Extracellular matrix fiber material as well as preparation method and application thereof

Abstract

The invention relates to the technical field of repair materials, and particularly discloses an extracellular matrix fiber material as well as a preparation method and application thereof. The invention discloses a preparation method of an extracellular matrix fiber material. The preparation method comprises the following steps: taking animal tissues, and carrying out virus inactivation, decellularization and antigen removal, degreasing, drying, grinding and sterilization; the decellularization and antigen removal method comprises the following specific steps: sequentially soaking a sample with a surfactant solution for h; then, soaking treatment is carried out by adopting a salt-alkali solution which contains 0.05-3 mol / L of salt and has the pH value of 8-11; then carrying out soaking treatment by adopting a 0.1-1wt% enzyme solution; and in degreasing, one or more of normal hexane, trichloromethane, dichloromethane, methanol, ethanol, acetone, diethyl ether and ethyl acetate are used for soaking the sample. According to the technical scheme, the ECM powder particle material with high growth factor content is prepared.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of repair materials, and in particular to an extracellular matrix fiber material and a preparation method and application thereof. BACKGROUND

[0002] The extracellular matrix (ECM) has been widely used in the field of biological medicine. As a natural material derived from biology, the decellularized matrix often uses animal-derived raw materials such as pig collagen and bovine cartilage collagen as raw materials for implantable degradable medical devices. These natural materials not only have good biocompatibility and biodegradability, but also have stable scaffold structures and contain growth active factors that can promote tissue regeneration. At present, there are already marketed materials such as dura mater, hernia repair patches, and nerve patches. A large amount of data has proved that they are safe and effective as implant materials. With the continuous progress of decellularization and antigen-removing technology and industrial processing technology, in addition to sheet materials, a variety of forms of decellularized extracellular matrix products including powders, gels, etc. have been put on the market.

[0003] Vaginal reconstruction surgery is a gynecological surgery aimed at providing a functional vagina for women, which is suitable for patients with congenital absence of vagina, trauma or after tumor resection. The new vagina in vaginal reconstruction surgery can increase the thickness of the repaired vaginal epithelium after using repair materials to guide tissue regeneration, increase the elasticity of the new vagina, and reduce the occurrence of other postoperative complications. There are many types of patch materials reported to be used, mainly including autologous tissue, allogeneic grafts, xenogeneic grafts and artificial synthetic patches.

[0004] At present, the animal-derived ECM used in vaginal reconstruction surgery is mainly in the form of sheet material, and the powder fiber material is only used for human mucosa transplantation (such as oral mucosa). At present, there is no related application of ECM powder fiber material for vaginal reconstruction surgery. Therefore, it is necessary to provide an animal-derived ECM powder fiber material for gynecological vaginal repair material. SUMMARY

[0005] In order to solve the above technical problems, the present application provides an extracellular matrix fiber material and a preparation method and application thereof.

[0006] In a first aspect, the present application provides a preparation method of an extracellular matrix fiber material, which specifically comprises sequentially performing the following steps: taking animal tissue, performing virus inactivation, decellularization and antigen removal, defatting, drying, powdering and sterilization. The specific steps of the decellularization and antigen removal are: under the condition of solid-liquid mass volume ratio of 1g:1-10ml, the sample is sequentially immersed and treated with 0.5-15wt% surfactant solution for 1-5 times, and each immersion treatment time is 0.5-6h; then immersed and treated with a salt alkaline solution containing 0.05-3mol / L salt and having a pH of 8-11 for 1-5 times, and each immersion treatment time is 1-10h; and then immersed and treated with 0.1-1wt% enzyme solution for 3-20h; The salt is selected from one or more of sodium chloride, sodium phosphate, and potassium chloride; The surfactant is selected from one or more of Triton X114, sodium dodecyl sulfonate, sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonate inner salt CHAPS, and sodium cholate; The enzyme is selected from one or more of trypsin, pepsin, and proteinase K; The defatting is performed by immersing and treating the sample with one or more of n-hexane, chloroform, dichloromethane, methanol, ethanol, acetone, and diethyl ether for 1-5 times, and each immersion treatment time is 1-8h.

[0007] During the experiment, the applicant found that the animal-derived decellularized matrix material has low mechanical strength before powdering during the powdering process, which makes the mechanical three-dimensional structure of the decellularized matrix material after powdering incomplete, and reduces the effective utilization of the decellularized matrix material. Through repeated experiments, the preparation process provided in the present application improves the method, and sequentially uses the processes of virus inactivation, decellularization and antigen removal, defatting, drying, powdering, and sterilization. The mechanical strength of the decellularized matrix material prepared before drying is excellent, facilitating the subsequent powdering operation steps, and can achieve large-scale standardized production while achieving powder particle fiber repair effect.

[0008] Through the above technical solutions, the ECM powder particle material with high growth factor content and slow degradation time is prepared, the ECM is close to the biological tissue structure near the vagina, the short-term repair effect is more significant, and the patient has a faster short-term curative effect; the ECM can perform in-situ physical space filling while repairing, prevent adhesion, supplement the vaginal tension, and relieve the symptoms of the patient; the large particle size granular solid injection prevents blood vessel blockage, so that the powder particles do not block the blood vessels after entering the blood vessels; large-scale standardized production can be achieved while achieving powder particle repair effect.

[0009] In the decellularization and antigen removal step, the sample is first treated with a surfactant to effectively destroy the cell membrane, promote cell lysis and component release, help remove cell residues, release more growth factors, and obtain pure extracellular matrix. Then the sample is treated with a salt solution of a specific concentration and a specific pH, which can induce intramolecular or intermolecular hydrogen bond crosslinking, increase β-pleated structure, and make fiber arrangement more compact. The removal of non-collagen fiber components such as short peptide chains, glycosaminoglycans and other impurities improves the overall rigidity and tensile strength of the material, enhances the mechanical properties of the dried ECM material, and further enables the grinding of the ECM fragments with more complete mechanical three-dimensional structure and more retained growth factors, promoting tissue repair in the human body. At the same time, alkaline treatment can effectively decompose DNA long chains and hydrolyze endotoxin glycoproteins, improving the biological safety of the final product. Finally, by precisely regulating the amount of enzyme added for synergistic decellularization treatment, the peptide chain is specifically cut to efficiently decompose the intracellular proteins, thereby promoting cell lysis and component release, helping to remove cell residues, and making the extracellular matrix more pure.

[0010] Preferably, the animal source is selected from one or more of bovine, porcine, and ovine; and the tissue is selected from one or more of dermis, muscle, tendon, heart, fat, small intestine, nerve, bone, and cartilage.

[0011] Preferably, the virus inactivation is performed using a disinfectant solution with a solid-liquid weight ratio of 1 g:1-10 ml; and the disinfectant solution is selected from one or more of hydrogen peroxide, peracetic acid solution, and ethanol.

[0012] Preferably, the specific method for virus inactivation is to use a mixed aqueous solution of peracetic acid with a concentration of 0.01-3 wt% and ethanol with a concentration of 1-20 wt% as a disinfectant solution, and soak the sample for 10-180 min, or to use a hydrogen peroxide solution with a concentration of 0.01-10 wt% to soak the sample for 1-12 h.

[0013] Further, the specific method for virus inactivation is to use a mixed aqueous solution of peracetic acid with a concentration of 1-3 wt% and ethanol with a concentration of 5-10 wt% as a disinfectant solution, and soak the sample for 60-120 min.

[0014] Preferably, the specific steps for decellularization and antigen removal are as follows: the sample is sequentially soaked with a 1-5 wt% sodium dodecyl sulfate solution for 2-4 times, with each soaking treatment time being 2-4 h; then the sample is soaked with a salt solution containing 1-2 mol / L sodium chloride and having a pH of 9-10 for 1-4 times, with each soaking treatment time being 1-7 h; and then the sample is soaked with a 0.2-0.8 wt% trypsin solution at a temperature of 2-8℃ for 6-15 h.

[0015] Further, the organic solvent in the defatting is composed of methanol and chloroform in a volume ratio of 1-5:1.

[0016] During the experiment, the organic solvent methanol in the defatting can fix proteins and dissolve lipids, and retain structural proteins (such as collagen and elastin) and growth factors in the ECM. While chloroform as a strong defatting agent can effectively remove cell membrane lipids and intracellular lipid droplets, and can ensure the complete destruction of the cell membrane, thereby more efficiently releasing the growth factors in the cells. The present application can realize efficient defatting and decellularization treatment by precisely controlling the ratio of methanol and chloroform, so as to prepare the extracellular matrix with excellent mechanical properties and rich growth factor content.

[0017] Preferably, the drying method is selected from any one of suction drying and freeze drying; The process parameters of the suction drying are: temperature 20-60℃, wind box pressure 1-20mbar, time 2-24h; The process parameters of the freeze drying are: freeze drying temperature -20℃ to -80℃, vacuum degree 0.1-100mbar, time 2-24h.

[0018] Preferably, the specific steps of the powder grinding are: using a cutting machine to cut under the conditions of a rotation speed of 2000-6000rpm and a distance between the cutting tool head and the edge of the cutting mold of 30-150μm, and collecting by sieving.

[0019] In the second aspect, the present application provides an extracellular matrix fiber material prepared by the above preparation method; The length of the extracellular matrix fiber material is 100-2000μm, the width is 50-1000μm, and the aspect ratio is 2-10:1; the melting point is 73-80℃.

[0020] Preferably, the length of the extracellular matrix fiber material is 300-1000μm, the width is 100-330μm, and the aspect ratio is 3-8:1; the extracellular matrix fiber material contains the following contents of growth factors: VEGF≥70000ng / g, bFGF≥14ng / g, TGF-β≥5ng / g, TNF-α≥11ng / g.

[0021] Preferably, the extracellular matrix fiber material contains the following contents of growth factors: VEGF≥80000ng / g, bFGF≥16ng / g, TGF-β≥6ng / g, TNF-α≥13ng / g.

[0022] Studies have shown that muscle fiber cells and vascular epithelial cells are easy to adhere to rough surfaces, while epithelial cells are more likely to grow on smooth surfaces. Compared with fine-grained ECM microparticles, the three-dimensional framework structure of the rough particle extracellular matrix fiber material provided in the application can promote the anchoring and proliferation of smooth muscle cells and vascular endothelial cells, and the collagen component has hemostatic and wound healing effects. The smooth surface not only facilitates the migration and proliferation of epithelial cells, but also plays an effective barrier function to inhibit adhesion and scar tissue formation.

[0023] The extracellular matrix fiber material provided in the application can be used for gynecological perivaginal repair material powder particle material, such as for vaginal reconstruction repair after vaginoplasty; the extracellular matrix (ECM) material is ground into a fragment fiber material, the ECM biological activity is retained, and the ECM is used for repairing after human vaginoplasty and filling repair around the vagina to function the vagina, plays an immediate support while promoting long-term fibroblast-driven collagen regeneration, and restores the physical and mechanical support function of the vagina; the ECM can perform in-situ physical space filling while repairing, prevent adhesion while supplementing vaginal tension, and relieve symptoms of patients. At the same time, when the extracellular matrix fiber material is used by injection, blood vessel blockage can be effectively prevented, so that the powder particles enter the blood vessels and cause blockage.

[0024] Animal experiments prove that in the rat animal experiment, the vaginal mucosa generation time of the rat vaginal natural repair process after the rat vaginal resection is 2 months, and the vaginal mucosa generation time of the beagle dog is 3 months. The repair material before the repair of the vaginal mucosa can play a role in occupying space, stimulating tissue regeneration, and preventing adhesion, and a material with longer-term degradation is more conducive to tissue repair. The powder particle material improves the processing technology, causes less damage to the collagen structure, can obtain a tissue repair material with longer degradation time, and is more suitable for soft tissue repair.

[0025] In a third aspect, the application provides a use of the extracellular matrix fiber material in the preparation of a medical repair material.

[0026] In summary, the technical scheme of the application has the following effects: The preparation process provided in the application adopts a process of virus inactivation, decellularization, antigen removal, defatting, drying, powdering, and sterilization, while the specific raw materials and process parameters in each step are controlled, so that an ECM fiber material with high growth factor content and slow degradation time is prepared. The ECM fiber material can be used for gynecological vaginal repair. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The morphology of the extracellular matrix fiber material in Example 1. DETAILED DESCRIPTION

[0028] The application will be further described in detail in connection with the following examples, comparative examples and performance test experiments, which cannot be understood as limiting the scope of the application claimed. Examples

[0029] Example 1 Example 1 provides an extracellular matrix fiber material and a preparation method thereof.

[0030] The preparation method of the extracellular matrix fiber material in Example 1 is specifically shown as follows.

[0031] (1) Material taking: fresh biological tissue pig small intestinal submucosa is taken, and washed repeatedly with water until no blood stains; then fat and fascia are removed, and cut into 20-50 cm strips.

[0032] (2) Virus inactivation: the sample is soaked in a disinfectant solution with a solid-liquid mass volume ratio of 1 g:5 ml, and a mixed aqueous solution with a concentration of 1.5 wt% peracetic acid and 8 wt% ethanol is used as the disinfectant solution, and the sample is soaked for 120 min.

[0033] (3) Decellularization and antigen removal: under the condition of a solid-liquid mass volume ratio of 1 g:5 ml, the sample is soaked in a 2.5 wt% sodium dodecyl sulfate surfactant solution for 3 times, and each soaking time is 3 h; then the sample is soaked in a salt alkali solution containing 1.5 mol / L sodium chloride and having a pH of 9.5 for 3 times, and each soaking time is 5 h; then the sample is soaked in a 0.6 wt% trypsin solution at a temperature of 5℃ for 10 h.

[0034] (4) Defatting: the sample is soaked in an organic solvent (composed of methanol and chloroform with a volume ratio of 1:1) for 3 times, and each soaking time is 5 h.

[0035] (5) Drying: pre-freezing at -20℃ for 8 h, freezing at -80℃ for 16 h, and vacuum drying for 12 h to obtain an extracellular matrix ECM sheet material sample.

[0036] (6) Powder grinding: a cutting machine is used to cut under the conditions of a rotation speed of 4000 rpm and a distance between the cutting knife head and the cutting mold edge of 30 μm-150 μm, and the fiber material is collected by sieving, with a length of 300-1000 μm, a width of 100-330 μm, and a length-width ratio of 3-8:1.

[0037] (7) Sterilization: the above ECM extracellular matrix fiber fragment material is selected, filled into a test tube, sealed and sterilized by irradiation, and then stored at room temperature.

[0038] As Figure 1The morphology of the extracellular matrix fiber material in Example 1 is shown, and it can be observed that the edges of the ECM powder particles exhibit a bristle fiber shape, and the surface is relatively flat; such a morphology can promote the proliferation of muscle fibers, new blood vessels and epithelial cells.

[0039] Examples 2-5 Examples 2-5 respectively provide an extracellular matrix powder material and a preparation method thereof.

[0040] The difference between the above example and Example 1 is specifically that the specific steps of decellularization and antigen removal are different, and are specifically as shown below.

[0041] In Example 2: under the condition of a solid-liquid mass-volume ratio of 1g:5ml, the sample is immersed in a 2.5wt% triton surfactant solution for 3 times, and each immersion treatment time is 3h; then the sample is immersed in a saline solution containing 0.05mol / L sodium chloride and having a pH of 11 for 3 times, and each immersion treatment time is 5h; then the sample is immersed in a 0.6wt% protease K solution at a temperature of 5℃ for 10h.

[0042] In Example 3: under the condition of a solid-liquid mass-volume ratio of 1g:5ml, the sample is immersed in a 2.5wt% sodium cholate surfactant solution for 3 times, and each immersion treatment time is 3h; then the sample is immersed in a saline solution containing 3mol / L sodium chloride and having a pH of 8 for 3 times, and each immersion treatment time is 5h; then the sample is immersed in a 0.6wt% trypsin solution at a temperature of 27℃ for 10h.

[0043] In Example 4: under the condition of a solid-liquid mass-volume ratio of 1g:5ml, the sample is immersed in a 1wt% sodium dodecyl sulfate surfactant solution for 3 times, and each immersion treatment time is 4h; then the sample is immersed in a saline solution containing 1mol / L sodium chloride and having a pH of 10 for 3 times, and each immersion treatment time is 5h; then the sample is immersed in a 0.2wt% trypsin solution at a temperature of 5℃ for 15h.

[0044] In Example 5: under the condition of a solid-liquid mass-volume ratio of 1g:5ml, the sample is immersed in a 5wt% sodium dodecyl sulfate surfactant solution for 3 times, and each immersion treatment time is 2h; then the sample is immersed in a saline solution containing 2mol / L sodium chloride and having a pH of 9 for 3 times, and each immersion treatment time is 5h; then the sample is immersed in a 0.8wt% trypsin solution at a temperature of 5℃ for 6h.

[0045] The other process parameters in the above examples are the same as those in Example 1.

[0046] Examples 6-9 Examples 6-9 respectively provide an extracellular matrix powder particle material and a preparation method thereof.

[0047] The difference between the above examples and Example 1 is that the composition of the organic solvent in the defatting is different, which is shown as follows.

[0048] In Example 6: the organic solvent in the defatting is composed of methanol and ethyl acetate with a volume ratio of 1:1.

[0049] In Example 7: the organic solvent in the defatting is composed of ethanol and chloroform with a volume ratio of 1:1.

[0050] In Example 8: the organic solvent in the defatting is composed of methanol and chloroform with a volume ratio of 1:5.

[0051] In Example 9: the organic solvent in the defatting is composed of methanol and chloroform with a volume ratio of 5:1.

[0052] The other process parameters in the above examples are the same as those in Example 1.

[0053] Comparative Examples Comparative Example 1 The present comparative example provides an extracellular matrix powder particle material and a preparation method thereof.

[0054] The preparation method of the extracellular matrix powder particle material in the present comparative example adopts the treatment mode of "defatting + virus inactivation + decellularization and antigen removal", which is shown as follows.

[0055] (1) Material taking: fresh biological tissue pig small intestinal submucosa is taken and repeatedly washed with water until no blood stains are left; then the fat and fascia are removed and cut into 20-50 cm strips.

[0056] (2) Defatting: the sample is immersed in an organic solvent (composed of methanol and chloroform with a volume ratio of 1:1) for 3 times of soaking treatment, and each soaking treatment time is 5h.

[0057] (3) Virus inactivation: the solid-liquid mass volume ratio of the sample and the disinfectant is 1g:5ml, and a mixed aqueous solution with a concentration of 1.5wt% peracetic acid and 8wt% ethanol is used as the disinfectant, and the sample is soaked for 120min.

[0058] (4) Decellularization and antigen removal: under the condition of solid-liquid mass volume ratio of 1 g:5 ml, the sample was immersed in a 2.5 wt% sodium dodecyl sulfate surfactant solution for 3 times, each time for 3 h; then the sample was immersed in a 1.5 mol / L sodium chloride solution with a pH of 9.5 for 3 times, each time for 5 h; then the sample was immersed in a 0.6 wt% trypsin solution at a temperature of 5°C for 10 h.

[0059] (5) Drying: pre-freezing at -20°C for 8 h, freezing at -80°C for 16 h, and vacuum drying for 12 h.

[0060] (6) Grinding: cutting at a rotation speed of 4000 rpm, with the distance between the cutting knife head and the cutting mold edge being 30-150 μm, and collecting the fiber material with a length of 300-1000 μm and a width of 100-330 μm, and a length-width ratio of 3-8:1.

[0061] (7) Sterilization: the ECM extracellular matrix fiber fragment material was selected, sealed in a test tube, and sterilized by irradiation, and then stored at room temperature.

[0062] Comparative Examples 2-5 Comparative Examples 2-5 each provide an extracellular matrix powder material and a preparation method thereof.

[0063] The differences between the above comparative examples and Example 1 are as follows.

[0064] In Comparative Example 2, the decellularization and antigen removal did not include a salt-alkali solution treatment, and the specific steps were as follows: under the condition of solid-liquid mass volume ratio of 1 g:5 ml, the sample was immersed in a 2.5 wt% sodium dodecyl sulfate surfactant solution for 3 times, each time for 3 h; then the sample was immersed in a 0.6 wt% trypsin solution at a temperature of 5°C for 10 h.

[0065] In Comparative Example 3, the decellularization and antigen removal included a salt-alkali solution treatment followed by a surfactant treatment, and the specific steps were as follows: under the condition of solid-liquid mass volume ratio of 1 g:5 ml, the sample was immersed in a 1.5 mol / L sodium chloride solution with a pH of 9.5 for 3 times, each time for 5 h; then the sample was immersed in a 2.5 wt% sodium dodecyl sulfate surfactant solution for 3 times, each time for 3 h; then the sample was immersed in a 0.6 wt% trypsin solution at a temperature of 5°C for 10 h.

[0066] In Comparative Example 4, the specific steps for decellularization and antigen removal were as follows: the sample was immersed in a surfactant solution containing 2.5 wt% sodium dodecyl sulfate and 50 wt% n-propanol at a solid-to-liquid mass-to-volume ratio of 1 g:5 ml for 3 times, each time for 3 h; then the sample was immersed in a salt solution containing 0.01 mol / L sodium chloride and having a pH of 11.5 for 3 times, each time for 5 h; and then the sample was immersed in a 0.05 wt% trypsin solution at a temperature of 5°C for 10 h.

[0067] In Comparative Example 5, the specific steps for decellularization and antigen removal were as follows: the sample was immersed in a surfactant solution containing 2.5 wt% sodium dodecyl sulfate and 40 wt% chloroform at a solid-to-liquid mass-to-volume ratio of 1 g:5 ml for 3 times, each time for 3 h; then the sample was immersed in a salt solution containing 4 mol / L sodium chloride and having a pH of 7.5 for 3 times, each time for 5 h; and then the sample was immersed in a 2 wt% trypsin solution at a temperature of 5°C for 10 h.

[0068] In the above comparative examples, the other process parameters were the same as in Example 1.

[0069] Performance test (1) Tensile mechanical test of extracellular matrix sheet material before grinding The dried extracellular matrix ECM sheet material sample (before grinding) was prepared into a 1 cm x 2.5 cm rectangular test sample, the clamp interval was 3 cm, the tensile strain rate was 5 mm / min at room temperature and normal pressure, and the maximum tensile strength was measured on a mechanical tester until fracture, and the axial tensile mechanical value was recorded, and the test was repeated 5 times.

[0070] (2) Growth factor content The extracellular matrix fiber materials prepared in the examples and comparative examples were used as the detection object, and the contents of VEGF, bFGF, TGF-β, and TNF-α were detected using an ELISA kit.

[0071] (3) Degradation time The extracellular matrix fiber materials prepared in the examples and comparative examples were used as the detection object, and tests were performed in vitro (PBS buffer, pH=10) and in vivo (0.2 ml was injected into the vagina of a rat), and the complete degradation time was detected. The test results are shown in Table 1.

[0072] In vitro test: prepare 0.01M PBS buffer (pH = 10), sterilize and then sub-pack into centrifuge tubes (10mL per tube); completely immerse 0.5g sample in the PBS solution, incubate in a 37°C water bath shaker (100rpm to simulate the dynamic environment of the body fluid), filter the liquid every 5 days, soak in clean water for 10 minutes, filter and repeat three times, freeze-dry the remaining solid, weigh, and record the complete degradation time.

[0073] In vivo test: select female SD rats (body weight 200±20g), n = 6 in each group, divided into example group, comparative example group, blank control group. After anesthesia (2% sodium pentobarbital, intraperitoneal injection), intravaginal injection of 0.2mL extracellular matrix fiber material suspension (resuspended in sterile saline, concentration 0.5g / L), the control group was injected with the same amount of saline.

[0074] After surgery, single-cage feeding. After 10 days, every 5 days, take the vagina and surrounding tissue, fix with 4% paraformaldehyde, paraffin-embedded section, and observe the retention of the implanted sample.

[0075] (4) Collagen melting point detection Take the ECM sample before grinding, and use the DSC method to detect the melting point of the extracellular matrix fiber material. Solid sample: accurately weigh 3mg, keep hydrated. Dissolve collagen in weak acid (0.1M acetic acid) at a concentration of 1mg / mL. The instrument temperature detection range is 10-100℃, and the temperature rising rate is 5℃ / min. Detect the denaturation process of the starting temperature, peak temperature and enthalpy change of the endothermic peak. According to the curve, the melting point of the extracellular matrix fiber material is determined.

[0076] Table 1 Performance test results of extracellular matrix fiber materials in examples and comparative examples According to the test results in the above table, in the preparation method of the extracellular matrix powder material in Comparative Example 1, the treatment method of "degreasing + virus inactivation + decellularization and antigen removal" is adopted, in Comparative Example 2, the salt alkali solution treatment is not carried out in the decellularization and antigen removal, in Comparative Example 3, the salt alkali solution treatment is carried out first and then the surfactant treatment is carried out in the decellularization and antigen removal, in Comparative Examples 4-5, the process parameters are not matched in the decellularization and antigen removal, the mechanical strength of the decellularized matrix material prepared before drying is poor, which is not conducive to the subsequent grinding operation step, and the performance of the ECM fiber powder material prepared is poor.

[0077] In contrast, by using the preparation process provided in the application, the processes of virus inactivation, decellularization and antigen removal, defatting, drying, grinding, sterilization are used in sequence, and the parameters of each process are optimized by screening, so that the mechanical strength of the decellularized matrix material prepared before drying is excellent, facilitating the subsequent grinding operation step, and the large-scale standardized production can be achieved while the powder and fiber repair effect can be achieved. At the same time, the ECM fiber powder and particle material with high growth factor content and slow degradation time is prepared.

[0078] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of protection required by the present application.

Claims

1. A method for the production of an extracellular matrix fibrous material, characterized in that, Specifically comprising the following steps in turn: taking animal tissue through virus inactivation, decellularization, defatting, drying, grinding, sterilization; The specific steps of the decellularization are: under the condition of solid-liquid mass volume ratio of 1 g: 1-10 ml, the sample is sequentially immersed and treated with 0.5-15wt% surfactant solution for 1-5 times, and each immersion treatment time is 0.5-6h; then immersed and treated with a salt alkaline solution containing 0.05-3mol / L salt and having a pH of 8-11 for 1-5 times, and each immersion treatment time is 1-10h; and then immersed and treated with 0.1-1wt% enzyme solution for 3-20h; The salt is selected from one or more of sodium chloride, sodium phosphate, and potassium chloride; The surfactant is selected from one or more of Triton X114, sodium dodecyl sulfonate, sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, 3-[3-(cholamide propyl) dimethylamino] propanesulfonic acid inner salt CHAPS, and sodium cholate; The enzyme is selected from one or more of trypsin, pepsin, and proteinase K; The defatting is performed by immersing the sample in one or more of n-hexane, trichloromethane, dichloromethane, methanol, ethanol, acetone, diethyl ether, and ethyl acetate for 1-5 times, and each immersion treatment time is 1-8h.

2. The method for preparing the extracellular matrix fiber material according to claim 1, characterized in that, The animal source is selected from one or more of cattle, pigs, and sheep; and the tissue is selected from one or more of dermis, muscle, tendon, heart, fat, small intestine, nerve, bone, and cartilage.

3. The method for preparing the extracellular matrix fiber material according to claim 1, characterized in that, The virus inactivation is performed by using a disinfectant solution with a solid-liquid weight ratio of 1g:1-10ml; and the disinfectant solution is selected from one or more of hydrogen peroxide, peracetic acid solution, or ethanol.

4. The method of claim 3, wherein the extracellular matrix fiber material is prepared by, The specific method of the virus inactivation is: using a mixed aqueous solution of peracetic acid with a concentration of 0.01-3wt% and ethanol with a concentration of 1-20wt% as the disinfectant solution, immersing the sample for 10-180min, or using a hydrogen peroxide solution with a concentration of 0.01-10wt% to immerse the sample for 1-12h.

5. The method for preparing the extracellular matrix fiber material according to claim 1, characterized in that, The specific steps of the decellularization are: sequentially immersing and treating the sample with 1-5wt% sodium dodecyl sulfate solution for 2-4 times, and each immersion treatment time is 2-4h; then immersing and treating with a salt alkaline solution containing 1-2mol / L sodium chloride and having a pH of 9-10 for 1-4 times, and each immersion treatment time is 1-7h; and then immersing and treating the sample with a trypsin solution with a concentration of 0.2-0.8wt% at a temperature of 2-8℃ for 6-15h.

6. The preparation method of the extracellular matrix fiber material according to claim 1, wherein the drying method is selected from any one of suction drying and freeze drying; The process parameters of the suction drying are: temperature 20-60℃, wind box pressure 1-20mbar, and time 2-24h; The process parameters of the freeze drying are: freeze drying temperature -20℃~-80℃, vacuum degree 0.1-100mbar, and time 2-24h.

7. The method of claim 1, wherein the extracellular matrix fiber material is prepared by a process comprising: The specific step of the grinding is: using a cutting machine, cutting at a rotating speed of 2000-6000 rpm, a distance of 30-150 μm between the cutting tool head and the edge of the cutting mold, and collecting by sieving.

8. An extracellular matrix fibrous material, characterized in that, The extracellular matrix fiber material is prepared by the preparation method of any one of claims 1-7, has a length of 100-2000 μm, a width of 50-1000 μm, a length-width ratio of 2-10:1, and a melting point of 73-80 ℃.

9. The extracellular matrix fibrous material according to claim 8, characterized in that, The extracellular matrix fiber material has a length of 300-1000 μm, a width of 100-330 μm, and a length-width ratio of 3-8:1, and contains growth factors in the following amounts: VEGF ≥ 70000 ng / g, bFGF ≥ 14 ng / g, TGF-β ≥ 5 ng / g, and TNF-α ≥ 11 ng / g.

10. Use of the extracellular matrix fiber material according to any one of claims 8-9 as a medical repair material.

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