Soft tissue repair patch and method of making same

Abstract

The application belongs to the technical field of medical materials, and discloses a soft tissue repair patch and a preparation method thereof, which comprises the following steps: S1, treating and cleaning animal soft tissue to obtain a first intermediate product; S2, adding the first intermediate product into a reagent to perform freeze-thaw circulation to obtain a second intermediate product; S3, soaking and treating the second intermediate product with an organic solvent and cleaning to obtain a third intermediate product; S4, soaking and treating the third intermediate product with an alkali solution and cleaning to obtain a fourth intermediate product; and S5, soaking and treating the fourth intermediate product with an acid solution, cleaning, drying and sterilizing to obtain the soft tissue repair patch. The application provides a more gentle and friendly decellularization scheme, and the decellularized matrix prepared by using the scheme is more completely decellularized, has no chemical residue, and maintains the natural scaffold structure of collagen.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical materials, and particularly relates to a soft tissue repair patch and a preparation method thereof. BACKGROUND

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

[0003] The raw materials for preparing xenogeneic decellularized tissues are very extensive, such as animal skin, peritoneal membrane, tendon, small intestinal mucosa, pericardium, and blood vessels. The decellularized tissue preparation process mainly removes the cellular components of fresh tissues through physical, chemical, and biological methods, and retains the extracellular matrix of the tissue. Physical methods mainly destroy cell membranes through physical means to release cell contents, including ultrasound, pressure, freeze-thaw, mechanical stirring, etc. Chemical methods mainly include acids, bases, and surfactants, which change the permeability of cell membranes through chemical reagents, ultimately causing cell swelling and rupture to achieve decellularization. Biological methods mainly use various enzymes, such as trypsin, nuclease, and DNAase.

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

[0005] To solve the above problems, the present application provides a soft tissue repair patch and a preparation method thereof, which combines physical and chemical decellularization methods to provide a preparation process that is more complete in decellularization, has no chemical residues, and maintains the natural scaffold structure of collagen.

[0006] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0007] A preparation method of a soft tissue repair patch, comprising: S1, treating and cleaning animal soft tissue to obtain a first intermediate product; S2, adding the first intermediate product into a reagent for freeze-thaw cycle to obtain a second intermediate product; S3, soaking and treating the second intermediate product with an organic solvent and cleaning to obtain a third intermediate product; S4, soaking and treating the third intermediate product with an alkali solution and cleaning to obtain a fourth intermediate product; S5, soaking and treating the fourth intermediate product with an acid solution, cleaning, drying and sterilizing to obtain the soft tissue repair patch; the composition of the reagent comprises: the mass percentage content of benzylsulfonyl 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.

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

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

[0010] Further, in step S2, the freeze-thaw cycle is 2-4 cycles, the freezing temperature of the freeze-thaw cycle is-60 to-80℃, the freezing time is 3-4 h, the thawing temperature is 30-40℃, and the thawing time is 30-60 min.

[0011] Further, in step S2, the freeze-thaw cycle is 2-4 cycles, the freezing temperature of the freeze-thaw cycle is-60 to-80℃, the freezing time is 3-4 h, the bubble generation temperature is 2-5℃, and the bubble generation temperature is maintained for 2-3 h, the thawing temperature is 15-25℃, and the thawing time is 10-20 min.

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

[0013] Further, in step S3, first, 50-70% ethanol is soaked for 0.5-1.0 h, then 99-100% ethanol is soaked for 0.5-1 h, and isopropyl alcohol is ultrasonically oscillated for 1-4 h.

[0014] Further, in step S4, the concentration of the alkali solution is 0.8-1.5 mol / L, the alkali solution further comprises 50-150 mmol / L EDTA, and the soaking time of the alkali solution is 1-4 h.

[0015] Further, 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, and the acid solution further contains sodium chloride, calcium chloride or potassium chloride, the concentration of the sodium chloride, calcium chloride or potassium chloride is 0.5-1.5 mol / L, and the acid solution soaking time is 1-4 h.

[0016] A soft tissue repair patch prepared by the preparation method.

[0017] The application has the following beneficial effects: the technical scheme provided by the application avoids damage to the ECM structure caused by dendrites generated in the freezing process by specific selection of reagent components in the freeze-thaw process and control of the content of each component. Although various decellularization technologies exist in the prior art, the degree of decellularization is relative, i.e., cell material cannot be completely removed, and a high removal rate of cell components can be achieved by combining various technologies, but this easily leads to a decrease in the structural integrity and activity of the patch, and the prepared patch is easily degraded. For the freeze-thaw technology, liquid solidification generates dendrites, such as snowflake-like structures generated in the solidification process of water, 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 processing, even if a high-strength elution process is used. The higher the content of cell tissue, the stronger the rejection or inflammatory reaction after the subsequent implantation of the patch. Secondly, if a high-strength elution process is used subsequently, the integrity of the patch changes, the activity decreases, and the degradation rate is high. The application adds benzylsulfonyl fluoride to the buffer solution to avoid damage to the protein structure caused by proteases released when the cells are broken, and adds a chelating agent, which can enter between the cells and the ECM, to improve the subsequent removal efficiency of the cell material. The benzylsulfonyl fluoride has a half-life, and during the freeze-thaw process, the chelating agent inhibits proteases on one hand, and on the other hand, the chelating agent prevents the cell material from being combined with the ECM at too high a strength, making it difficult to effectively remove the cell material subsequently. However, benzylsulfonyl fluoride and the chelating agent can strengthen the growth trend of dendrites. To solve the above problems, the application adds a certain amount of soluble sodium salt or potassium salt to reduce the growth trend of dendrites, but the sodium salt or potassium salt affects the activity of the chelating agent. To solve the above problems, the application limits the content of various components to solve the above technical problems and prevent the late activity of benzylsulfonyl fluoride and the combination of the cell material and the ECM at too high a strength, and reduce the trend of generating dendrites. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort based on these drawings.

[0019] Figure 1 The HE staining diagram of the section of the patch prepared in the present application and the comparative example, a: example 1, b: example 4, c: comparative example 1, d: comparative example 5, e: comparative example 7;

[0020] Figure 2 The transmission electron microscope diagram of the patch prepared in the present application and the comparative example, a: example 1, b: example 4, c: comparative example 1, d: comparative example 5, e: comparative example 7;

[0021] Figure 3 The initial diagram of the patch prepared in the present application and the comparative example immersed in the degradation solution, a0: example 1, b0: example 4, c0: comparative example 1, d0: comparative example 5, e0: comparative example 7;

[0022] Figure 4 The diagram after 64 hours of the patch prepared in the present application and the comparative example immersed in the degradation solution, 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 application more clear, the present application will be further described in detail with the drawings and specific embodiments.

[0024] In order to avoid errors caused by single test, the quantitative test in the embodiments of the present application is set to three repeated experiments, and the average value is taken.

[0025] The embodiment of the present application provides a kind of soft tissue repair patch and preparation method, comprising: S1, animal soft tissue is handled and cleaned, and first intermediate product is obtained;S2, first intermediate product is added to reagent and freeze-thaw cycle is carried out, and second intermediate product is obtained;S3, second intermediate product is treated by organic solvent soaking and cleaned, and third intermediate product is obtained;S4, third intermediate product is treated by alkali solution soaking and cleaned, and fourth intermediate product is obtained;S5, fourth intermediate product is treated by acid solution soaking, cleaning, drying, sterilization, and the soft tissue repair patch is obtained;The composition of the reagent includes: the mass percentage content of benzylsulfonyl fluoride is 0.2-0.8mmol / L, the content of chelating agent is 12-18mmol / L, the content of Tris is 22-25mmol / L, and the content of soluble sodium salt or potassium salt is 0.1-0.5mmol / L.

[0026] The technical scheme provided by the present application avoids the damage of ECM structure caused by dendrites generated in the freezing process by specific selection of the reagent composition in the freeze-thaw process and control of the content of each component. Although there are various decellularization technologies in the prior art, the degree of decellularization is relative, i.e. it is impossible to completely remove cell material. High removal rate of cell components can be achieved by combining various technologies, but it is easy to cause the decrease of patch structure integrity and activity, and the prepared patch is easy to be degraded. For freeze-thaw technology, during the freeze-thaw process, liquid solidification generates dendrites, such as snowflake structure generated by water during solidification. 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 processing. Even if high-strength 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 high-strength elution process is used subsequently, it will cause changes in patch integrity, decrease in activity and high degradation rate. By adding benzylsulfonyl fluoride to the buffer solution, the present application avoids the damage of protein structure caused by proteases released when cells are broken. At the same time, a chelating agent is added. The chelating agent can enter between the cells and the ECM, improving the subsequent removal efficiency of cell material. At the same time, benzylsulfonyl fluoride has a half-life. During the freeze-thaw process, the chelating agent inhibits proteases on one hand, and on the other hand, the chelating agent prevents the cell material from being combined with the ECM at too high a strength, making it difficult to effectively remove the cell material subsequently. However, benzylsulfonyl fluoride and the chelating agent will strengthen the growth trend of dendrites. In order to solve the above problems, a certain amount of soluble sodium salt or potassium salt is added to reduce the growth trend of dendrites. However, sodium salt or potassium salt will affect the activity of the chelating agent. In order to solve the above problems, the content of various components is limited to solve the above technical problems and prevent the late activity of benzylsulfonyl fluoride and the combination strength of cell material and ECM from being too high, and reduce the trend of generating dendrites.

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

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

[0029] In step S1, the animal soft tissue refers to one or more of the dermis of an animal, the pericardium of an animal, the intestinal mucosa of an animal, and the bladder tissue of an animal, and the animal is a mammal such as a cow, a horse, or a pig. The animal soft tissue is cut into a certain size, and at least one direction of the size is not more than 5 mm, the fat and muscle tissue are removed, and the blood and dirt are cleaned, so that the size of the cut tissue is limited, the inside and outside of the tissue can be cooled or heated as synchronously as possible, the structural damage to the soft tissue is reduced, the anti-degradation ability of the prepared product is increased, and if the size is too large, the outside tissue freezes first, the inside tissue freezes later, and the inside tissue expands in the freezing process, which causes damage to the outside tissue.

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

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

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

[0033] In step S3, first, 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, 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 is ultrasonically agitated for 1-4h, such as 1h, 2h, 3h or 4h. Soaking with low-concentration ethanol first can avoid the use of high-concentration ethanol directly, which causes the cells to lose water rapidly and thus cannot achieve effective dehydration effect; isopropanol has good degreasing effect, on the other hand, isopropanol combined with the freeze-thaw process can facilitate the subsequent removal of cell material, isopropanol has high permeability, which reduces the binding strength of cell fragments and ECM after freeze-thaw, facilitating subsequent removal.

[0034] In step S4, the concentration of the alkali 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 alkali solution further comprises 50-150 mmol / L 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 soaking time of the alkali solution is 1-4 h, such as 1 h, 2 h, 3 h, 4 h. The alkali solution can cause the swelling and loosening of collagen fibers and can saponify lipids, and the EDTA in the alkali solution can effectively remove cells and fragments from the extracellular matrix by reducing the concentration of divalent cations. However, the concentration, time and frequency of the alkali solution treatment should not be too long, otherwise the collagen scaffold structure can be easily destroyed, and the mechanical strength of the final material can be significantly reduced. In the present application, the improved freeze-thaw process reduces the treatment strength of the alkali solution, i.e. reduces the concentration and treatment time of the alkali solution, and maintains the integrity of the collagen scaffold structure. The alkali 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, 2.0 mol / L; the acid solution further comprises 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, 1.5 mol / L, and the soaking time of the acid solution is 1-4 h, such as 1 h, 2 h, 3 h, 4 h. The acid solution can cause the swelling and loosening of collagen fibers, can remove glycoprotein and glycosaminoglycan, non-collagen protein and nucleic acid substances, and the addition of the salt solution in the acid solution can control the swelling of the collagen tissue matrix during the acid treatment and remove glycoprotein and proteoglycan from the collagen matrix.

[0036] The preparation method of the present application is carried out at room temperature, i.e. 20-25℃, unless otherwise specified.

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

[0038] The application also protects a soft tissue repair patch prepared by the above preparation method.

[0039] The soft tissue repair patch provided by the application can be applied in oral soft tissue repair, dura repair, burn and scald wound repair and breast reconstruction.

[0040] In order to characterize the soft tissue repair patch prepared by the examples and the comparative examples of the application, the following test items are used:

[0041] (1) Decellularization detection:

[0042] The middle position of the sample of each example and the comparative example is sampled longitudinally, embedded, sliced (about 5 microns thick), dried, HE stained, sealed, and observed under a microscope.

[0043] DNA content: detected according to YY / T0606.25-2014 "Animal-derived biological material DNA residual amount determination method: fluorescence staining method".

[0044] (2) Transmission electron microscopy analysis:

[0045] The middle position of the sample of each example and the comparative example is sampled longitudinally, embedded, sliced (about 70 nm thick), dried, and observed under a transmission electron microscope.

[0046] (3) Product in vitro degradation test: each example sample is uniformly processed to 2cm*3cm size, accurately weighed, the initial weight is recorded as m0, according to the extraction ratio of 0.1g:15ml, the degradation liquid (0.2mg / ml collagenase type I physiological saline solution) is added, the processed example samples are placed in a constant temperature incubator at 37℃, taken out after 64 hours, and freeze-dried, the freeze-dried sample is accurately weighed, the weight is recorded as m. The degradation rate of each example sample after 64 hours is calculated. And the degradation morphology change of the sample from 0 to 64 hours is observed.

[0047] The degradation rate calculation formula is:

[0048]

[0049] In order to better illustrate the embodiments of the application, the application will be further described in detail through specific examples.

[0050] Example 1

[0051] The example of the application provides a soft tissue repair patch and a preparation method, which comprises:

[0052] S1, treating and cleaning animal soft tissue to obtain a first intermediate product. Fresh animal skin with a thickness of not more than 5 mm is removed of subcutaneous fat, muscle and hair, and cleaned with water to obtain the first intermediate product.

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

[0054] S3, soaking and treating the second intermediate product with an organic solvent and cleaning 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 isopropanol is ultrasonically vibrated for 1 h. After the soaking is completed, the third intermediate product is cleaned with purified water.

[0056] S4, soaking and treating the third intermediate product with an alkali solution and cleaning to obtain a fourth intermediate product. The concentration of the alkali solution is 0.8 mol / L, and the alkali solution further comprises 50 mmol / L EDTA. The soaking time of the alkali solution is 1 h, and the fourth intermediate product is cleaned with purified water.

[0057] S5, soaking and treating the fourth intermediate product with an acid solution, cleaning, drying and sterilizing 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 soaking time of the acid solution is 1 h. The acid solution is cleaned with PBS, freeze-dried and sterilized to obtain the acellular dermal matrix.

[0059] Example 2

[0060] The embodiment of the present application provides a soft tissue repair patch and a preparation method, which comprises:

[0061] S1, treating and cleaning animal soft tissue to obtain a first intermediate product. Fresh animal skin with a thickness of not more than 5 mm is removed of subcutaneous fat, muscle and hair, and cleaned with water to obtain the first intermediate product.

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

[0063] S3, the second intermediate product is treated by soaking in an organic solvent and cleaned to obtain a third intermediate product.

[0064] Firstly, the second intermediate product is soaked in 60% ethanol for 0.8h, then is soaked in 99-100% ethanol for 0.8h, isopropanol is ultrasonically vibrated for 3h, and after the soaking is completed, the third intermediate product is obtained by cleaning with purified water.

[0065] S4, the third intermediate product is treated by soaking in an alkali solution and cleaned to obtain a fourth intermediate product. The concentration of the alkali solution is 1.0 mol / L, the alkali solution further comprises 100 mmol / L EGTA, and the soaking time of the alkali solution is 3h. The fourth intermediate product is cleaned with purified water.

[0066] S5, the fourth intermediate product is treated by soaking in an acid solution, 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 soaking time of the acid solution is 3h. The acid solution is cleaned with PBS, freeze-dried and sterilized to obtain the acellular dermal matrix.

[0068] Example 3

[0069] The embodiment of the application provides a soft tissue repair patch and a preparation method thereof, which comprises the following steps:

[0070] S1, an animal soft tissue is treated and cleaned to obtain a first intermediate product. Fresh animal skin with a thickness of not more than 5mm is removed of subcutaneous fat, muscle and hair, and is cleaned with water to obtain the first intermediate product.

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

[0072] S3, the second intermediate product is treated by soaking in an organic solvent and cleaned to obtain a third intermediate product.

[0073] Firstly, the second intermediate product is soaked in 70% ethanol for 1.0 h, then in 99-100% ethanol for 1 h, and in isopropyl alcohol for ultrasonic oscillation for 4 h. After the soaking, the third intermediate product is obtained by cleaning with purified water.

[0074] S4, the third intermediate product is treated by soaking in an alkali solution and cleaned to obtain a fourth intermediate product. The concentration of the alkali solution is 1.5 mol / L, the alkali solution further comprises 100 mmol / L EDTA, and the soaking time of the alkali solution is 4 h. The fourth intermediate product is cleaned with purified water.

[0075] S5, the fourth intermediate product is treated by soaking in an acid solution, 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, the soaking time of the acid solution is 4 h. The acid-treated product is cleaned with PBS, freeze-dried, and sterilized to obtain the acellular dermal matrix.

[0077] Example 4

[0078] Different from example 1, in step S2 of the present example, the freeze-thaw cycle is 2 cycles, the freezing temperature of the freeze-thaw cycle is -60℃, the freezing time is 3 h, the bubble generation temperature is 2℃, the bubble generation temperature is maintained for 2 h, the thawing temperature is 15℃, the thawing time is 10 min, and the chelating agent is EDTA.

[0079] Example 5

[0080] Different from example 1, in step S2 of the present example, the freeze-thaw cycle is 2 cycles, the freezing temperature of the freeze-thaw cycle is -70℃, the freezing time is 3.5 h, the bubble generation temperature is 4℃, the bubble generation temperature is maintained for 2 h, the thawing temperature is 20℃, the thawing time is 15 min, and the chelating agent is EDTA.

[0081] Example 6

[0082] Different from example 1, in step S2 of the present example, the freeze-thaw cycle is 2 cycles, the freezing temperature of the freeze-thaw cycle is -80℃, the freezing time is 4h, the bubble generating temperature is 5℃, and the holding time at the bubble generating temperature is 3h, the thawing temperature is 25℃, the thawing time is 20min, and the chelating agent is EDTA.

[0083] Comparative example 1

[0084] Different from example 1, in step S2 of the present comparative example, the reagent does not contain NaCl.

[0085] Comparative example 2

[0086] Different from example 1, in step S2 of the present comparative example, the reagent contains: benzylsulfonyl fluoride with a mass percentage of 0.1mmol / L, chelating agent with a content of 10mmol / L, Tris with a content of 20mmol / L, and NaCl with a content of 0.1mmol / L.

[0087] Comparative example 3

[0088] Different from example 1, in step S2 of the present comparative example, the reagent contains: benzylsulfonyl fluoride with a mass percentage of 0.9mmol / L, chelating agent with a content of 19mmol / L, Tris with a content of 26mmol / L, and NaCl with a content of 0.1mmol / L.

[0089] Comparative example 4

[0090] Different from example 1, in step S2 of the present comparative example, the reagent contains: benzylsulfonyl fluoride with a mass percentage of 0.2mmol / L, Tris with a content of 22mmol / L, and NaCl with a content of 0.1mmol / L.

[0091] Comparative example 5

[0092] Different from example 1, in step S2 of the present comparative example, the reagent contains: chelating agent with a content of 12mmol / L, Tris with a content of 22mmol / L, and NaCl with a content of 0.1mmol / L.

[0093] Comparative example 6

[0094] Different from example 1, in step S3 of the present comparative example, the second intermediate product is first soaked in 50% ethanol for 0.5h, then soaked in 99-100% ethanol for 0.5h, and then subjected to ultrasonic oscillation in a mixture of hexane and acetone for 1h, the mass ratio of hexane to acetone being 1:1.

[0095] Comparative example 7

[0096] Different from Example 1, in step S4 of the present comparative example, the concentration of the alkaline solution is 1.6 mol / L, the alkaline solution further comprises 50 mmol / L EDTA, and the soaking time of the alkaline solution is 8 h.

[0097] Comparative Example 8

[0098] Different from Example 1, in step S5 of the present comparative example, the concentration of the acid solution is 2.2 mol / L, the concentration of NaCl is 0.5 mol / L, and the soaking 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 determined, and the test results are as follows:

[0100] The DNA content of the samples of each example and comparative example was determined, and the results are shown in Table 1.

[0101] Table 1: Decellularization results of each example

[0102]

[0103]

[0104] Figure 1 Examples 1, 4 and Comparative Examples 1, 5 and 7 were selected as typical representatives for display. The HE staining diagrams of Examples 1-3 were similar, the HE staining diagrams of Examples 4-6 were similar, the HE staining diagrams of Comparative Examples 1, 3, 4 and 6 were similar, and the HE staining diagrams of Comparative Examples 2, 5, 7 and 8 were similar.

[0105] It can be seen that none of Examples 1-6 has obvious cell nucleus components, and Examples 4-6 have higher efficiency of removing cell material relative to Examples 1-3 due to the use of bubble generation temperature. Taking DNA content as an index, it can be seen that the DNA content has been greatly reduced compared to Example 1. Secondly, as can be seen from Example 1 and Comparative Example 1, without NaCl, the amount of residual cells is large, mainly because the dendrites generated during freezing and thawing increase the firmness of the cell material combined with ECM, resulting in that even if a high-strength decellularization process is used, the cell material cannot be effectively removed. As can be seen from Example 1, Comparative Examples 2 and 3, when the content of benzyl sulfonyl fluoride, chelating agent and Tris is low, on the one hand, due to the addition of NaCl, the activity is reduced, and because the content of benzyl sulfonyl fluoride and chelating agent is low, the inhibition of proteinase released after cell rupture is low, part of the collagen is decomposed, and the decomposition of collagen will increase the residual amount of trace cell material to some extent, and when the content of benzyl sulfonyl fluoride, chelating agent and Tris is high, the solution dendrite trend is strengthened, which increases the firmness of the cell material combined with ECM, resulting in a small amount of cell nucleus residue; As can be seen from Example 1, Comparative Examples 4 and 5, when the chelating agent is not present in the reagent, the cell components cannot be effectively eluted, resulting in a small amount of cell nucleus residue in the prepared patch, and when benzyl sulfonyl fluoride is not present in the reagent, the proteinase generated in the later stage of freezing and thawing cannot be effectively inhibited, which damages the ECM and also easily causes trace cell nucleus to remain in the ECM scaffold; As can be seen from Example 1 and Comparative Example 6, isopropyl alcohol has good degreasing effect, on the other hand, isopropyl alcohol can be combined with the freezing and thawing process to facilitate the subsequent removal of cell material. Isopropyl alcohol has high permeability, which reduces the binding strength of cell fragments and ECM after freezing and thawing, facilitating subsequent removal, while other degreasing reagents cannot meet the requirements of high-efficiency cell material removal in combination with the freezing and thawing process.

[0106] (2) Analysis of collagen scaffold structure of patches prepared in each example.

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

[0108] It is known that collagen fibers have light and dark cross striations, which are produced by the lateral aggregation of collagen molecules with triple helix structure. The transmission electron microscopy results of Examples 1-6 and Comparative Examples 1, 3, 4, 6 show that the prepared collagen has triple helix structure and complete collagen fiber bundles. The transmission electron microscopy of Comparative Examples 2, 5, 7 and 8 shows light and dark cross striations, but the collagen fiber bundles are not complete, and it is analyzed that the collagen fiber bundles are damaged during the preparation of the patch. 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 and 8 are damaged.

[0109] (3) Analysis of the collagen scaffold structure of the patch prepared in each example.

[0110] Figure 3 The initial state diagram of the patch of Example 1, 4 and Comparative Examples 1, 5 and 7 placed in the degradation liquid, Figure 4 The state diagram of the patch of Example 1, 4 and Comparative Examples 1, 5 and 7 after being placed in the degradation liquid for 64 hours. The specific 64h patch degradation rate is shown in Table 3. It can be seen that due to the damage of the collagen fibers of Comparative Examples 2, 5, 7 and 8, the degradation rate is relatively high. And Examples 4-6 use bubble generation temperature in the freeze-thaw process, so that the decellularization efficiency is improved while the damage to collagen protein is reduced.

[0111] Table 3: Degradation rate of collagen scaffold of each example

[0112]

[0113]

[0114] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method of making a soft tissue repair patch, characterized by, The preparation method comprises the following steps: S1, treating and cleaning animal soft tissue to obtain a first intermediate product; S2, adding the first intermediate product into a reagent for freeze-thaw cycle to obtain a second intermediate product; S3, soaking and treating the second intermediate product with an organic solvent and cleaning to obtain a third intermediate product; S4, soaking and treating the third intermediate product with an alkali solution and cleaning to obtain a fourth intermediate product; S5, soaking the fourth intermediate product with an acid solution, cleaning, drying and sterilizing to obtain the soft tissue repair patch. The reagent comprises the following components: 0.2-0.8 mmol / L of benzylsulfonyl fluoride, 12-18 mmol / L of chelating agent, 22-25 mmol / L of Tris, and 0.1-0.5 mmol / L of soluble sodium salt or potassium salt. In step S1, the animal soft tissue is cut into a certain size, and the size of at least one direction is not more than 5 mm, the fat and muscle tissue are removed, and the blood and dirt are cleaned. The chelating agent is EDTA or EGTA. In step S3, the third intermediate product is first soaked in 50-70% ethanol for 0.5-1.0 h, then soaked in 99-100% ethanol for 0.5-1 h, and finally ultrasonically oscillated in isopropyl alcohol for 1-4 h. In step S4, the alkali solution has a concentration of 0.8-1.5 mol / L, and further comprises 50-150 mmol / L EDTA, and the soaking time of the alkali solution is 1-4 h. In step S5, the acid solution is one of hydrochloric acid, sulfuric acid and acetic acid, and the acid solution has a concentration of 1.2-2.0 mol / L, and further comprises 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, and the soaking time of the acid solution is 1-4 h.

2. The preparation method according to claim 1, wherein the animal soft tissue is one or more of animal dermis, animal pericardium, animal intestinal mucosa and animal bladder tissue.

3. The preparation method according to claim 1, wherein in step S2, the freeze-thaw cycle is 2-4 cycles, the freezing temperature of the freeze-thaw cycle is -60 to -80℃, the freezing time is 3-4 h, the thawing temperature is 30-40℃, and the thawing time is 30-60 min.

4. The preparation method according to claim 1, wherein in step S2, the freeze-thaw cycle is 2-4 cycles, the freezing temperature of the freeze-thaw cycle is -60 to -80℃, the freezing time is 3-4 h, the freeze-thaw cycle further comprises a bubble generation temperature, the bubble generation temperature is 2-5℃, the bubble generation temperature is maintained for 2-3 h, the thawing temperature is 15-25℃, and the thawing time is 10-20 min. The soft tissue repair patch is prepared by the preparation method of any one of claims 1-4. ​ ​ 5. A soft tissue repair patch characterized by, ​

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