A double cross-linked acellular matrix hydrogel and its preparation method and application

Abstract

The invention provides a double-crosslinked decellular matrix hydrogel and a preparation method and application thereof, belonging to the technical field of hydrogels; the preparation method comprises: 1) performing decellularization treatment on a porcine dermis layer to obtain a decellularized porcine dermal matrix; 2) placing the decellularized porcine dermal matrix freeze-dried powder in an acidic pepsin solution, stirring and digesting, adjusting the concentration, adding 10xPBS, and adjusting the pH to obtain a pregel solution; 3) placing a gel formed by the pregel solution in a tannic acid solution for crosslinking to obtain a single-crosslinked decellular matrix hydrogel; 4) crushing the single-crosslinked decellular matrix hydrogel and mixing it with a glucose oxidase solution and a ferrous ion solution with a concentration of 0.1-10 mg / mL to obtain a double-crosslinked decellular matrix hydrogel precursor; 5) placing the double-crosslinked decellular matrix hydrogel precursor in a glucose concentration of 10-200 mg / dl to form a double-crosslinked decellular matrix hydrogel; the hydrogel prepared by the invention can achieve a balance among safety, support and degradability, and has a wide range of applications.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogels, and particularly relates to a double-crosslinked acellular matrix hydrogel and a preparation method and application thereof. Background Art

[0002] In the medical field, soft tissue repair is a clinical problem that is both clinically important and extremely challenging. This problem is mainly caused by critical-sized full-thickness local soft tissue defects caused by acute or chronic conditions such as burns, traumatic brain injury, myocardial infarction, cerebral stroke, and senile degeneration. The damage to the parts covers multiple areas such as the heart, elbows, brain, skeletal muscle, lower urinary tract, esophagus, cartilage, and dura mater.

[0003] At present, the main methods used in clinical practice for soft tissue repair are artificial material replacement or material filling, aiming to restore the function or shape of the tissue and make it as close to the healthy state as possible. The materials used for soft tissue repair need to have good biocompatibility and the ability to promote tissue growth, so as to improve the appearance and function of the patient's injured part; among them, the injection methods for soft tissue filling are mainly: autologous fat injection and hyaluronic acid injection. Although autologous fat injection has good biocompatibility and high safety, it also has the disadvantages of high operation requirements, unstable survival rate of fat cells, uneven absorption, and poor correction effect of large-area deep depressions; hyaluronic acid injection, although the injection process is relatively simple and the immediate effect is good, It may also cause non-allergic reactions and serious complications in the local skin. It is difficult to achieve a suitable balance in terms of support, biodegradability, safety and effectiveness when using inorganic synthetic materials such as polylactic acid, hydroxyapatite, and tricalcium phosphate for repair and filling. Most materials can only provide a simple support, which compresses the growth space of the tissue. The recurrence symptoms are more obvious after the material is degraded. In addition, the existing artificial substitutes for valves, blood vessels, dura mater and other parts are generally prepared with synthetic polymers or cross-linked animal tissues, which are difficult to guide soft tissue repair and regeneration, and are non-degradable and permanently retained in the body, which will cause chronic inflammatory reactions, resulting in repair failure or failure of the substitute function. Therefore, it is urgent to develop new biomaterials that have good tissue repair growth promotion and are degradable to repair or induce new tissue growth.

[0004] Decellularized matrix is ​​a biomaterial that has been specially treated to remove immunogenic cellular components from tissues or organs while retaining the three-dimensional spatial structure of the extracellular matrix (mainly composed of collagen fibers, proteoglycans, glycoproteins, etc.) and functional proteins (such as growth factors). It has good biocompatibility and biodegradability, can promote cell adhesion, proliferation, migration and differentiation, and is a good candidate material for tissue repair and regeneration. In recent years, studies have found that after the separated decellularized matrix is ​​dissolved, the formation of decellularized matrix hydrogel can be induced by adjusting the temperature and pH, or adding a cross-linking agent. This material not only retains the main components, structure and biological activity of the extracellular matrix, but also has the characteristics of water swelling, fluidity and injectability of hydrogel, showing great potential in the field of biomedical materials. However, the decellularized matrix hydrogel prepared by the existing technology generally has problems such as poor mechanical properties, easy degradation, relatively unstable structure, poor mechanical properties, difficulty in playing the original supporting role after injection, and the introduction of toxic chemicals during the cross-linking process. Summary of the invention

[0005] The purpose of the present invention is to address the deficiencies in the above-mentioned prior art and provide a double-crosslinked acellular matrix hydrogel and a preparation method and application thereof. By improving the decellularization and gel synthesis methods, the ordinary acellular extracellular matrix hydrogel is initially crosslinked to make it close to the limited range of injectability. Finally, through special in vivo secondary crosslinking, the injected gel is reorganized to strengthen the gel's supporting properties, ultimately achieving a balance between safety, support, and degradability.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] The first aspect of the present invention provides a method for preparing a double-crosslinked acellular matrix hydrogel, comprising the following steps:

[0008] 1) Proteolysis, cleaning, degreasing, cleaning, and deep decellularization of the pig dermis fragments are performed in sequence to obtain acellular pig dermal matrix;

[0009] 2) freeze-drying the decellularized porcine dermal matrix obtained in step 1) and grinding it into matrix powder, placing the matrix powder in an acidic pepsin solution and stirring to digest, and after digestion is completed, adding an appropriate amount of RO water under ice bath conditions to adjust the solute concentration in the solution to more than 10 mg / mL, and then adding 1 / 10 volume of 10xPBS, and then adjusting the pH of the solution to neutral to obtain a pregel solution;

[0010] 3) placing the pregel solution obtained in step 2) at 37°C to form a gel, and then placing the gel in a tannic acid solution with a concentration of 1-10 mg / mL to crosslink for 10-48 h to obtain a single cross-linked acellular matrix hydrogel;

[0011] 4) sterilizing the single cross-linked acellular matrix hydrogel obtained in step 3), and then physically breaking it into millimeter scale, adding a glucose oxidase solution with a concentration of 1-10u / mL and a ferrous ion solution with a concentration of 0.1-10mg / mL to the broken hydrogel, and stirring evenly to obtain a double cross-linked acellular matrix hydrogel precursor;

[0012] 5) The double-crosslinked acellular matrix hydrogel precursor obtained in step 4) is placed in a glucose concentration of 10-200 mg / dl to form a double-crosslinked acellular matrix hydrogel by in situ spontaneous crosslinking.

[0013] Preferably, the specific steps of step 1) include:

[0014] 1-1): Soak the pig dermis fragments in 0.25% trypsin solution for 8-24 hours, and then wash them with deionized water for at least three times;

[0015] 1-2): After cleaning, soak in 70% ethanol solution for 12-24 hours, and then rinse with deionized water;

[0016] 1-3): After cleaning, soak in 3% hydrogen peroxide solution for 15-30 minutes, rinse with deionized water at least twice, and then soak in decellularization solution for 24 hours, and rinse with deionized water after soaking;

[0017] 1-4): After cleaning, place it in a disinfectant containing 0.1% peracetic acid and 4% ethanol for disinfection for 2-6 hours. After disinfection, soak it in 75% ethanol for 12-24 hours. Finally, rinse it with DPBS and wash it with deionized water to obtain the acellular porcine dermal matrix.

[0018] Further preferably, the decellularization solution in step 1-3) comprises: 1% Triton X-100, 0.26% EDTA, and 0.69% Tris solution.

[0019] Preferably, the acidic pepsin solution in step 2) is prepared by dissolving pepsin in a hydrochloric acid solution, wherein the concentration of pepsin in the acidic pepsin solution is 1 mg / mL and the concentration of hydrochloric acid is 0.1 mol / L.

[0020] Preferably, in step 2), the solid-liquid mass ratio of the decellularized porcine dermal matrix powder to the acidic pepsin solution is in the range of 0.04-0.1:1, and the digestion stirring time is 2-4 d.

[0021] Preferably, in step 3), the pre-gel solution is allowed to stand for 30 min to 90 min to form a gel.

[0022] Preferably, the disinfection and sterilization step in step 4) is: soaking the single cross-linked acellular matrix hydrogel in a 75% ethanol solution for 3 times, each time for 6 hours, and then washing and soaking in PBS for 3 times, each time for 6 hours.

[0023] Preferably, the volume ratio of the single cross-linked decellularized matrix hydrogel, the glucose oxidase solution and the ferrous ion solution in step 4) is 10:1-5:1, and the ferrous ion solution is one or more of ferrous sulfate solution, ferrous chloride solution or ferrous nitrate solution.

[0024] The present invention also provides a double-crosslinked decellular matrix hydrogel precursor prepared by the above method. The hydrogel precursor can spontaneously crosslink under glucose conditions in vitro to form a double-crosslinked decellular matrix hydrogel, which can be used for modifying heart valves or other artificial tissue substitutes. It can also be injected into the body and spontaneously crosslink under glucose conditions in vivo to form a double-crosslinked decellular matrix hydrogel for soft tissue filling.

[0025] The second aspect of the present invention provides a double-crosslinked acellular matrix hydrogel prepared by the above-mentioned preparation method.

[0026] The third aspect of the present invention provides the use of the double-crosslinked acellular matrix hydrogel as a material for preparing soft tissue repair.

[0027] Preferably, the double-crosslinked acellular matrix hydrogel can be used to prepare heart valve repair materials, vascular repair materials, tissue depression defect treatment materials, etc.

[0028] The beneficial effects of the present invention are:

[0029] 1. The present invention innovatively adopts a double cross-linking preparation method. The first cross-linking forms a stable chemical bond between tannic acid and the components in the decellularized matrix to construct a single cross-linked decellularized matrix hydrogel with a unique fibrous structure, thereby enhancing the mechanical properties and stability of the hydrogel. The second cross-linking is carried out under glucose conditions through a glucose oxidase-ferrous ion system, using glucose oxidase to catalyze the oxidation of glucose to produce hydrogen peroxide and Fe 2+ Ion reaction produces Fe 3+ Ions trigger the secondary cross-linking reaction of the decellularized matrix hydrogel, enhance the interaction between the decellularized matrix particles, and make the originally dispersed gel particles reunite and form a more compact network structure, which not only improves the mechanical strength of the hydrogel, but also gives it good biocompatibility, effectively preventing the gel from being rapidly degraded or dispersed in the body, so that it can maintain its shape and function for a long time;

[0030] 2. The present invention prepares a double-crosslinked acellular matrix hydrogel precursor with good fluidity, so that the hydrogel can be injected into the part of the body that needs to be repaired or filled, and then uses the physiological conditions of glucose in the body to trigger the second crosslinking reaction, so that the hydrogel spontaneously crosslinks in situ at the injection site to form a double-crosslinked acellular matrix hydrogel, thereby ensuring that the hydrogel can be adaptively formed according to the shape and space requirements of the injection site, so as to better fit the tissue defect site, improve the treatment effect, and be simple to operate. It has a wide range of applications and good flexibility in clinical applications;

[0031] 3. The present invention uses the extracellular matrix of porcine dermis, which is more similar to the extracellular matrix components of human tissue, as the main component of the hydrogel, retains most of the bioactive components and structural characteristics of the extracellular matrix, is beneficial to cell adhesion, proliferation and differentiation, and enables the double-cross-linked acellular matrix hydrogel to better interact with surrounding tissues in the body, providing a good microenvironment for tissue repair and regeneration, and reducing the body's immune rejection reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a storage modulus / loss modulus diagram of the decellularized matrix hydrogel C1 prepared in Example 1;

[0033] Figure 2 This is a storage modulus / loss modulus diagram of the decellularized matrix hydrogel C2 prepared in Example 6;

[0034] Figure 3 Storage modulus / loss modulus diagram of the single cross-linked acellular matrix hydrogel B1 prepared in Example 1;

[0035] Figure 4 Storage modulus / loss modulus diagram of the single cross-linked acellular matrix hydrogel B3 prepared in Example 3;

[0036] Figure 5 Storage modulus / loss modulus diagram of the double-crosslinked acellular matrix hydrogel A4 prepared in Example 4;

[0037] Figure 6 Storage modulus / loss modulus diagram of the double-crosslinked acellular matrix hydrogel A5 prepared in Example 5;

[0038] Figure 7 Shear stress diagrams of the single cross-linked decellular matrix hydrogel B1 prepared in Example 1, the single cross-linked decellular matrix hydrogel B3 prepared in Example 3, the double cross-linked decellular matrix hydrogel A4 prepared in Example 4, and the double cross-linked decellular matrix hydrogel A5 prepared in Example 5;

[0039] Figure 8Degradation rate diagrams of the decellularized matrix hydrogel C1 and the double-crosslinked decellularized matrix hydrogel A1 prepared in Example 1, and the decellularized matrix hydrogel C2 and the double-crosslinked decellularized matrix hydrogel A6 prepared in Example 6;

[0040] Fig. 9 The swelling ratio results of the single cross-linked decellular matrix hydrogel B2 and the double cross-linked decellular matrix hydrogel A2 prepared in Example 2, and the single cross-linked decellular matrix hydrogel B3 and the double cross-linked decellular matrix hydrogel A3 prepared in Example 3;

[0041] Fig.10 Cell viability graphs of the single cross-linked decellular matrix hydrogel B1 and the double cross-linked decellular matrix hydrogel A1 prepared in Example 1, and the single cross-linked decellular matrix hydrogel B3 and the double cross-linked decellular matrix hydrogel A3 prepared in Example 3;

[0042] Fig.11 This is a SEM image of the single cross-linked acellular matrix hydrogel B1 prepared in Example 3;

[0043] Fig.12 This is the SEM image of the decellularized matrix hydrogel C1 prepared in Example 1. DETAILED DESCRIPTION

[0044] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned content of the present invention. In the following examples, reagents and instruments not specifically described are all commercially available, and experimental operations not specifically described are implemented according to the manufacturer's instructions or conventional techniques in the field. Unless otherwise defined, all professional and scientific terms used in the text have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the recorded contents can be applied to the present invention; the endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in this article;

[0045] The present invention provides a method for preparing a double-crosslinked acellular matrix hydrogel, comprising the following steps:

[0046] 1) Proteolysis, cleaning, degreasing, cleaning, and deep decellularization of the pig dermis fragments are performed in sequence to obtain acellular pig dermal matrix;

[0047] 2) freeze-drying the decellularized porcine dermal matrix obtained in step 1) and grinding it into matrix powder, placing the matrix powder in an acidic pepsin solution and stirring to digest, and after digestion is completed, adding an appropriate amount of RO water under ice bath conditions to adjust the solute concentration in the solution to more than 10 mg / mL, and then adding 1 / 10 volume of 10xPBS, and then adjusting the pH of the solution to neutral to obtain a pregel solution;

[0048] 3) placing the pregel solution obtained in step 2) at 37°C to form a gel, and then placing the gel in a tannic acid solution with a concentration of 1-10 mg / mL to crosslink for 10-48 h to obtain a single cross-linked acellular matrix hydrogel;

[0049] 4) sterilizing the single cross-linked acellular matrix hydrogel obtained in step 3), and then physically breaking it into millimeter scale, adding a glucose oxidase solution with a concentration of 1-10u / mL and a ferrous ion solution with a concentration of 0.1-10mg / mL to the broken hydrogel, and stirring evenly to obtain a double cross-linked acellular matrix hydrogel precursor;

[0050] 5) The double-crosslinked acellular matrix hydrogel precursor obtained in step 4) is placed in a glucose concentration of 10-200 mg / dl to form a double-crosslinked acellular matrix hydrogel by in situ spontaneous crosslinking.

[0051] Among them, pig dermis fragments can be prepared in a conventional manner, such as selecting the back skin of a domestic pig with a thicker dermis, removing the epidermis, subcutaneous fascia and connective tissue and most of the adipose tissue through physical and mechanical stratification, rinsing with deionized water to obtain the dermis of the pig skin, and then cutting the dermis into pieces of approximately 0.5 cm×0.5 cm in size.

[0052] In some preferred embodiments of the present invention, the specific steps of step 1) include:

[0053] 1-1): Soak the pig dermis fragments in 0.25% trypsin solution for 8-24 hours, and then wash them with deionized water for at least three times;

[0054] 1-2): After cleaning, soak in 70% ethanol solution for 12-24 hours, and then rinse with deionized water;

[0055] 1-3): After cleaning, soak in 3% hydrogen peroxide solution for 15-30 minutes, rinse with deionized water at least twice, and then soak in decellularization solution for 24 hours, and rinse with deionized water after soaking;

[0056] 1-4): After cleaning, place it in a disinfectant containing 0.1% peracetic acid and 4% ethanol for disinfection for 2-6 hours. After disinfection, soak it in 75% ethanol for 12-24 hours. Finally, rinse it with DPBS and wash it with deionized water to obtain the acellular porcine dermal matrix.

[0057] In some further preferred embodiments of the present invention, the decellularization solution in step 1-3) comprises: 1% Triton X-100, 0.26% EDTA, and 0.69% Tris solution.

[0058] In some preferred embodiments of the present invention, the acidic pepsin solution in step 2) is prepared by dissolving pepsin in a hydrochloric acid solution, the concentration of pepsin in the acidic pepsin solution is 1 mg / mL, the concentration of hydrochloric acid is 0.1 mol / L, the solid-liquid mass ratio of the decellularized porcine dermal matrix powder to the acidic pepsin solution is in the range of 0.04-0.1:1, and the digestion stirring time is 2-4 d; the solid-liquid mass ratio range can also be selected as 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1; the digestion stirring time can also be selected as 2.5d, 3d, 3.5d.

[0059] In some preferred embodiments of the present invention, the concentration of the tannic acid solution in step 3) can also be selected from 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, and 9 mg / mL; the static cross-linking time can also be selected from 15 h, 20 h, 24 h, 30 h, 36 h, 40 h, and 44 h.

[0060] In some preferred embodiments of the present invention, the pre-gel solution in step 3) is allowed to stand for 30-90 minutes to form a gel. The standing time can also be 45 minutes, 55 minutes, 65 minutes, 75 minutes, or 85 minutes.

[0061] In some preferred embodiments of the present invention, the disinfection and sterilization step in step 4) is: soaking the single cross-linked acellular matrix hydrogel in a 75% ethanol solution for 3 times, each time for 6 hours, and then washing and soaking in PBS for 3 times, each time for 6 hours.

[0062] In some preferred embodiments of the present invention, the volume ratio of the single cross-linked decellularized matrix hydrogel, the glucose oxidase solution and the ferrous ion solution in step 4) is 10:1-5:1, and the ferrous ion solution is one or more of ferrous sulfate solution, ferrous chloride solution or ferrous nitrate solution; the volume ratio can also be selected as 10:2:1, 10:3:1, 10:4:1.

[0063] The present invention also provides a double-crosslinked decellularized matrix hydrogel precursor prepared by the above method. The hydrogel precursor can spontaneously crosslink under glucose conditions in vitro to form a double-crosslinked decellularized matrix hydrogel, which can be used for modifying heart valves or other artificial tissue substitutes; it can also be injected into the body and spontaneously crosslink under glucose conditions in vivo to form a double-crosslinked decellularized matrix hydrogel.

[0064] The present invention also provides a double-crosslinked acellular matrix hydrogel prepared by the above method, and the hydrogel can achieve a balance among safety, support and degradation.

[0065] The present invention also provides the use of the double-crosslinked acellular matrix hydrogel as a material for preparing soft tissue repair; specifically, it can be used to prepare heart valve repair materials, blood vessel repair materials, tissue depression defect treatment materials, etc.

[0066] Hereinafter, the double-crosslinked acellular matrix hydrogel and its preparation method and application will be further described through the following specific examples.

[0067] Example 1

[0068] The pig dermis fragments were soaked in a 0.25% trypsin solution for 8 h, washed with deionized water for 3 times, each time for 15 min, then soaked in a 70% ethanol solution for 12 h, washed with deionized water for 15 min, then soaked in a 3% hydrogen peroxide solution for 15 min, washed with deionized water twice, each time for 15 min, and then soaked in a decellularization solution containing 1% Triton X-100, 0.26% EDTA and 0.69% Tris for 24 h, washed with deionized water overnight, and then disinfected in a disinfectant containing 0.1% peracetic acid and 4% ethanol for 2 h. After disinfection, they were soaked in 75% ethanol overnight, and finally rinsed with DPBS and washed in deionized water overnight to obtain a decellularized pig dermal matrix;

[0069] The decellularized porcine dermal matrix prepared in step 1) was frozen at -80°C, freeze-dried in a freeze dryer for 3 days, and then ground into fine powder or particles using a freeze grinder. The ground powders were placed in a 1 mg / mL pepsin acid pepsin solution and stirred for 2-4 days until the contents were completely dissolved (the solution turned white and there were no visible particles, indicating digestion was complete). The digested liquid was taken out, and an appropriate amount of RO water was added to adjust the solute concentration in the solution to 10 mg / mL. Then, 1 / 10 volume of 10xPBS was added, and the pH was adjusted to 6.8 to obtain a pre-gel solution.

[0070] The pre-gel solution prepared in step 2) is placed in an incubator at 37°C and allowed to stand for 30 min-1.5 h to allow the solution to self-assemble into acellular matrix hydrogel C1;

[0071] The gel formed in step 3) was placed in a tannic acid solution (TA solution) with a concentration of 1 mg / mL and allowed to crosslink for 10 h to prepare a single cross-linked acellular matrix hydrogel B1;

[0072] The single cross-linked decellularized matrix hydrogel B1 prepared in step 4) was soaked in 75% ethanol solution for three times, each time for 6 hours, and then washed and soaked in PBS for 3 times, each time for 6 hours. The washed cross-linked decellularized matrix hydrogel was physically broken into millimeter level, and a glucose oxidase solution with a concentration of 10u / mL and a ferrous chloride solution with a concentration of 1mg / mL were added to the broken hydrogel and stirred evenly. Then, a glucose solution with a concentration of 10mg / dl was added, and the mixture was allowed to stand for 1 hour to obtain a double cross-linked decellularized matrix hydrogel A1.

[0073] Example 2

[0074] In this example, the single cross-linked acellular matrix hydrogel B2 and the injectable double cross-linked acellular matrix hydrogel A2 were prepared according to the method of Example 1, with the only difference being that the concentration of the tannic acid solution in step 3) was 5 mg / mL.

[0075] Example 3

[0076] In this example, the single cross-linked decellularized matrix hydrogel B3 and the double cross-linked decellularized matrix hydrogel A3 were prepared according to the method of Example 1, with the only difference being that the concentration of the tannic acid solution in step 3) was 10 mg / mL.

[0077] Example 4

[0078] In this example, the double-crosslinked acellular matrix hydrogel A4 was prepared according to the method of Example 1, with the only difference being that the concentration of the ferrous chloride solution in step 4) was 0.1 mg / mL.

[0079] Example 5

[0080] In this example, the double-crosslinked acellular matrix hydrogel A5 was prepared according to the method of Example 1, with the only difference being that the concentration of the ferrous chloride solution in step 4) was 10 mg / mL.

[0081] Example 6

[0082] In this example, the method in Example 1 was used to prepare the decellularized matrix hydrogel C2 and the double-crosslinked decellularized matrix hydrogel A6, with the only difference being that the concentration of the solute in the solution in step 2) was 5 mg / mL.

[0083] Test Example 1

[0084] The decellularized matrix hydrogel C1, the single-crosslinked decellularized matrix hydrogel B1, and the double-crosslinked decellularized matrix hydrogel A1 prepared in Example 1, the single-crosslinked decellularized matrix hydrogel B3 prepared in Example 3, the double-crosslinked decellularized matrix hydrogel A4 prepared in Example 4, the double-crosslinked decellularized matrix hydrogel A5 prepared in Example 5, and the decellularized matrix hydrogel C2 prepared in Example 6 were subjected to dynamic frequency scanning analysis;

[0085] Test method: After standing for 1 hour in an environment of 37°C, the samples were placed in a rotational rheometer, and the angular velocity ω was adjusted to a range of 0.1-100 rad / s. The dynamic frequency scanning analysis was performed at a tension of 2%. The storage modulus (G', expressed as ESM) and loss modulus (G'', expressed as LM) of B1, B3, A4, A5, C1 and C2 were measured, and the shear stress (shear stress) of B1, B3, A4 and A5 was measured.

[0086] The experimental results are as follows Figure 1-Figure 7 As shown:

[0087] Depend on Figure 1 and Figure 2 It can be seen that the storage modulus of the decellularized matrix hydrogel C1 prepared in Example 1 is greater than the loss modulus when the angular velocity is lower than 50 rad / s, and the hydrogel mainly exhibits solid-state properties. However, when the angular velocity exceeds 50 rad / s, the loss modulus is greater than the storage modulus, which means that the viscous behavior of the hydrogel gradually dominates, the solid-state properties weaken, and the material tends to exhibit fluid properties. The loss modulus of the decellularized matrix hydrogel C2 prepared in Example 6 is substantially equal to the storage modulus when the angular velocity is lower than 30 rad / s, and the loss modulus is greater than the storage modulus when the angular velocity exceeds 30 rad / s, and its solid-state properties are significantly poor, which further indicates that the uncross-linked hydrogel structure has poor stability and is easily deformed by external forces.

[0088] Depend on Figure 3-Figure 6 It can be seen that the storage modulus of the single-crosslinked decellular matrix hydrogels B1 and B3, and the double-crosslinked decellular matrix hydrogels A4 and A5 are much larger than the loss modulus, and therefore they all exhibit significant solid-state properties, among which the solid-state properties of the double-crosslinked decellular matrix hydrogels A4 and A5 are more obvious. This result indicates that the double-crosslinked decellular matrix hydrogel prepared after the second crosslinking has better mechanical strength and can better resist external deformation and damage.

[0089] Depend on Figure 7It can be seen that the shear stress of the double-crosslinked decellular matrix hydrogels A4 and A5 is greater than the shear stress of the single-crosslinked decellular matrix hydrogel B1. This shows that the mechanical strength of the double-crosslinked decellular matrix hydrogel obtained after the second crosslinking is stronger than that of the single-crosslinked decellular matrix hydrogel, and as the concentration of ferrous ions increases, the mechanical strength of the hydrogel also increases.

[0090] Test Example 2

[0091] The decellularized matrix hydrogel C1 and the double-crosslinked decellularized matrix hydrogel A1 prepared in Example 1, and the decellularized matrix hydrogel C2 and the double-crosslinked decellularized matrix hydrogel A6 prepared in Example 6 were subjected to in vitro degradation tests;

[0092] Experimental methods:

[0093] Experimental group: Hydrogels C1, C2, A1 and A6 were washed and soaked in deionized water for 3 times, and then freeze-dried in a 24-well plate. The original weight (M0) of the freeze-dried hydrogels was measured respectively, and then the freeze-dried hydrogels were placed in a 1 mg / mL collagenase I solution for incubation for 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 days. At each time point, part of the hydrogel was taken out, washed with deionized water, freeze-dried, and the remaining weight (Md) was weighed. Finally, the degradation rate was calculated according to the formula: Degradation rate (%) = (M0-Md) / M0*100%.

[0094] Control group: Hydrogels A1 and A6 were freeze-dried and weighed according to the above method, and then the freeze-dried hydrogels were incubated in PBS solution. The incubation time point and treatment method were the same as those in collagenase I solution. Samples were also taken out at each time point for washing, freeze-drying and weighing, and the degradation rate was calculated.

[0095] The results are as follows Figure 8 Shown by: Figure 8 It can be seen that the degradation time of the double-crosslinked decellularized matrix hydrogel prepared by the present invention is longer than that of the uncrosslinked decellularized matrix hydrogel, which further indicates that the hydrogel prepared by the double-crosslinking mechanism of the present invention has higher stability.

[0096] Test Example 3

[0097] The single cross-linked decellular matrix hydrogel B2 and the double cross-linked decellular matrix hydrogel A2 prepared in Example 2, and the single cross-linked decellular matrix hydrogel B3 and the double cross-linked decellular matrix hydrogel A3 prepared in Example 3 were subjected to a swelling ratio experiment;

[0098] Experimental method: Soak in PBS and place at 37°C for 48 hours. Use filter paper to gently absorb and weigh (Wb). Then freeze-dry and weigh the dry weight (We). Analyze the swelling ratio (SR) of the hydrogel according to the formula: swelling ratio (%) = (Wb-We) / We*100%.

[0099] The results are as follows Fig. 9 Shown by: Fig. 9 It can be seen that the swelling ratio of the single-crosslinked decellularized matrix hydrogel and the double-crosslinked decellularized matrix hydrogel is about 1500-2000, which further indicates that the hydrogels with single crosslinking and double crosslinking have excellent water absorption properties.

[0100] Test Example 4

[0101] The single cross-linked decellular matrix hydrogel B1 and the double cross-linked decellular matrix hydrogel A1 prepared in Example 1, and the single cross-linked decellular matrix hydrogel B3 and the double cross-linked decellular matrix hydrogel A3 prepared in Example 3 were subjected to in vivo cytotoxicity tests;

[0102] The experimental method is as follows: L929 cells were cultured in DMEM medium, and L929 cells in the growth period with good growth were selected, washed with PBS, digested with 0.25% trypsin, suspended in DMEM medium, and 10ul of the suspension was placed on a hemocytometer. Cells were counted under a microscope using a cell counter, and the cell concentration of the medium was adjusted to 5×104 cells / well.

[0103] Add culture media containing B1, B3, A1 and A3 hydrogels, as well as culture media without gel, to a 24-well plate, and then add 1 mL of cell suspension to each well. After inoculation, place the culture plate in a 37°C, 5% CO2 incubator for co-culture, and change the medium every 2 days.

[0104] Take out the co-culture 24-well plate, aspirate the original culture medium, dilute the CCK-8 solution ten times with the culture medium, add 200ul CCK-8 diluent to each well, and take 4 blank wells in the fourth row as blank controls with only CCK-8 diluent added. Incubate at 37°C for about 1.5h, and test the absorbance at 450nm using an enzyme reader. Perform the entire experiment once at 4d and 7d, respectively, and calculate the cell viability according to the following formula.

[0105] Cell viability = (OD1-OD3) / (OD2-OD3)

[0106] Wherein, OD1 is the absorbance value of the cell group containing gel at 450 nm, OD2 is the absorbance value of the blank cell group without gel at 450 nm, and OD3 is the absorbance value of the CCK-8 reagent at 450 nm.

[0107] The results are as follows Fig.10 Shown by: Fig.10 It can be seen that the survival rate of cells cultured in the culture medium containing B1, B3, A1 and A3 hydrogels decreased slightly after 7 days, but was basically above 90%, indicating that the single-crosslinked decellularized matrix hydrogel and double-crosslinked decellularized matrix hydrogel prepared by the method of the present invention have good biocompatibility.

[0108] In summary, it should be noted that the above is only a preferred embodiment of the present invention and does not overly limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, for those skilled in the art, they can still make some simple deductions, replacements, or equivalent replacements for some of the technical features of the technical solutions recorded in the aforementioned embodiments without departing from the concept of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a double-crosslinked acellular matrix hydrogel, characterized in that: The steps include: 1) Proteolysis, cleaning, degreasing, cleaning, and deep decellularization of the pig dermis fragments are performed in sequence to obtain acellular pig dermal matrix; 2) freeze-drying the decellularized porcine dermal matrix obtained in step 1) and grinding it into matrix powder, placing the matrix powder in an acidic pepsin solution and stirring to digest, and after digestion is completed, adding an appropriate amount of RO water under ice bath conditions to adjust the solute concentration in the solution to more than 10 mg / mL, and then adding 1 / 10 volume of 10xPBS, and then adjusting the pH of the solution to neutral to obtain a pregel solution; 3) placing the pregel solution obtained in step 2) at 37°C to form a gel, and then placing the gel in a tannic acid solution with a concentration of 1-10 mg / mL to crosslink for 10-48 h to obtain a single cross-linked acellular matrix hydrogel; 4) sterilizing the single cross-linked acellular matrix hydrogel obtained in step 3), and then physically breaking it into millimeter scale, adding a glucose oxidase solution with a concentration of 1-10u / mL and a ferrous ion solution with a concentration of 0.1-10mg / mL to the broken hydrogel, and stirring evenly to obtain a double cross-linked acellular matrix hydrogel precursor; 5) The double-crosslinked acellular matrix hydrogel precursor obtained in step 4) is placed in situ in a glucose concentration of 10-200 mg / dl to spontaneously crosslink to form a double-crosslinked acellular matrix hydrogel.

2. The method for preparing a double-crosslinked acellular matrix hydrogel according to claim 1, characterized in that: The specific steps of step 1) include: 1-1): Soak the pig dermis fragments in 0.25% trypsin solution for 8-24 hours, and then wash them with deionized water for at least three times; 1-2): After cleaning, soak in 70% ethanol solution for 12-24 hours, and then rinse with deionized water; 1-3): After cleaning, soak in 3% hydrogen peroxide solution for 15-30 minutes, rinse with deionized water at least twice, and then soak in decellularization solution for 24 hours, and rinse with deionized water after soaking; 1-4): After cleaning, place it in a disinfectant containing 0.1% peracetic acid and 4% ethanol for disinfection for 2-6 hours. After disinfection, soak it in 75% ethanol solution for 12-24 hours, and finally rinse it with DPBS and wash it with deionized water to obtain the acellular porcine dermal matrix.

3. The method for preparing a double-crosslinked acellular matrix hydrogel according to claim 2, characterized in that: The decellularization solution in step 1-3) includes: 1% Triton X-100, 0.26% EDTA, and 0.69% Tris solution.

4. The method for preparing a double-crosslinked acellular matrix hydrogel according to claim 1, characterized in that: The acidic pepsin solution in step 2) is prepared by dissolving pepsin in a hydrochloric acid solution. The concentration of pepsin in the acidic pepsin solution is 1 mg / mL, and the concentration of hydrochloric acid is 0.1 mol / L.

5. The method for preparing a double-crosslinked acellular matrix hydrogel according to claim 1, characterized in that: In the step 2), the solid-liquid mass ratio of the decellularized porcine dermal matrix powder to the acidic pepsin solution is in the range of 0.04-0.1:1, and the digestion stirring time is 2-4 d.

6. The method for preparing a double-crosslinked acellular matrix hydrogel according to claim 1, characterized in that: In the step 3), the pre-gel solution is allowed to stand for 30 min to 90 min to form a gel.

7. The method for preparing a double-crosslinked acellular matrix hydrogel according to claim 1, characterized in that: The disinfection and sterilization steps in step 4) are as follows: soaking the single cross-linked acellular matrix hydrogel in a 75% ethanol solution for 3 times, each time for 6 hours, and then washing and soaking in PBS for 3 times, each time for 6 hours.

8. The method for preparing a double-crosslinked acellular matrix hydrogel according to claim 1, characterized in that: The volume ratio of the single cross-linked decellularized matrix hydrogel, the glucose oxidase solution and the ferrous ion solution in step 4) is 10:1-5:1, and the ferrous ion solution is one or more of ferrous sulfate solution, ferrous chloride solution or ferrous nitrate solution.

9. A double cross-linked acellular matrix hydrogel, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 8.

10. An application of the double cross-linked acellular matrix hydrogel as claimed in claim 9, characterized in that: The double-crosslinked acellular matrix hydrogel is used as a material for preparing soft tissue repair.

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