CN109364299B - Biological pelvic floor repairing mesh and preparation method thereof - Google Patents

Abstract

The invention discloses a pelvic floor biological repairing mesh and a preparation method thereof. The acellular material is twisted into threads to obtain higher strength, so that the acellular material is not easy to break, and the threads are interwoven after being woven, so that the external force of tensile tearing is not applied to one point but applied to a plurality of interweaving points, so that the acellular material has good mechanical property, the defects that the acellular material pelvic floor biological patch is poor in mechanical strength and needs to introduce a cross-linking agent or a synthetic material are overcome, the mesh structure is prepared by a weaving process, and the size of the mesh aperture can be easily adjusted through weaving parameters so as to meet the requirements of practical application. Meanwhile, the pelvic floor biological patch reserves the original three-dimensional structure, non-collagen, growth factor and other components in the extracellular matrix, and promotes the functional reconstruction and postoperative healing of tissues.

Description

Biological pelvic floor repairing mesh and preparation method thereof

Technical Field

The invention relates to the field of biological materials, in particular to a biological repairing mesh for a pelvic floor and a preparation method thereof.

Background

Female pelvic floor dysfunction is mainly manifested by pelvic organ prolapse, stress urinary incontinence and the like, and 50% of women who pass through the body have been reported to have pelvic floor dysfunction in different degrees. Surgical operations are the main methods for treating the serious pelvic floor dysfunction, and the operation methods are mainly repair and suspension operations of various tissues and physiological structures. Often, materials such as patches are implanted to enhance the surgical effect. Tension-free pelvic floor repair is the current main surgical mode of the pelvic floor. With the rapid development of medical biomaterials, various pelvic floor repair materials have been widely used in clinical applications.

The pelvic floor patch is mainly divided into a synthetic material patch and a biological material patch.

Composite material patch

The basin bottom patch made of synthetic material mainly comprises multiple polymers including degradable type and non-degradable type. The non-degradable synthetic material patch has good tensile strength, but cannot be organically combined with body tissues because of being not degraded by the tissues, so that local tissue inflammation and infection after operation are easily caused after the patch is placed in the body, the structural integrity after long-term in vivo retention is damaged, the patch can be transferred to other tissues to cause chronic inflammation and foreign body reaction, and revision operation is needed; secondly, due to the difference of mechanical compliance between the in vivo tissue and the surrounding tissue, the fibrosis reaction of the surrounding tissue can be gradually caused, and a fibrous envelope is gradually formed, so that the erosion risk of the product to the adjacent tissue organ is increased after long-term use.

With the development of the technology, degradable synthetic material patches are derived, and are generally synthesized by polylactic acid and the like, although the mechanical properties are still good, the degradable synthetic material patches are implanted into the body to cause acute inflammatory reaction, then chronic inflammation is caused, and finally granulation tissues and fiber packages are formed; and high-concentration lactic acid and glycolic acid which are locally formed in the degradation process of materials such as polylactic acid and the like are easy to cause cytotoxicity.

The synthetic material has good mechanical strength but poor biological performance, and can not induce tissue regeneration and healing. Therefore, the development direction of the synthetic patch is bionic, namely different weaving methods are adopted, natural biological materials such as collagen, fibrin and the like are added, and the tissue characteristics are simulated, so that the healing potential is enhanced, and the success rate of the pelvic floor repair operation is further improved.

② biological material patch

The biological material patch is mainly derived from tissue materials and can be divided into allogeneic acellular tissue materials, xenogeneic acellular tissue materials and the like.

Allogeneic materials are mainly derived from products of human dermal skin tissues, and although they have the ability to promote tissue healing after pelvic floor repair, they have the risks of lack of sources, susceptibility to infectious diseases (such as AIDS) and the like, so the application is limited to a certain extent.

The xenogenic acellular material is mainly derived from tissues such as animal dermis, small intestine, bladder, pericardium, and the like, and is a material which retains a three-dimensional structure and collagen fiber components originally in an extracellular matrix by treating immune components such as cells, DNA, and the like. The three-dimensional structure, collagen, non-collagen, growth factor and other components in the extracellular matrix provide adaptive environment for adhesion, proliferation and differentiation of host cells, and contribute to functional reconstruction of tissues, thereby promoting healing of tissues after pelvic floor repair.

In practical application, because the acellular material patch has insufficient mechanical strength, epoxide or glutaraldehyde and other chemical cross-linking agents need to be introduced in the processing process, and the acellular material patch has the following defects: has potential cytotoxicity, slow degradation rate and mismatch with tissue regeneration, and can cause reactions such as fibrosis, chronic inflammation and the like. The invention patent of China with the authorization number of CN103800942, a pelvic floor repair patch, is prepared by the technology of electrostatic spinning and the like to prepare non-degradable and degradable synthetic materials into the patch; although the patch is made of biomaterial, the chinese patent application No. 201610678065.0 only uses a clamp to tightly attach multiple layers, or uses an adhesive or suture to firmly attach the layers together, and does not substantially increase the mechanical strength of the biomaterial.

Therefore, the invention is developed on the basis of not introducing synthetic materials or cross-linking agents and aims to enhance the mechanical strength of the pelvic floor biological patch material.

Disclosure of Invention

The invention aims to provide a biological pelvic floor repairing mesh aiming at the defects of the biological patch in the prior art in terms of structural strength.

Therefore, the biological repairing mesh for the pelvic floor is provided by the invention, wherein the mesh is woven by heterogeneous acellular matrix materials.

Furthermore, the xenogenic acellular matrix material is prepared by washing and cutting xenogenic acellular matrix, inactivating viruses, degreasing, acellular, removing DNA and alpha-Gal antigens, shaping and freeze-drying.

Further, the acellular matrix includes, but is not limited to, one or more combinations of small intestine submucosa, bladder submucosa, stomach submucosa, dermal matrix, pericardium, meninges, amnion, visceral membrane, peritoneum of mammals.

Further, the acellular matrix is small intestine submucosa of animals.

The invention also provides a preparation method of the biological repairing mesh for the pelvic floor, which comprises the following steps:

(1) the pretreatment is carried out in a pre-treatment way,

cleaning and cleaning animal tissues of fresh slaughtered animals, soaking in acetic acid solution, scraping off mucosa, muscle layer, serosa layer and lymph node of the animal tissues, separating submucosa, longitudinally and uniformly cutting into fine strips, and washing with purified water to obtain the pelvic floor biological repair material;

(2) the virus is inactivated, and the virus is inactivated,

soaking the biological repairing material of the basin bottom in a mixed aqueous solution of peroxyacetic acid and ethanol at the ultrasonic room temperature, inactivating viruses, and ultrasonically cleaning with purified water;

(3) degreasing the mixture, namely degreasing the mixture,

soaking the raw materials in an ethanol solution under the conditions of ultrasound and normal temperature, and then ultrasonically cleaning the raw materials by using water for injection;

(4) decellularizing, DNA removing and alpha-Gal antigen removing,

soaking the mixture in mixed water solution containing trypsin and EDTA under ultrasonic condition, and ultrasonic cleaning with PBS;

soaking in water solution containing DNA enzyme under ultrasonic condition; then rinsing with PBS and ultrasonically cleaning;

soaking the raw materials in an aqueous solution containing alpha-galactosidase under an ultrasonic condition; then performing ultrasonic cleaning by using PBS;

soaking the substrate with NaOH aqueous solution at normal temperature under an ultrasonic condition, and then ultrasonically cleaning the substrate with PBS until the substrate is neutral;

(5) shaping, freeze-drying, weaving and sterilizing

Twisting the treated fine strip submucosa into lines, fixing the lines on a mould, freezing and drying the lines, weaving into a net-shaped pelvic floor biological repair net sheet, packaging by a PET packaging bag, and then carrying out irradiation sterilization.

Further, in the pretreatment step, the concentration of acetic acid is 0.01-0.5%, the soaking time is 10-120min, and the ratio of animal tissues to the acetic acid solution is 1:2-1: 10.

Further, in the virus inactivation step, the concentration of the peroxyacetic acid is 0.5-1.5%, the concentration of the ethanol is 15-25%, and the ratio of the pelvic floor biological repair material to the mixed aqueous solution is 1:2-1:10, the soaking time is 30-120 min.

Further, in the degreasing step, the concentration of the ethanol is 90-100%, the ratio of the biological repairing material for the basin bottom to the ethanol is 1:2-1:10, and the soaking time at normal temperature is 0.5-12 h.

Further, in the steps of decellularizing, DNA removing and a-Gal antigen removing:

the content of trypsin and EDTA in the mixed water solution is 0.01-0.10% and 0.01-0.05%, respectively, the ratio of the pelvic floor biological repair material to the trypsin/EDTA solution is 1:2-1:10, and the mixed water solution is soaked for 15-40min at 36 +/-2 ℃ under the ultrasonic condition;

the content of the DNase in the aqueous solution containing the DNase is 0.05-10U/ml, the ratio of the biological repair material of the pelvic floor to the aqueous solution containing the DNase is 1:2-1:10, the soaking temperature is 36 +/-2 ℃, and the soaking time is 15-40 min;

the alpha-galactosidase content in the alpha-galactosidase-containing water solution is 0.05-10U/ml, the ratio of the basin bottom biological repairing material to the alpha-galactosidase solution is 1:2-1:10, the soaking temperature is 20-37 ℃, and the soaking time is 15-40 min;

the concentration of the NaOH aqueous solution is 5-40mM, the ratio of the biological repair material on the basin bottom to the NaOH solution is 1:5-1:50, and the soaking time at normal temperature is 20-60 min.

Compared with the prior art, the invention has the following remarkable advantages and beneficial effects:

the invention aims to solve the technical problem of providing a novel pelvic floor biological repairing mesh aiming at the defects of a pelvic floor biological repairing patch.

1. The biological repairing mesh for pelvic floor provided by the invention takes tissues of animal dermis, small intestine, pericardium and the like as raw materials, immune components such as cells, DNA and the like are removed, the raw materials are cut into strips, the strips are twisted into lines, and the mesh is woven. The heterogenous acellular material is twisted into threads to obtain higher mechanical strength, so that the heterogenous acellular material is not easy to break, and after the mesh-shaped patch is woven into a mesh shape, because the threads are interwoven, the external force of stretching and tearing does not act on one point but a plurality of interwoven points, so that the mesh has good mechanical property, the defect of poor mechanical strength of the heterogenous acellular material pelvic floor biological patch is overcome, the structure of the mesh is prepared by a weaving process, and the pore size of the mesh can be easily adjusted by weaving parameters, so that the requirement of practical application is met.

2. The pelvic floor biological repairing mesh provided by the invention reserves the original three-dimensional structure, collagen fiber, non-collagen, growth factor and other components in the extracellular matrix, has the healing promoting effect, and accelerates the functional reconstruction of tendon and the healing after pelvic floor repairing.

3. The ECM three-dimensional structure of the biological repairing mesh has the functions of inducing cells and blood vessels to grow in, the mesh can be gradually degraded when new tissues grow in, the polypeptide component of a degradation product has antibacterial performance, and the incidence rate of inflammation and infection after implantation can be reduced.

4. The biological pelvic floor repairing mesh provided by the invention does not introduce a cross-linking agent and a synthetic material, does not have potential cytotoxicity, and does not cause reactions such as fibrosis and chronic inflammation.

5. According to clinical requirements, the biological pelvic floor repairing mesh provided by the invention can be added with healing promoting substances or antibiotics in the preparation process, and can also be loaded with the healing promoting substances or antibiotics in a soaking mode before being implanted into a body, so that the healing of wounds is further promoted and the infection incidence rate is reduced.

Drawings

Fig. 1 is a real object diagram of a biological mesh for repairing a pelvic floor.

Fig. 2 is a structural schematic diagram of the pelvic floor biological repair mesh.

FIG. 3 is a staining diagram of a slice HE of a biological mesh for pelvic floor repair.

FIG. 4 is a microscopic view of a biological mesh for pelvic floor repair.

FIG. 5 is a flow chart of the process for preparing a biological mesh for pelvic floor repair.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Example 1

Preparation of biological repair mesh for porcine small intestine submucosa pelvic floor, please refer to fig. 5:

(1) pretreatment of

Cleaning and cleaning small intestine tissues of a fresh slaughtered animal pig, soaking in a 0.5% acetic acid solution for 30min, wherein the ratio of the small intestine to the acetic acid solution is 1:5, removing a mucous membrane layer, a muscular layer, a serous membrane layer and a lymph node of the small intestine jejunum of the pig by using a physical scraping method, separating out a submucosa layer, uniformly cutting the submucosa layer into thin strips longitudinally, and washing for 3 times by using purified water to obtain a biological repairing material, namely a small intestine submucosa material, which is hereinafter referred to as SIS material for short.

(2) Inactivation of viruses

A mixed aqueous solution containing 1.0% of peroxyacetic acid and 15% of ethanol is used, the ratio of the SIS material to the mixed aqueous solution is 1:10, and the mixture is soaked for 100min at room temperature under the ultrasonic condition for virus inactivation. Followed by 3 ultrasonic washes with purified water.

(3) Degreasing

Ethanol with the concentration of 95% is used, the ratio of the SIS material to the ethanol is 1:10, and the soaking is carried out for 2 hours at normal temperature under the ultrasonic condition. Then ultrasonically cleaning for 3 times by using water for injection.

(4) Decellularized, DNA-removed and alpha-Gal antigens

A mixed aqueous solution containing 0.02% of trypsin and 0.02% of EDTA is used, the ratio of the SIS material to the trypsin/EDTA solution is 1:5, and the mixture is soaked for 30min at 37 ℃ under the ultrasonic condition. Followed by 3 ultrasonic washes with PBS.

An aqueous solution containing 5U/ml of DNase is used, the ratio of the SIS material to the DNase solution is 1:5, and the mixture is soaked for 20min at 37 ℃ under the ultrasonic condition. Followed by 3 ultrasonic washes using PBS rinsing.

An aqueous solution containing 5U/ml of alpha-galactosidase is used, the ratio of the SIS material to the alpha-galactosidase solution is 1:5, and the mixture is soaked for 20min at 30 ℃ under the ultrasonic condition. Followed by 3 ultrasonic washes with PBS.

Using 25mM NaOH aqueous solution, wherein the ratio of the SIS material to the NaOH solution is 1:20, and soaking for 50min at normal temperature under the ultrasonic condition. Followed by sonication with PBS until neutral.

(5) Shaping, freeze-drying, weaving and sterilizing

Twisting the treated strip-shaped fine-strip submucosa into lines, fixing the lines on a mould, freezing and drying the lines, weaving the net-shaped pelvic floor biological repair mesh sheet into the mesh sheet in the figure 1 by a weaving machine, packaging by a PET packaging bag, and then irradiating and sterilizing the mesh sheet, wherein the schematic diagram of the pelvic floor biological repair mesh sheet is shown in figure 2.

Example 2

Preparation of biological repairing net sheet for pig small intestine submucosa pelvic floor

(1) Pretreatment of

Cleaning small intestine tissue of fresh slaughtered animal, soaking in 0.01% acetic acid solution at a ratio of 1:10 for 120min, removing mucosa, muscle layer, serosal layer and lymph node of small intestine by physical scraping, separating submucosa, cutting into segments, and washing with purified water for 3 times.

(2) Inactivation of viruses

A mixed aqueous solution containing 0.5% of peroxyacetic acid and 25% of ethanol is used, the ratio of the SIS material to the mixed aqueous solution is 1:15, and the mixture is soaked for 120min at room temperature under the ultrasonic condition for virus inactivation. Followed by 3 ultrasonic washes with purified water.

(3) Degreasing

Ethanol with the concentration of 90% is used, the ratio of the SIS material to the ethanol is 1:15, and the soaking is carried out for 4 hours at normal temperature under the ultrasonic condition. Then ultrasonically cleaning for 3 times by using water for injection.

(4) Decellularized, DNA-removed and alpha-Gal antigens

A mixed aqueous solution containing 0.05% of trypsin and 0.01% of EDTA is used, the ratio of the SIS material to the trypsin/EDTA solution is 1:10, and the mixture is soaked for 20min at 37 ℃ under the ultrasonic condition. Followed by 3 ultrasonic washes with PBS.

An aqueous solution containing 1U/ml of DNase is used, the ratio of the SIS material to the DNase solution is 1:10, and the SIS material and the DNase solution are soaked for 30min at 37 ℃ under the ultrasonic condition. Followed by 3 ultrasonic washes using PBS rinsing.

An aqueous solution containing 1U/ml of alpha-galactosidase is used, the ratio of the SIS material to the alpha-galactosidase solution is 1:10, and the mixture is soaked for 30min at 30 ℃ under the ultrasonic condition. Followed by 3 ultrasonic washes with PBS.

Using 40mM NaOH aqueous solution, wherein the ratio of the SIS material to the NaOH solution is 1:10, and soaking for 30min at normal temperature under the ultrasonic condition. Followed by sonication with PBS until neutral.

(5) Shaping, freeze-drying, weaving and sterilizing

Twisting the treated small intestinal submucosa into lines, fixing on a mould, after freeze-drying, weaving into a mesh-shaped pelvic floor biological repair mesh sheet by a weaving machine, and after packaging by a PET packaging bag, carrying out irradiation sterilization.

Example 3

For the safety of the sample, the sample prepared in example 1-2 was subjected to the detection of the immunogenic substance.

(1) The cell residue detection method comprises the following steps: fixing with 10% neutral formalin, embedding in paraffin, cutting into 0.4 micrometer slices, dewaxing with xylene, dehydrating with alcohol, staining with hematoxylin-eosin, and observing cell residue and matrix fiber structure under microscope.

(2) The DNA content detection method comprises the following steps: according to YY/T0606.25-2014 animal-derived biomaterial DNA residue determination method: fluorescence staining method for detection.

(3) The alpha-Gal antigen content detection method comprises the following steps: after fixing the sample with paraformaldehyde, the sections were embedded in normal paraffin with a slice thickness of 3 μm. The specific affinity characteristic of the biotin label BSI-B4 and alpha-Gal antigen is utilized to carry out an immunohistochemical reaction. And (3) judging a dyeing result: dark brown yellow particles are strongly positive (+++), brown yellow particles are positive (++), yellow particles are weakly positive (+), and no coloration is negative (-).

(4) The lipid content detection method comprises the following steps: the measurement was carried out by referring to Soxhlet extraction method in GB/T5009.6 measurement of fat in foods.

The results are shown in the following table:

example 4

The samples prepared in examples 1-2 were tested for biological, histological, and bacterial endotoxin and antibacterial properties.

(1) Biological Performance testing

The method comprises the following steps: the test was carried out with reference to the GB/T16886 series of methods.

As a result: the cytotoxicity reactions are all grade 1; no delayed hypersensitivity reaction; the intradermal reaction showed that the difference between the mean scores of the test sample and the solvent control was less than 1.0; no pyrogenicity; no hemolytic reaction; the result of the genotoxicity test shows that the salmonella typhimurium back mutation (Ames) test shows negative reaction, the mouse lymphoma test shows negative reaction and no chromosome aberration; no acute systemic toxic reaction; no sub-chronic systemic toxicity; the tissue reaction of the muscle implanted for 30 days, 60 days and 90 days is not obviously different from that of the negative control.

(2) Histological examination

1) Observation with an optical microscope

The method comprises the following steps: fixing with 10% neutral formalin, embedding in paraffin, cutting into 0.4 micrometer slices, dewaxing with xylene, dehydrating with alcohol, staining with hematoxylin-eosin, and observing cell residue and matrix fiber structure under microscope.

As a result: no cells and cell debris remain; the collagen fibers were continuous without breaks, as shown in fig. 3.

2) Ultrastructural observation

The method comprises the following steps: scanning is performed using an electronic scanning mirror.

As a result: the material is in a porous structure, and collagen fibers are not broken, as shown in figure 4.

(3) Bacterial endotoxin detection

The method comprises the following steps: the detection is carried out according to a relevant method in GB/T14233.

As a result: are all less than 2.15 EU/unit.

(4) Detection of antibacterial Properties

The samples prepared in examples 1 and 2 were separately ground in 0.01M hydrochloric acid with a grinding rod until no particles were visible to the naked eye, and the concentration was adjusted to 100mg/10 mL. Adding pepsin for digestion, wherein the ratio of the pepsin to the samples is 1: 10. Stirring is continued for 48h at 25 ℃ and then cooled to 4 ℃ and 1/10 volumes of 0.1M sodium hydroxide are added to adjust the pH to 7.2-7.4.

Preparing a mixed bacteria culture medium plate, and respectively picking a small amount of cultured staphylococcus aureus and escherichia coli slant culture medium substances in 5ml of sterile physiological saline by using an inoculating loop to prepare bacterial suspension. Adding 1.0ml of the bacterial suspension and 1ml of the degraded sample into a sterilized and dried culture dish, adding a common nutrient broth agar culture medium cooled to about 50 ℃, shaking uniformly, fully condensing for later use, performing inverted culture at 35-37 ℃ for 24 hours, and observing the growth condition of bacteria; meanwhile, the addition of the antibacterial materials and the addition of 5 mu g/mL of antibacterial peptide are compared, and the results are shown in the following table:

example 5

The samples prepared in examples 1 to 2 and samples 1 to 2 which were not cut and woven were subjected to mechanical property testing.

(1) Suture strength

The method comprises the following steps: sewing the center of the two sides of the sample by using a 3-0 non-absorbent suture line at a position 2mm away from the edge, respectively fixing the other end of the suture line and the other end of the sample on the two ends of a tension meter, stretching at the speed of 20mm/min until the sewing point is torn, and recording the maximum force value.

(2) Tensile strength

The method comprises the following steps: respectively cutting the sample into a shape with the width of 10mm along two directions; after cutting, the test is carried out after the glass is placed in an environment with the relative humidity of 40-60% and the temperature of 22 +/-2 ℃ for 2 hours. The distance between the clamps is 25mm, the two ends of the sample are fixed on a chuck of a tensile testing machine, the sample is stretched at the speed of 100mm/min, and the maximum force value during fracture is recorded.

(3) Burst strength

The method comprises the following steps: a9.5 mm diameter probe is selected for detection according to a measuring method of rupture strength of an 8.3.3.2 probe of YY 0500-2004 cardiovascular implant artificial blood vessel, and the results are shown in the following table:

many modifications may be made by one of ordinary skill in the art in light of the above teachings. Therefore, it is intended that the invention not be limited to the particular details of the embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.

Claims (8)

1. A biological net piece of repairing of pelvic floor which characterized in that: the mesh is woven by a heterogeneous acellular matrix material, the acellular matrix material is obtained by using a mixed solution containing trypsin and EDTA, the concentrations of the mixed solution are 0.01-0.10% and 0.01-0.05%, and the mesh is soaked for 15-40 minutes under the ultrasonic condition; the acellular matrix material is woven by cutting the acellular matrix material into thin strips, twisting the strips into threads, and weaving the strips into net sheets by using a weaving machine.

2. The biological pelvic floor repair mesh according to claim 1, wherein: the mesh structure is prepared by a weaving process, and the size of the mesh aperture can be adjusted by weaving parameters.

3. The biological pelvic floor repair mesh according to claim 1, wherein: the acellular matrix comprises one or more of the combination of small intestine submucosa, bladder submucosa, stomach submucosa, dermal matrix, pericardium, meninges, amnion, organ membrane and peritoneum of mammals.

4. A preparation method of a biological repairing mesh for a pelvic floor is characterized by comprising the following steps:

(1) the pretreatment is carried out in a pre-treatment way,

cleaning and cleaning animal tissues of fresh slaughtered animals, soaking in acetic acid solution, scraping off mucosa, muscle layer, serosa layer and lymph node of the animal tissues, separating submucosa, longitudinally and uniformly cutting into fine strips, and washing with purified water to obtain the pelvic floor biological repair material;

(2) the virus is inactivated, and the virus is inactivated,

soaking the biological pot bottom repairing material in mixed water solution containing peroxyacetic acid and ethanol at room temperature by using ultrasonic waves, inactivating viruses, and ultrasonically cleaning the materials by using purified water;

(3) degreasing the mixture, namely degreasing the mixture,

soaking the raw materials in an ethanol solution under the conditions of ultrasound and normal temperature, and then ultrasonically cleaning the raw materials by using water for injection;

(4) decellularizing, DNA removing and alpha-Gal antigen removing,

soaking the mixture in mixed water solution containing trypsin and EDTA under ultrasonic condition, and ultrasonic cleaning with PBS;

soaking in water solution containing DNA enzyme under ultrasonic condition; then rinsing with PBS and ultrasonically cleaning;

soaking the raw materials in an aqueous solution containing alpha-galactosidase under an ultrasonic condition; then performing ultrasonic cleaning by using PBS;

soaking the substrate with NaOH aqueous solution at normal temperature under an ultrasonic condition, and then ultrasonically cleaning the substrate with PBS until the substrate is neutral;

(5) shaping, freeze-drying, weaving, sterilizing,

twisting the treated fine strip submucosa into lines, fixing the lines on a mould, freezing and drying the lines, weaving into a net-shaped pelvic floor biological repair net sheet, packaging by a PET packaging bag, and then carrying out irradiation sterilization.

5. The method according to claim 4, wherein in the pretreatment step, the concentration of acetic acid is 0.01 to 0.5%, the soaking time is 10 to 120min, and the ratio of the animal tissue to the acetic acid solution is 1:2 to 1: 10.

6. The method according to claim 4, wherein in the virus inactivation step, the concentration of peracetic acid is 0.5 to 1.5%, the concentration of ethanol is 15 to 25%, the ratio of the pelvic floor biological repair material to the mixed aqueous solution is 1:2 to 1:10, and the soaking time is 30 to 120 min.

7. The preparation method according to claim 4, wherein in the degreasing step, the concentration of ethanol is 90-100%, the ratio of the biological repair material for the pelvic floor to ethanol is 1:2-1:10, and the soaking time at normal temperature is 0.5-12 h.

8. The method of claim 5, wherein in the steps of decellularizing, DNA removing and α -Gal antigen removing:

the concentrations of trypsin and EDTA in the mixed water solution are 0.01-0.10% and 0.01-0.05%, respectively, the ratio of the pelvic floor biological repair material to the trypsin/EDTA solution is 1:2-1:10, and the mixture is soaked for 15-40min at 36 +/-2 ℃ under the ultrasonic condition;

the content of the DNase in the aqueous solution containing the DNase is 0.05-10U/ml, the ratio of the biological repair material of the pelvic floor to the aqueous solution containing the DNase is 1:2-1:10, the soaking temperature is 36 +/-2 ℃, and the soaking time is 15-40 min;

the alpha-galactosidase content in the alpha-galactosidase-containing water solution is 0.05-10U/ml, the ratio of the basin bottom biological repairing material to the alpha-galactosidase solution is 1:2-1:10, the soaking temperature is 20-37 ℃, and the soaking time is 15-40 min;

the concentration of the NaOH aqueous solution is 5-40mM, the ratio of the biological repair material on the basin bottom to the NaOH solution is 1:5-1:50, and the soaking time at normal temperature is 20-60 min.

Images