Soft tissue repair patch and preparation method thereof

Abstract

The invention belongs to the technical field of medical materials, and discloses a soft tissue repair patch and a preparation method thereof.The preparation method comprises the steps that S1, animal soft tissue is treated and cleaned, and a first intermediate product is obtained; s2, adding a reagent into the first intermediate product, and performing freeze-thaw cycle to obtain a second intermediate product; s3, soaking the second intermediate product with an organic solvent, and cleaning to obtain a third intermediate product; s4, the third intermediate product is soaked in an alkali solution and cleaned, and a fourth intermediate product is obtained; and S5, soaking the fourth intermediate product in an acid solution, cleaning, drying, and sterilizing to obtain the soft tissue repair patch. The invention provides a milder and friendly decellularization scheme, and the decellularization matrix prepared by using the scheme is more complete in decellularization and free of chemical residues, and maintains a natural scaffold structure of collagen.

Description

Technical Field

[0001] The invention belongs to the technical field of medical materials, and in particular relates to a soft tissue repair patch and a preparation method thereof. Background Art

[0002] Tissue decellularization is a promising method for preparing biological scaffolds for regenerative medicine. Removing cellular components from tissues or organs will produce a scaffold material composed of active structural proteins, which can be used for a variety of soft tissue repairs. The source of allogeneic tissue is very limited, and xenogeneic decellularized matrix materials have become a key research direction in soft tissue repair technology. The extracellular matrix (ECM) mainly includes collagen, elastin, proteoglycans, aminoglycans, non-collagenous proteins, cytokines, etc. In mammals, the main component of ECM is collagen.

[0003] The sources of raw materials used to prepare heterologous decellularized tissues are very wide, such as animal skin, peritoneal membrane, tendon, small intestinal mucosa, pericardium, blood vessels, etc. The decellularized tissue preparation process mainly removes cell components from fresh tissues through physical, chemical, and biological methods to retain the extracellular matrix of the tissue. Physical methods mainly use physical means to destroy the cell membrane and release the cell contents, including ultrasound, pressure, freeze-thaw, mechanical stirring, etc.; chemical methods mainly include acids, alkalis, surfactants, etc., which change the permeability of the cell membrane through chemical reagents, and finally make the cells swell and rupture to achieve the purpose of decellularization; biological methods are mainly various enzymes, such as trypsin, nuclease, DNA enzyme, etc.

[0004] In the decellularization process, residual chemical reagents can cause inflammatory reactions, so after decellularization, it is necessary to undergo long-term repeated washing to remove residual reagents in the tissue. However, long-term washing will cause changes in the structure of the tissue matrix and affect the product performance of the extracellular matrix. Reasonable immunogenicity removal technology is the core of the decellularization process and is also the key and difficulty of the decellularized matrix material preparation process. Therefore, it is crucial to find a process that can completely remove cells and ensure that the tissue is not damaged and has good degradation performance. Summary of the invention

[0005] To solve the above problems, the present invention provides a soft tissue repair patch and a preparation method thereof. By combining physical and chemical decellularization methods, a preparation process is provided that is more completely decellularized, free of chemical residues, and maintains the natural scaffold structure of collagen.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing a soft tissue repair patch comprises: S1, treating and cleaning animal soft tissue to obtain a first intermediate product; S2, adding a reagent to the first intermediate product for freeze-thaw cycle to obtain a second intermediate product; S3, soaking the second intermediate product in an organic solvent and cleaning it to obtain a third intermediate product; S4, soaking the third intermediate product in an alkaline solution and cleaning it to obtain a fourth intermediate product; S5, soaking the fourth intermediate product in an acid solution, cleaning, drying and sterilizing it to obtain the soft tissue repair patch; the components of the reagent include: a mass percentage of phenylmethylsulfonyl fluoride of 0.2-0.8 mmol / L, a content of a chelating agent of 12-18 mmol / L, a content of Tris of 22-25 mmol / L, and a content of a soluble sodium salt or potassium salt of 0.1-0.5 mmol / L.

[0008] Furthermore, the animal soft tissue refers to one or more of animal dermis, animal pericardium, animal intestinal mucosa, and animal bladder tissue.

[0009] Furthermore, in step S1, the animal soft tissue is cut into a certain size, and the size in at least one direction in each direction does not exceed 5 mm, and fat and muscle tissue are removed, and blood and dirt are cleaned.

[0010] Furthermore, in step S2, 2 to 4 freeze-thaw cycles are performed, the freezing temperature of the freeze-thaw cycle is -60 to -80°C, the freezing time is 3 to 4 hours, the thawing temperature is 30 to 40°C, and the thawing time is 30 to 60 minutes.

[0011] Furthermore, in step S2, 2-4 freeze-thaw cycles are performed, the freezing temperature of the freeze-thaw cycle is -60 to -80°C, the freezing time is 3 to 4 hours, and the bubble generation temperature is also included, the bubble generation temperature is 2 to 5°C, and is maintained at the bubble generation temperature for 2 to 3 hours, the thawing temperature is 15 to 25°C, and the thawing time is 10 to 20 minutes.

[0012] Furthermore, the chelating agent is EDTA or EGTA.

[0013] Furthermore, in step S3, firstly, 50-70% ethanol is used for soaking for 0.5-1.0 h, then 99-100% ethanol is used for soaking for 0.5-1 h, and isopropanol is ultrasonically shaken for 1-4 h.

[0014] Furthermore, in step S4, the concentration of the alkaline solution is 0.8-1.5 mol / L, the alkaline solution also includes 50-150 mmol / L EDTA, and the immersion time in the alkaline solution is 1-4 hours.

[0015] Furthermore, the acid solution in step S5 is one of hydrochloric acid, sulfuric acid, and acetic acid; the concentration of the acid solution is 1.2 to 2.0 mol / L, and the acid solution also contains sodium chloride, calcium chloride or potassium chloride, the concentration of the sodium chloride, calcium chloride or potassium chloride is 0.5 to 1.5 mol / L, and the acid solution immersion time is 1 to 4 hours.

[0016] A soft tissue repair patch is prepared by the above-mentioned preparation method.

[0017] Beneficial effects of the present invention: The technical solution proposed by the present invention avoids the damage of the ECM structure caused by the dendrites generated during the freezing process by specifically selecting the reagent components in the freeze-thaw process and controlling the content of each component. Although there are a variety of decellularization technologies in the prior art, the degree of decellularization is relative, that is, the cell material cannot be completely removed. A high removal rate of cell components can be achieved by combining a variety of technologies, but it is easy to cause the integrity and activity of the patch structure to decrease, and the prepared patch is easy to be degraded. For the freeze-thaw technology, during the freeze-thaw process, the liquid solidifies to produce dendrites, such as water generates a snowflake-like structure during the solidification process, and the sharp dendrites pierce the cells, and the cell material is tightly embedded in the ECM matrix, making it difficult to remove the cell components in subsequent treatment. Even if a high-intensity elution process is used, it is difficult to fundamentally reduce the content of cell tissue. The higher the content of cell tissue, the stronger the rejection or inflammatory reaction after the subsequent patch implantation; secondly, if a high-intensity elution process is used in the subsequent process, the integrity of the patch will change, the activity will decrease, and the degradation rate will be high. The present invention adds phenylmethylsulfonyl fluoride to the buffer to prevent the protease released when the cell ruptures from damaging the protein structure. At the same time, a chelating agent is added. The chelating agent can enter between the cell and the ECM to improve the subsequent removal efficiency of the cell material. At the same time, the phenylmethylsulfonyl fluoride has a half-life. During the freeze-thaw process, the chelating agent inhibits the protease on the one hand, and on the other hand, the chelating agent prevents the cell material from being too strongly bonded to the ECM, resulting in the subsequent inability to effectively remove the cell material. However, the phenylmethylsulfonyl fluoride and the chelating agent will enhance the growth tendency of dendrites. In order to solve the above problems, the present invention adds a certain amount of soluble sodium salt or potassium salt to reduce the growth tendency of dendrites, but the sodium salt or potassium salt will affect the activity of the chelating agent. In order to solve the above problems, the present application limits the content of various components to solve the above technical problems, thereby preventing the low activity of phenylmethylsulfonyl fluoride in the later stage and the excessively high bonding strength between the cell material and the ECM, and reducing the tendency of generating dendrites. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 HE staining images of the slices of the patches prepared in the examples and comparative examples of the present invention, a: Example 1, b: Example 4, c: Comparative Example 1, d: Comparative Example 5, e: Comparative Example 7;

[0020] Figure 2 Transmission electron microscopy images of the patches prepared in the examples and comparative examples of the present invention, a: Example 1, b: Example 4, c: Comparative Example 1, d: Comparative Example 5, e: Comparative Example 7;

[0021] Figure 3 Initial diagrams of the patches prepared in the examples and comparative examples of the present invention immersed in degradation liquid, a0: Example 1, b0: Example 4, c0: Comparative Example 1, d0: Comparative Example 5, e0: Comparative Example 7;

[0022] Figure 4 These are the pictures of the patches prepared according to the examples and comparative examples of the present invention after being immersed in degradation liquid for 64 hours, a64: Example 1, b64: Example 4, c64: Comparative Example 1, d64: Comparative Example 5, e64: Comparative Example 7. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] In order to avoid errors caused by a single test, the quantitative test in the embodiment of the present invention is set up with three repeated experiments, and the results are averaged.

[0025] The embodiment of the present invention provides a soft tissue repair patch and a preparation method, comprising: S1, processing and cleaning animal soft tissue to obtain a first intermediate product; S2, adding a reagent to the first intermediate product for freeze-thaw cycle to obtain a second intermediate product; S3, soaking the second intermediate product in an organic solvent and cleaning it to obtain a third intermediate product; S4, soaking the third intermediate product in an alkaline solution and cleaning it to obtain a fourth intermediate product; S5, soaking the fourth intermediate product in an acid solution, cleaning, drying, and sterilizing to obtain the soft tissue repair patch; the components of the reagent include: a mass percentage of phenylmethylsulfonyl fluoride of 0.2-0.8 mmol / L, a chelating agent content of 12-18 mmol / L, a Tris content of 22-25 mmol / L, and a soluble sodium salt or potassium salt content of 0.1-0.5 mmol / L.

[0026] The technical solution proposed by the present invention avoids the damage of the ECM structure caused by the dendrites generated during the freezing process by specifically selecting the reagent components in the freeze-thaw process and controlling the content of each component. Although there are a variety of decellularization technologies in the prior art, the degree of decellularization is relative, that is, the cell material cannot be completely removed. A high removal rate of cell components can be achieved by combining a variety of technologies, but it is easy to cause the integrity and activity of the patch structure to decrease, and the prepared patch is easy to be degraded. For the freeze-thaw technology, during the freeze-thaw process, the liquid solidifies to produce dendrites, such as water generates a snowflake-like structure during the solidification process, and after the sharp dendrites pierce the cells, the cell material is tightly embedded in the ECM matrix, making it difficult to remove the cell components in subsequent treatments. Even if a high-intensity elution process is used, it is difficult to fundamentally reduce the content of cell tissue. The higher the content of cell tissue, the stronger the rejection or inflammatory reaction after the subsequent patch implantation; secondly, if a high-intensity elution process is used in the subsequent process, the integrity of the patch will change, the activity will decrease, and the degradation rate will be high. The present invention adds phenylmethylsulfonyl fluoride to the buffer to prevent the protease released when the cell ruptures from damaging the protein structure. At the same time, a chelating agent is added. The chelating agent can enter between the cell and the ECM to improve the subsequent removal efficiency of the cell material. At the same time, the phenylmethylsulfonyl fluoride has a half-life. During the freeze-thaw process, the chelating agent inhibits the protease on the one hand, and on the other hand, the chelating agent prevents the cell material from being too strongly bonded to the ECM, resulting in the subsequent inability to effectively remove the cell material. However, the phenylmethylsulfonyl fluoride and the chelating agent will enhance the growth tendency of dendrites. In order to solve the above problems, the present invention adds a certain amount of soluble sodium salt or potassium salt to reduce the growth tendency of dendrites, but the sodium salt or potassium salt will affect the activity of the chelating agent. In order to solve the above problems, the present application limits the content of various components to solve the above technical problems, thereby preventing the low activity of phenylmethylsulfonyl fluoride in the later stage and the excessively high bonding strength between the cell material and the ECM, and reducing the tendency of generating dendrites.

[0027] It should be noted that the soluble sodium salt or potassium salt may be NaCl, KCl, NaNO3 or KNO3.

[0028] Among the components of the reagent, the mass percentage of phenylmethylsulfonyl fluoride is 0.2-0.8mmol / L, which can be 0.2mmol / L, 0.3mmol / L, 0.4mmol / L, 0.5mmol / L, 0.6mmol / L, 0.7mmol / L, and 0.8mmol / L; the content of the chelating agent is 12-18mmol / L, which can be 12mmol / L, 13mmol / L, 14mmol / L, 15mmol / L, 16mmol / L, 17mmol / L, and 18mmol / L; the content of Tris is 22-25mmol / L, which can be 22mmol / L, 23mmol / L, 24mmol / L, and 25mmol / L; the content of soluble sodium salt or potassium salt is 0.1-0.5mmol / L, which can be 0.1mmol / L, 0.2mmol / L, 0.3mmol / L, 0.4mmol / L, and 0.5mmol / L.

[0029] In step S1, the animal soft tissue refers to one or more of the animal's dermis, animal pericardium, animal intestinal mucosa, and animal bladder tissue, and the animal is a mammal such as a cow, horse, or pig. The animal soft tissue is cut into a certain size, and the size of at least one direction in each direction does not exceed 5 mm, and fat and muscle tissue are removed, and blood and dirt are cleaned. The size after cutting is limited so that the inside and outside of the tissue can be cooled or heated synchronously as much as possible, reducing structural damage to the soft tissue and increasing the degradation resistance of the prepared product. If the size is too large, the external tissue freezes first, and the internal tissue freezes later. The volume of the internal tissue increases during the freezing process, and stress is applied to the external tissue to cause tissue damage.

[0030] In step S2, freeze-thaw cycles are performed for 2 to 4 times, the freezing temperature of the freeze-thaw cycles is -60 to -80°C, such as -60°C, -65°C, -70°C, -75°C, -80°C, the freezing time is 3 to 4h, such as 3h, 3.5h or 4h, the thawing temperature is 30 to 40°C, such as 30°C, 35°C or 40°C, and the thawing time is 30 to 60min, such as 30min, 40min, 50min, 60min.

[0031] In a preferred embodiment, in step S2, freeze-thaw cycles are performed for 2-4 times, the freezing temperature of the freeze-thaw cycle is -60 to -80°C, such as -60°C, -65°C, -70°C, -75°C, and -80°C, the freezing time is 3 to 4h, such as 3h, 3.5h, and 4h, and also includes a bubble generation temperature, the bubble generation temperature is 2 to 5°C, such as 2°C, 3°C, 4°C or 5°C, and the bubble generation temperature is maintained for 2 to 3h, such as 2h, 2.5h, and 3h, the thawing temperature is 15 to 25°C, such as 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, and 25°C, and the thawing time is 10 to 20min, such as 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min, and 20min. Under this process, bubbles can be generated in the tissue, which further separate the cellular material from the ECM, thereby improving the separation effect of the cellular material and the ECM. In order to achieve a higher bubble generation efficiency and bubble generation effect, the present application limits the bubble generation temperature and time. If the bubbles are generated densely, the separation effect is good, but if the density is too high, the bubbles will fuse, resulting in a poor separation effect.

[0032] The chelating agent is EDTA or EGTA, wherein EDTA is ethylenediaminetetraacetic acid, and EGTA is ethylene glycol bis-α-aminoethyl ether tetraacetic acid.

[0033] In step S3, firstly, 50-70% ethanol is used for soaking for 0.5-1.0h, which can be 50%, 55%, 60%, 65%, 70% ethanol, and the soaking time can be 0.5h, 0.6h, 0.7h, 0.8h, 0.9h or 1.0h, and then 99-100% ethanol is used for soaking for 0.5-1h, such as 0.5h, 0.6h, 0.7h, 0.8h, 0.9h or 1h, and isopropanol ultrasonic oscillation is used for 1-4h, such as 1h, 2h, 3h or 4h. Soaking with low concentration ethanol first can avoid the direct use of high concentration ethanol to make the cells lose water rapidly, so that effective dehydration effect cannot be achieved; isopropanol has a good degreasing effect. On the other hand, isopropanol is combined with freeze-thaw process to facilitate the removal of subsequent cell substances. Isopropanol has high permeability, which reduces the binding strength of cell fragments and ECM after freeze-thaw, and facilitates subsequent removal.

[0034] In step S4, the concentration of the alkaline solution is 0.8-1.5 mol / L, such as 0.8 mol / L, 0.9 mol / L, 1.0 mol / L, 1.1 mol / L, 1.2 mol / L, 1.3 mol / L, 1.4 mol / L, 1.5 mol / L, and the alkaline solution also includes 50-150 mmol / EDTA, such as 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L, 110 mmol / L, 120 mmol / L, 130 mmol / L, 140 mmol / L, 150 mmol / L; the immersion time of the alkaline solution is 1-4 h, such as 1 h, 2 h, 3 h, 4 h. The alkaline solution can cause the expansion and loosening of collagen fibers and saponify lipids, and the EDTA in the alkaline solution can effectively reduce the concentration of divalent cations and improve the removal of cells and debris from the extracellular matrix. However, the treatment concentration, time and number of times of the alkaline solution should not be too long, otherwise it will easily lead to the destruction of the collagen scaffold structure and significantly reduce the mechanical strength of the final material. The present application reduces the intensity of the alkaline solution treatment through an improved freeze-thaw process, that is, reduces the concentration of the alkaline solution and the treatment time, thereby maintaining the integrity of the collagen scaffold structure. The alkaline solution is one or more of sodium hydroxide, potassium hydroxide, sodium carbonate or sodium bicarbonate.

[0035] The acid solution in step S5 is one of hydrochloric acid, sulfuric acid and acetic acid; the concentration of the acid solution is 1.2-2.0 mol / L, such as 1.2 mol / L, 1.3 mol / L, 1.4 mol / L, 1.5 mol / L, 1.6 mol / L, 1.7 mol / L, 1.8 mol / L, 1.9 mol / L and 2.0 mol / L; the acid solution further contains sodium chloride, calcium chloride or potassium chloride, and the concentration of the sodium chloride, calcium chloride or potassium chloride is 0.5-1.5 mol / L, such as 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1.0 mol / L, 1.1 mol / L, 1.2 mol / L, 1.3 mol / L, 1.4 mol / L and 1.5 mol / L, and the soaking time of the acid solution is 1-4 h, such as 1 h, 2 h, 3 h and 4 h. Acid solution can cause the collagen fibers to swell and loosen, and can remove glycoproteins and glycosaminoglycans, non-collagenous proteins and nucleic acid substances. Adding salt solution to the acid solution can control the swelling of the collagen tissue matrix during acid treatment and remove glycoproteins and proteoglycans from the collagen matrix.

[0036] Unless otherwise specified, the preparation methods proposed in the present invention are all carried out at room temperature, i.e., 20-25°C.

[0037] The cleaning, drying and sterilization in step S5 are all existing technologies and are not limited here.

[0038] The present application also protects a soft tissue repair patch, which is prepared by the above-mentioned preparation method.

[0039] The soft tissue repair patch provided in the present application can be used in oral soft tissue repair, dura mater repair, burn wound repair, and breast reconstruction.

[0040] In order to characterize the performance of the soft tissue repair patches prepared in the embodiments and comparative examples of the present invention, the following test items were used:

[0041] (1) Decellularization detection:

[0042] A longitudinal sample was taken from the middle position of the sample of each embodiment and comparative example, embedded, sliced ​​(thickness of about 5 microns), dried, HE-stained, sealed, and observed under a microscope.

[0043] DNA content: Detected in accordance with YY / T0606.25-2014 "Determination of DNA Residue in Animal-derived Biological Materials: Fluorescence Staining Method".

[0044] (2) Transmission electron microscopy analysis:

[0045] A longitudinal sample was taken from the middle position of the sample of each embodiment and comparative example, embedded, sliced ​​(thickness about 70 nm), dried, and observed under a transmission electron microscope.

[0046] (3) Product in vitro degradation test: The samples of each embodiment were uniformly processed into a size of 2cm*3cm, accurately weighed, and the initial weight was recorded, recorded as m0. According to the extraction ratio of 0.1g:15ml, the degradation solution (physiological saline solution containing 0.2mg / ml type I collagenase) was added, and the treated samples of each embodiment were placed in a constant temperature incubator at 37°C. After 64 hours, they were taken out and freeze-dried. The freeze-dried samples were accurately weighed and the weight was recorded, recorded as m. The degradation rate of each example sample after 64 hours was calculated. The degradation morphological changes of the samples from 0 to 64 hours were observed.

[0047] The degradation rate calculation formula is:

[0048]

[0049] In order to better illustrate the embodiments of the present invention, the present invention is further described in detail below through specific examples.

[0050] Example 1

[0051] The embodiment of the present invention provides a soft tissue repair patch and a preparation method thereof, comprising:

[0052] S1. Process and clean the animal soft tissue to obtain a first intermediate product. Remove subcutaneous fat, muscle and hair from fresh animal skin with a thickness not exceeding 5 mm, and clean it with water to obtain a first intermediate product.

[0053] S2, adding the first intermediate product to a reagent for freeze-thaw cycle to obtain a second intermediate product. The first intermediate product is added to a reagent for freeze-thaw cycle, wherein the reagent is: the mass percentage of phenylmethylsulfonyl fluoride is 0.2mmol / L, the content of the chelating agent is 12mmol / L, the content of Tris is 22mmol / L, and the content of NaCl is 0.1mmol / L. Freeze and thaw for 2 cycles, the freezing temperature of the freeze-thaw cycle is -60°C, the freezing time is 3h, the thawing temperature is 30°C, the thawing time is 30min, and the chelating agent is EDTA.

[0054] S3, soaking the second intermediate product in an organic solvent and washing it to obtain a third intermediate product.

[0055] First, the second intermediate product is soaked in 50% ethanol for 0.5 h, then soaked in 99-100% ethanol for 0.5 h, and ultrasonically shaken in isopropanol for 1 h. After the soaking, it is cleaned with purified water to obtain a third intermediate product.

[0056] S4, soaking the third intermediate product in an alkaline solution and washing it to obtain a fourth intermediate product. The concentration of the alkaline solution is 0.8 mol / L, the alkaline solution also includes 50 mmol / L EDTA, the soaking time is 1 hour, and the product is rinsed with purified water.

[0057] S5. The fourth intermediate product is treated by soaking in an acid solution, and then cleaned, dried and sterilized to obtain the soft tissue repair patch.

[0058] The concentration of the acid solution is 1.2 mol / L, the concentration of NaCl is 0.5 mol / L, and the acid solution is immersed for 1 hour. The tissue is rinsed with PBS, freeze-dried, and sterilized to obtain an acellular dermal matrix.

[0059] Example 2

[0060] The embodiment of the present invention provides a soft tissue repair patch and a preparation method thereof, comprising:

[0061] S1. Process and clean the animal soft tissue to obtain a first intermediate product. Remove subcutaneous fat, muscle and hair from fresh animal skin with a thickness not exceeding 5 mm, and clean it with water to obtain a first intermediate product.

[0062] S2, adding the first intermediate product to a reagent for freeze-thaw cycle to obtain a second intermediate product. The first intermediate product is added to a reagent for freeze-thaw cycle, wherein the reagent is: the mass percentage of phenylmethylsulfonyl fluoride is 0.4mmol / L, the content of the chelating agent is 15mmol / L, the content of Tris is 23mmol / L, and the content of NaCl is 0.3mmol / L. Freeze and thaw for 3 cycles, the freezing temperature of the freeze-thaw cycle is -70°C, the freezing time is 3h, the thawing temperature is 35°C, the thawing time is 50min, and the chelating agent is EGTA.

[0063] S3, soaking the second intermediate product in an organic solvent and washing it to obtain a third intermediate product.

[0064] First, the second intermediate product was soaked in 60% ethanol for 0.8 h, then soaked in 99-100% ethanol for 0.8 h, and ultrasonically vibrated in isopropanol for 3 h. After the soaking, it was cleaned with purified water to obtain a third intermediate product.

[0065] S4, soaking the third intermediate product in an alkaline solution and washing it to obtain a fourth intermediate product. The concentration of the alkaline solution is 1.0 mol / L, and the alkaline solution also includes 100 mmol / L EGTA. The soaking time of the alkaline solution is 3 hours, and then the product is rinsed with purified water.

[0066] S5. The fourth intermediate product is treated by soaking in an acid solution, and then cleaned, dried and sterilized to obtain the soft tissue repair patch.

[0067] The concentration of the acid solution is 1.6 mol / L, the concentration of NaCl is 1.0 mol / L, and the acid solution immersion time is 3 hours. The tissue is rinsed with PBS, freeze-dried, and sterilized to obtain an acellular dermal matrix.

[0068] Example 3

[0069] The embodiment of the present invention provides a soft tissue repair patch and a preparation method thereof, comprising:

[0070] S1. Process and clean the animal soft tissue to obtain a first intermediate product. Remove subcutaneous fat, muscle and hair from fresh animal skin with a thickness not exceeding 5 mm, and clean it with water to obtain a first intermediate product.

[0071] S2, adding the first intermediate product to a reagent for freeze-thaw cycle to obtain a second intermediate product. The first intermediate product is added to a reagent for freeze-thaw cycle, wherein the reagent is: the mass percentage of phenylmethylsulfonyl fluoride is 0.8mmol / L, the content of the chelating agent is 18mmol / L, the content of Tris is 25mmol / L, and the content of NaCl is 0.5mmol / L. Freeze and thaw for 3 cycles, the freezing temperature of the freeze-thaw cycle is -80°C, the freezing time is 4h, the thawing temperature is 40°C, the thawing time is 60min, and the chelating agent is EDTA.

[0072] S3, soaking the second intermediate product in an organic solvent and washing it to obtain a third intermediate product.

[0073] First, the second intermediate product was soaked in 70% ethanol for 1.0 h, then soaked in 99-100% ethanol for 1 h, and ultrasonically shaken in isopropanol for 4 h. After the soaking, it was cleaned with purified water to obtain a third intermediate product.

[0074] S4, soaking the third intermediate product in an alkaline solution and washing it to obtain a fourth intermediate product. The concentration of the alkaline solution is 1.5 mol / L, the alkaline solution also includes 100 mmol / L EDTA, the soaking time is 4 hours, and the product is rinsed with purified water.

[0075] S5. The fourth intermediate product is treated by soaking in an acid solution, and then cleaned, dried and sterilized to obtain the soft tissue repair patch.

[0076] The concentration of the acid solution is 2.0 mol / L, the concentration of NaCl is 1.5 mol / L, and the acid solution immersion time is 4 hours. The tissue is rinsed with PBS, freeze-dried, and sterilized to obtain the acellular dermal matrix.

[0077] Example 4

[0078] Different from Example 1, in step S2 of this embodiment, two freeze-thaw cycles are performed, the freezing temperature of the freeze-thaw cycle is -60°C, the freezing time is 3 hours, the bubble generation temperature is 2°C, and it is maintained at the bubble generation temperature for 2 hours, the thawing temperature is 15°C, the thawing time is 10 minutes, and the chelating agent is EDTA.

[0079] Example 5

[0080] Different from Example 1, in step S2 of this embodiment, two freeze-thaw cycles are performed, the freezing temperature of the freeze-thaw cycle is -70°C, the freezing time is 3.5 hours, the bubble generation temperature is 4°C, and it is maintained at the bubble generation temperature for 2 hours, the thawing temperature is 20°C, the thawing time is 15 minutes, and the chelating agent is EDTA.

[0081] Example 6

[0082] Different from Example 1, in step S2 of this embodiment, two freeze-thaw cycles are performed, the freezing temperature of the freeze-thaw cycle is -80°C, the freezing time is 4 hours, the bubble generation temperature is 5°C, and it is maintained at the bubble generation temperature for 3 hours, the thawing temperature is 25°C, the thawing time is 20 minutes, and the chelating agent is EDTA.

[0083] Comparative Example 1

[0084] Different from Example 1, in step S2 of this comparative example, no NaCl is added to the reagent.

[0085] Comparative Example 2

[0086] Different from Example 1, in step S2 of this comparative example, the reagents are: the mass percentage content of phenylmethylsulfonyl fluoride is 0.1 mmol / L, the content of chelating agent is 10 mmol / L, the content of Tris is 20 mmol / L, and the content of NaCl is 0.1 mmol / L.

[0087] Comparative Example 3

[0088] Different from Example 1, in step S2 of this comparative example, the reagents are: the mass percentage content of phenylmethylsulfonyl fluoride is 0.9 mmol / L, the content of the chelating agent is 19 mmol / L, the content of Tris is 26 mmol / L, and the content of NaCl is 0.1 mmol / L.

[0089] Comparative Example 4

[0090] Different from Example 1, in step S2 of this comparative example, the reagents are: the mass percentage content of phenylmethylsulfonyl fluoride is 0.2 mmol / L, the content of Tris is 22 mmol / L, and the content of NaCl is 0.1 mmol / L.

[0091] Comparative Example 5

[0092] Different from Example 1, in step S2 of this comparative example, the reagents are as follows: the content of the chelating agent is 12 mmol / L, the content of Tris is 22 mmol / L, and the content of NaCl is 0.1 mmol / L.

[0093] Comparative Example 6

[0094] Different from Example 1, in step S3 of this comparative example, the second intermediate product is first soaked in 50% ethanol for 0.5 h, then soaked in 99-100% ethanol for 0.5 h, and ultrasonically shaken with a mixed solution of hexane and acetone for 1 h, and the mass ratio of hexane to acetone is 1:1.

[0095] Comparative Example 7

[0096] Different from Example 1, in step S4 of this comparative example, the concentration of the alkaline solution is 1.6 mol / L, the alkaline solution also includes 50 mmol / L EDTA, and the immersion time in the alkaline solution is 8 h.

[0097] Comparative Example 8

[0098] Different from Example 1, in step S5 of this comparative example, the concentration of the acid solution is 2.2 mol / L, the concentration of NaCl is 0.5 mol / L, and the immersion time of the acid solution is 5 h.

[0099] The appearance and performance of the soft tissue repair patches prepared in the above examples and comparative examples were measured, and the test results are as follows:

[0100] The results of DNA content determination of each example and comparative example sample are shown in Table 1.

[0101] Table 1 Decellularization results of each example

[0102]

[0103]

[0104] Figure 1 Examples 1 and 4 and comparative examples 1, 5 and 7 are selected as typical representatives for demonstration. The HE staining images of Examples 1-3 are similar, the HE staining images of Examples 4-6 are similar, the HE staining images of Comparative Examples 1, 3, 4 and 6 are similar, and the HE staining images of Comparative Examples 2, 5, 7 and 8 are similar.

[0105] It can be seen that there is no obvious nuclear component in Examples 1-6, and Examples 4-6 have a higher efficiency in removing cell substances than Examples 1-3 due to the use of bubble generation temperature. Taking DNA content as an indicator, it can be seen that the DNA content has been greatly reduced compared with Example 1. Secondly, it can be seen from Example 1 and Comparative Example 1 that the absence of NaCl leads to a large amount of residual cells, mainly because the dendrites generated during the freeze-thaw process increase the firmness of the combination of cell substances and ECM, resulting in the inability to effectively remove cell substances even if a high-intensity decellularization process is used subsequently. From Example 1, Comparative Examples 2 and 3, it can be seen that when the contents of phenylmethylsulfonyl fluoride, chelating agent and Tris are low, on the one hand, due to the addition of NaCl, the activity thereof is reduced, and due to the low contents of phenylmethylsulfonyl fluoride and chelating agent, the superposition of the two has a low degree of inhibition of the protease released after cell rupture, and part of the collagen is decomposed. The decomposition of collagen will increase the residual amount of trace cellular material to a certain extent, and when the contents of phenylmethylsulfonyl fluoride, chelating agent and Tris are high, the dendrite tendency of the solution is strengthened, the firmness of the binding of cellular material to ECM is increased, and a small amount of cell nuclei remain; from Example 1, Comparative Examples 4 and 5, it can be seen that when there is no chelating agent in the reagent, The cell components cannot be effectively eluted, resulting in a small amount of cell nuclei remaining in the prepared patch. When phenylmethylsulfonyl fluoride is not present in the reagent, the protease generated in the late freeze-thaw period cannot be effectively inhibited, causing damage to the ECM and easily causing a trace amount of cell nuclei to remain in the ECM scaffold. It can be seen from Example 1 and Comparative Example 6 that isopropanol has a good degreasing effect. On the other hand, isopropanol combined with the freeze-thaw process can facilitate the subsequent removal of cell substances. Isopropanol has a high permeability, which reduces the binding strength of cell fragments and ECM after freeze-thaw, facilitating subsequent removal. Other degreasing agents cannot meet the effect of combining with the freeze-thaw process to achieve efficient cell substance removal.

[0106] (2) Structural analysis of the collagen patch scaffold prepared in each embodiment.

[0107] Figure 2 These are TEM images of the patches of Examples 1 and 4 and Comparative Examples 1, 5 and 7. The TEM images of Examples 1-3 are similar, the TEM images of Examples 4-6 are similar, the TEM images of Comparative Examples 1, 3, 4 and 6 are similar, the TEM images of Comparative Examples 2 and 5 are similar, and the TEM images of Comparative Examples 7 and 8 are similar.

[0108] As is known to all, collagen fibers have light and dark stripes, which are produced by the lateral aggregation of collagen molecules with a triple helical structure. The transmission electron microscopy results of Examples 1-6 and Comparative Examples 1, 3, 4, and 6 show that the prepared collagen has a triple helical structure and has complete collagen fiber bundles. Although the transmission electron microscopy of Comparative Examples 2, 5, 7, and 8 shows light and dark stripes, the collagen fiber bundles are not complete. Analysis shows that the collagen fiber bundles were damaged during the patch preparation process. It can be seen that Comparative Examples 2, 5, 7, and 8 can achieve the same decellularization effect as Example 1, but the collagen fibers of Comparative Examples 7-8 are damaged.

[0109] (3) Structural analysis of the collagen patch scaffold prepared in each embodiment.

[0110] Figure 3 The initial state diagram of the patches of Examples 1, 4 and Comparative Examples 1, 5 and 7 after being placed in the degradation solution, Figure 4 The state diagram of the patches of Examples 1, 4 and Comparative Examples 1, 5 and 7 after being placed in the degradation solution for 64 hours is shown in Table 3. The specific degradation rate of the patches after 64 hours is shown in Table 3. It can be seen that the degradation rate is high due to the destruction of collagen fibers in Comparative Examples 2, 5, 7 and 8. However, since the bubble generation temperature is used in the freeze-thaw process of Examples 4-6, the decellularization efficiency is improved while the damage to collagen is reduced.

[0111] Table 3 Degradation rate of collagen scaffolds in various examples

[0112]

[0113]

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a soft tissue repair patch, characterized in that: include: S1. Processing and cleaning the animal soft tissue to obtain a first intermediate product; S2, adding the first intermediate product to a reagent to perform a freeze-thaw cycle to obtain a second intermediate product; S3, soaking the second intermediate product with an organic solvent and washing it to obtain a third intermediate product; S4, soaking the third intermediate product in an alkaline solution and washing it to obtain a fourth intermediate product; S5, soaking the fourth intermediate product in an acid solution, washing, drying, and sterilizing to obtain the soft tissue repair patch; The reagent comprises the following components: the mass percentage content of phenylmethylsulfonyl fluoride is 0.2-0.8 mmol / L, the content of chelating agent is 12-18 mmol / L, the content of Tris is 22-25 mmol / L, and the content of soluble sodium salt or potassium salt is 0.1-0.5 mmol / L.

2. The preparation method according to claim 1, characterized in that: The animal soft tissue refers to one or more of animal dermis, animal pericardium, animal intestinal mucosa, and animal bladder tissue.

3. The preparation method according to claim 1, characterized in that: In step S1, the animal soft tissue is cut into a certain size, and the size of at least one direction in each direction does not exceed 5 mm, and fat and muscle tissue are removed, and blood and dirt are cleaned.

4. The preparation method according to claim 1, characterized in that: In step S2, 2 to 4 freeze-thaw cycles are performed, the freezing temperature of the freeze-thaw cycle is -60 to -80°C, the freezing time is 3 to 4 hours, the thawing temperature is 30 to 40°C, and the thawing time is 30 to 60 minutes.

5. The preparation method according to claim 1, characterized in that: In step S2, freeze-thaw cycles are performed for 2-4 times, the freezing temperature of the freeze-thaw cycle is -60 to -80°C, the freezing time is 3 to 4 hours, and the bubble generation temperature is also included. The bubble generation temperature is 2 to 5°C and is maintained at the bubble generation temperature for 2 to 3 hours. The thawing temperature is 15 to 25°C and the thawing time is 10 to 20 minutes.

6. The preparation method according to claim 1, characterized in that: The chelating agent is EDTA or EGTA.

7. The preparation method according to claim 1, characterized in that: In step S3, firstly, the sample is immersed in 50-70% ethanol for 0.5-1.0 h, then immersed in 99-100% ethanol for 0.5-1 h, and ultrasonically vibrated with isopropanol for 1-4 h.

8. The preparation method according to claim 1, characterized in that: In step S4, the concentration of the alkaline solution is 0.8-1.5 mol / L, the alkaline solution also includes 50-150 mmol / LEDTA, and the immersion time of the alkaline solution is 1-4 hours.

9. The preparation method according to claim 1, characterized in that: The acid solution in step S5 is one of hydrochloric acid, sulfuric acid, and acetic acid; the concentration of the acid solution is 1.2 to 2.0 mol / L, and the acid solution also contains sodium chloride, calcium chloride or potassium chloride, the concentration of the sodium chloride, calcium chloride or potassium chloride is 0.5 to 1.5 mol / L, and the acid solution immersion time is 1 to 4 hours.

10. A soft tissue repair patch, characterized in that: The soft tissue repair patch is prepared by the preparation method described in any one of claims 1-9.

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