added dropwise from the edge, cultured in a cell incubator at 37°C, and the medium was changed every day; USCs were seeded on the SIS membrane as a control group.

d. Take the cell-material complex that has been cultured for 21 days, and add PBS dropwise from the edge to wash the cells. After adding 4% paraformaldehyde to fix for 20min, wash with PBS for 3 times. E-Cadherin immunofluorescence staining and CK18 immunofluorescence staining were performed according to the method of Test Example 1, respectively.

2. Experimental results

Figure 8 shows the expression of E-Cadherin in the co-culture of USCs and ECM _EC -SIS for 21 days. It can be seen from Figure 8 that the USCs inoculated on ECM _EC -SIS were positive for E-Cadherin antibody, the cytoplasm was red, the nucleus was blue, and the cells were mostly oval and full of cytoplasm, forming sheets on the SIS membrane Growth, the cells are closely arranged, and the adhesion grows well. However, USCs seeded on SIS membranes were negative for E-Cadherin antibody.

Figure 9 shows the expression of CK18 in the co-culture of USCs and ECM _EC -SIS for 21 days. CK18 fluorescence appears red, marking endometrial epithelial cells. DAPI fluorescence appears blue, marking the nuclei. It can be seen from Figure 9 that the USCs inoculated on ECM _EC -SIS were positive for CK18 antibody, the cytoplasm was red, and the nucleus was blue. Most of the cells were oval, full of cytoplasm, and grew in sheets on the SIS membrane. The cells were closely arranged and adhered well. However, USCs seeded on SIS membranes were negative for CK18 antibody.

The above experimental results indicated that ECM _EC -SIS could induce USCs to differentiate into epithelial cells.

Test Example 4 Compound Culture of Urine-derived Stem Cells (USCs) and ECM _SMC -PU/SIS

1. Experimental method

(1) Primary culture of USCs

The USCS primary culture method is as described in "Experimental Method (1)" in Test Example 3.

(2) Co-cultivation and immunofluorescence staining of USCs and ECM _SMC -PU/SIS

a. Pretreatment of materials: ECM _SMC -PU/SIS prepared in Example 2 was sterilized with ethylene oxide, placed in a 24-well plate, and soaked overnight in DMEM/F12 medium containing FBS.

b. Inoculation of cells: discard the medium, place the 24-well plate in the cell incubator for 20 minutes, and further volatilize the medium.

c. Combined culture of USCs and ECM _SMC -PU/SIS: USCs in good growth state and with a fusion rate of 70-80% were digested, centrifuged and counted. The cell density was adjusted to 5×10 ⁶ cells/mL, and 10 μL of cell suspension was uniformly inoculated on the surface of ECM _SMC -PU/SIS in each well, and cultured in a cell incubator for 2 hours. 500 μL of DMEM/F12 medium containing FBS was added dropwise from the edge, cultured in a cell incubator at 37°C, and the medium was changed every day; USCs were seeded on the PU/SIS composite material as a control group.

d. Take the cell-material complex that has been cultured for 21 days, and add PBS dropwise from the edge to wash the cells. After adding 4% paraformaldehyde to fix for 20min, wash with PBS 3 times. According to the method of Test Example 1, α-SMA immunofluorescence staining, Desmin immunofluorescence staining and Myosin immunofluorescence staining were respectively performed.

2. Experimental results

Figure 10 shows the α-SMA expression of USCs and ECM _SMC -PU/SIS co-cultured for 21 days. It can be seen from Figure 10 that the USCs inoculated on ECM _SMC -PU/SIS were positive for α-SMA antibody, the cytoplasm was red, the nucleus was blue, the cells were spindle-shaped or oval, and the bundled smooth muscle cells were in the material The edges overlap to grow well, and the growth is good on the material. However, USCs seeded on PU/SIS composites were negative for α-SMA antibody.

Figure 11 shows the expression of Desmin in USCs and ECM _SMC -PU/SIS co-cultured for 21 days. It can be seen from Figure 11 that the USCs seeded on ECM _SMC -PU/SIS were weakly positive to Desmin antibody, the cytoplasm was red, the nucleus was blue, the cells were spindle-shaped or oval, and the smooth muscle cells in bundles were on the edge of the material Overlapping growth, good adhesion growth on material. However, USCs seeded on PU/SIS composites were negative for Desmin antibody.

Figure 12 shows the Myosin expression of USCs and ECM _SMC -PU/SIS co-cultured for 21 days. It can be seen from Figure 12 that the USCs inoculated on ECM _SMC -PU/SIS were weakly positive for Myosin antibody, the cytoplasm was red, the nucleus was blue, the cells were spindle-shaped or oval, and the smooth muscle cells were mainly distributed on the edge of the material. Adhesive growth is good. However, USCs seeded on PU/SIS composites were negative for Myosin antibody.

The above experimental results indicated that ECM _SMC -PU/SIS has certain smooth muscle inducing activity.

In summary, the present invention firmly bonds the SIS film and the PU/SIS composite material together through the water-based polyurethane emulsion to form a multi-layer soft tissue repair material, wherein the PU/SIS composite material acts as the muscle layer and supports the SIS film layer As the intima layer, the SIS membrane is more conducive to the adhesion and growth of cells, and can promote the repair of soft tissues. Simultaneously, the extracellular matrix-modified endometrial layer scaffold material (ECM _EC -SIS) and the extracellular matrix-modified myometrium scaffold material (ECM _SMC -PU/SIS) prepared after the decellularization treatment of the present invention can respectively promote Differentiation of urine-derived stem cells to epithelial and smooth muscle cells. It shows that the extracellular matrix-modified multi-layer soft tissue repair material prepared by the present invention can promote the differentiation of urine-derived stem cells to specific cells. It further demonstrates that the repair material prepared by the present invention has a certain activity of inducing cell differentiation. The repair material prepared by the invention is not only expected to be used to construct soft tissue models for various pathological or biological studies, but also to provide new methods and ideas for soft tissue defect repair.

Claims (17)

1. An extracellular matrix-modified multi-layer soft tissue repair material, comprising: it is a double-layer composite material modified by polyurethane-bonded extracellular matrix; wherein, one layer of the double-layer composite material is a small intestine submucosa film, and the other layer is a polyurethane/small intestine submucosa composite material formed by cross-linking polyurethane and small intestine submucosa powder after mixing; the extracellular matrix modification is an extracellular matrix modified small intestine submucosa membrane and/or an extracellular matrix modified polyurethane/small intestine submucosa composite material; the thickness ratio of the lower intestinal submucosa layer to the polyurethane/lower intestinal submucosa composite material layer is 1: 10-1: 1000; the using amount of the polyurethane for bonding is 0.02-1 mL/cm 2; the polyurethane is aqueous polyurethane emulsion; the solid content of the aqueous polyurethane emulsion is 9-40 wt%.

2. The repair material of claim 1, wherein: the preparation method of the extracellular matrix modified double-layer composite material comprises the following steps:

(1) Coating a layer of polyurethane emulsion on the small intestine submucosa membrane, sticking the polyurethane emulsion on the polyurethane/small intestine submucosa composite material, and drying at 37 ℃ to obtain the double-layer composite material; wherein the dosage of the polyurethane emulsion is 0.02-1 mL/cm 2;

(2) compounding cells on the double-layer composite material; 1 × 104-1 × 108 cells are compounded on each square centimeter of the double-layer composite material;

(3) taking the double-layer composite material compounded with the cells, and carrying out cell removal treatment to obtain the composite material.

3. The repair material of claim 2, wherein: the cell removing treatment method comprises the following steps:

and (3) dropwise adding the cell-removing solution on the double-layer composite material compounded with the cells, treating for 10-20 min, cleaning, and freeze-drying.

4. the repair material of claim 3, wherein: the cell removing solution is 0.5 percent Triton X-100 solution containing 20mM NH4 OH; the treatment time is 15 min; the cleaning solution is PBS; the freeze-drying is freeze-drying, the temperature of the freeze-drying is-40 ℃, and the time of the freeze-drying is 24 hours.

5. The repair material of claim 2, wherein: the cell-compounded double-layer composite material is formed by compounding cells with a small intestine submucosa membrane and/or compounding cells with a polyurethane/small intestine submucosa composite material layer.

6. The repair material of claim 2, wherein: the preparation method of the small intestine submucosa membrane comprises the following steps:

removing serosal layer and muscular layer from small intestine, defatting, removing cells, removing scale, lyophilizing, and sterilizing;

the degreasing step comprises the steps of soaking for 10-14 hours by using a mixed solution of methanol and trichloromethane with the volume ratio of 1:1, and washing with deionized water;

And/or the decellularization is digested by enzyme, and is soaked in a pancreatin solution with the concentration of 0.25% overnight at the temperature of 4 ℃, and the pancreatin is removed by flushing with physiological saline;

And/or the descaling is carried out by soaking in a 0.5% sodium dodecyl sulfate solution for 4-6 h and washing with deionized water;

and/or the sterilization is sterilization using ethylene oxide.

7. The repair material of claim 2, wherein: the preparation method of the polyurethane/small intestinal submucosa composite material comprises the following steps:

Blending the polyurethane emulsion and the powder of the lower layer of the small intestinal mucosa, introducing the suspension solution into a mold, and performing freeze-drying molding, crosslinking, washing and freeze-drying to obtain the polyurethane emulsion;

Wherein the mass ratio of the polyurethane emulsion to the powder of the lower layer of the small intestinal mucosa is 5: 1-9: 1;

and/or, the freeze-drying is carried out after pre-freezing for 24 hours in a low-temperature refrigerator at the temperature of-40 ℃;

and/or the crosslinking solution used for crosslinking is a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide;

And/or the crosslinking is carried out for 30-40 h at 37 ℃ in a dark reaction mode.

8. the repair material of claim 7, wherein: the preparation method of the small intestine submucosa powder comprises the following steps:

removing serosal layer and muscular layer from small intestine, defatting, removing cells, removing scale, lyophilizing, pulverizing at low temperature, and sterilizing;

the degreasing step comprises the steps of soaking for 10-14 hours by using a mixed solution of methanol and trichloromethane with the volume ratio of 1:1, and washing with deionized water;

and/or the decellularization is digested by enzyme, the cells are soaked in pancreatin solution with the concentration of 0.25% overnight at the temperature of 4 ℃, and the pancreatin is removed by flushing with physiological saline;

And/or the descaling is carried out by soaking in a 0.5% sodium dodecyl sulfate solution for 4-6 h and washing with deionized water;

and/or the low-temperature crushing is carried out by cooling through liquid nitrogen, then using a ball mill for low-temperature crushing, and sieving through a 200-mesh sieve;

And/or the sterilization is sterilization using ethylene oxide.

9. the repair material of claim 7, wherein: the preparation method of the polyurethane emulsion comprises the following steps:

(1) pre-polymerization: taking a hydroxyl donor, isocyanate and a catalyst with a catalytic amount for prepolymerization;

Wherein the molar ratio of the hydroxyl donor to the isocyanate is 1: 0.9-1: 2;

The hydroxyl donor is one or more of polytetrahydrofuran ether, 1, 2-pentanediol, polyethylene glycol and glycerol;

The isocyanate is one or more of isophorone diisocyanate, methyl diisocyanate and 1, 6-hexamethylene diisocyanate;

The catalyst is stannous octoate or dibutyltin dilaurate;

(2) Chain extension: adding a chain extender for chain extension;

Wherein the molar ratio of the chain extender to the hydroxyl donor in the step (1) is 1: 8-8: 1;

The chain extender is any one or more of 2, 2-dimethylolpropionic acid, methyldiethanolamine, dihydroxymethylpropanoic acid, butanediol sulfate and ethylenediamine ethanesulfonic acid sodium;

(3) neutralization and emulsification: adding a neutralizer, uniformly stirring, dropwise adding the reaction system into a 20% (v/v) acetone aqueous solution, stirring at a high speed for 1-2 h, and removing small molecular organic matter residues to obtain the aqueous solution;

Wherein, the mole ratio of the neutralizer to the chain extender in the step (2) is as follows: 0.5: 1-3: 1; the neutralizing agent is triethylamine.

10. The repair material of claim 9, wherein:

In the step (1), the molar ratio of the hydroxyl donor to the isocyanate is 1: 1.05-1: 1.85;

the hydroxyl donor is polytetrahydrofuran ether;

The isocyanate is isophorone diisocyanate;

The catalyst is stannous octoate;

In the step (2), the molar ratio of the chain extender to the hydroxyl donor is 1: 1;

the chain extender is 2, 2-dimethylolpropionic acid;

In the step (3), the molar ratio of the neutralizing agent to the chain extender is 1.5: 1.

11. the repair material of claim 9, wherein:

in the step (1), the prepolymerization temperature is 74 ℃; the prepolymerization time is 2.5 h-3.5 h;

And/or in the step (2), the temperature of chain extension is 50-55 ℃; the chain extension time is 3-4 h;

And/or, in the step (3), the method for removing the small molecular organic substance residues is rotary evaporation and dialysis;

And/or in the step (3), the rotation speed of high-speed stirring is 1300 rpm.

12. a method of producing a repair material according to any one of claims 1 to 11, characterized in that: it comprises the following steps:

(1) coating a layer of polyurethane emulsion on the small intestine submucosa membrane, sticking the polyurethane emulsion on the polyurethane/small intestine submucosa composite material, and drying at 37 ℃ to obtain the double-layer composite material; wherein the dosage of the polyurethane emulsion is 0.02-1 mL/cm 2;

(2) compounding cells on the double-layer composite material; 1 × 104-1 × 108 cells are compounded on each square centimeter of the double-layer composite material;

(3) taking the double-layer composite material compounded with the cells, and carrying out cell removal treatment to obtain the composite material.

13. the method of claim 12, wherein: the cell removing treatment method comprises the following steps:

And (3) dropwise adding the cell-removing solution on the double-layer composite material compounded with the cells, treating for 10-20 min, cleaning, and freeze-drying.

14. The method of claim 13, wherein:

The cell removing solution is 0.5 percent Triton X-100 solution containing 20mM NH4 OH; the treatment time is 15 min; the cleaning solution is PBS; the freeze-drying is freeze-drying, the temperature of the freeze-drying is-40 ℃, and the time of the freeze-drying is 24 hours.

15. The method of claim 12, wherein: the cell-compounded double-layer composite material is formed by compounding cells with a small intestine submucosa membrane and/or compounding cells with a polyurethane/small intestine submucosa composite material layer.

16. Use of an extracellular matrix-modified multilayered soft tissue repair material according to any one of claims 1 to 11 for the preparation of a soft tissue repair material.

17. use according to claim 16, characterized in that: the soft tissue repair material is a uterine wall soft tissue repair material.