A sterile decellularized matrix warm sensitive gel scaffold and methods of making and using same

Abstract

The application belongs to the technical field of biological tissue engineering materials, and particularly relates to a sterile decellularized matrix warm-sensitive gel support and a preparation method and application thereof. The application obtains a sterile decellularized matrix freeze-dried fiber with high biological activity and large pore diameter through optimization of a preparation process, the sterile decellularized matrix freeze-dried fiber can be rapidly fused with water phase to form a uniform sol solution, in the sol state, various cells or drug compositions with different properties can be freely loaded, the solution is injected into a treatment area in a solution form, and under the temperature stimulation of body temperature, the sol-gel phase transition can occur at the drug application site, and the sterile decellularized matrix warm-sensitive gel support is used for treatment of related diseases. Meanwhile, the preparation method of the sterile decellularized matrix warm-sensitive gel support is simple, convenient and low in production cost, and is helpful to large-scale industrial production, and therefore has good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering materials technology, specifically relating to a sterile decellularized matrix thermosensitive gel scaffold, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Current research on the preparation of drug- and cell-loaded collagen thermosensitive gel matrices primarily employs aseptic processing and aseptic filtration. Aseptic processing from raw material handling to finished product yields high costs, while aseptic filtration can save on these costs. Aseptic filtration technology involves forcing solutes and solvents through a membrane smaller than 0.22 μm under pressure, while preventing bacteria and large molecular solutes from passing through, thus achieving sterilization. However, due to collagen's high viscosity and tendency to form films, a gel layer easily forms on the membrane surface, causing a sharp decrease in membrane flux and low aseptic filtration efficiency, which limits the application of aseptic filtration to some extent.

[0004] Vacuum freeze-drying technology can effectively prevent changes in the physicochemical and biological properties of products, and effectively protect the stability of the active ingredients in many heat-sensitive pharmaceutical and biological products. Secondly, freeze-dried products have a loose morphology and their color remains largely unchanged after drying. Furthermore, the very low moisture content after freeze-drying improves product stability and reduces the chance of contamination, which not only facilitates transportation but also extends shelf life. However, how to rapidly dissolve high-concentration collagen freeze-dried matrix into the aqueous phase is a problem that urgently needs to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a sterile decellularized matrix thermosensitive gel scaffold, its preparation method, and its applications. Specifically, this invention optimizes the preparation process to obtain a highly bioactive and large-pore sterile decellularized matrix lyophilized fiber that can rapidly fuse with an aqueous phase to form a homogeneous sol solution and undergo a sol-gel phase transition under body temperature stimulation, thereby successfully obtaining the aforementioned sterile decellularized matrix thermosensitive gel scaffold. Based on the above research results, this invention is thus completed.

[0006] To achieve the above objectives, the present invention relates to the following technical solutions:

[0007] In a first aspect, the present invention provides a sterile decellularized matrix thermosensitive gel scaffold, which comprises at least sterile decellularized matrix lyophilized fibers dissolved in an aqueous phase.

[0008] The weight percentage of the sterile decellularized matrix freeze-dried fiber is 0.2-2%, further 0.5-1.5%, and even further 1%.

[0009] The decellularized matrix freeze-dried fiber is obtained by enzymatic hydrolysis of non-human mammalian extracellular matrix with 0.5-2% (preferably 1%) citric acid-pepsin-urea solution, sterile filtration, removal of pepsin and urea, neutralization, and freeze-drying.

[0010] In a second aspect, the present invention provides a method for preparing the above-mentioned sterile decellularized matrix thermosensitive gel scaffold, the method comprising: adding an aqueous phase to a sterile decellularized matrix lyophilized fiber and mixing it thoroughly to obtain the scaffold.

[0011] The aqueous phase can be sterile water for injection. In actual production, a sterile syringe can be used to inject the aqueous phase, i.e., sterile water for injection, into the sterile decellularized matrix freeze-dried fiber.

[0012] Furthermore, the preparation method also includes adding at least one of cells, drugs, and non-drug active ingredients.

[0013] A third aspect of the present invention provides the use of the above-described sterile decellularized matrix thermosensitive gel scaffold in at least one of the following:

[0014] (a) Drug release or preparation of drug-releasing products;

[0015] (b) As a tissue cell scaffold or for the preparation of tissue cell scaffolds;

[0016] (c) Tissue repair or preparation of tissue repair products.

[0017] The beneficial technical effects of one or more of the above technical solutions:

[0018] (1) The method provided by the above technical solution involves transferring extracellular matrix from mammals into a 1% citric acid-pepsin-urea solution, and then placing it in a reaction vessel for enzymatic hydrolysis at room temperature with shearing and stirring for 12-48 hours. The introduction of intermittent shearing during the hydrolysis process increases the contact area between the material and pepsin, significantly improving the hydrolysis speed, shortening the hydrolysis time, and saving production costs. Furthermore, the addition of urea to the hydrolysate utilizes the principle that urea can effectively disrupt the secondary structure of proteins, causing them to unfold and expose ionizable structural groups, thereby enhancing their solubility. This reduces viscosity and increases the aseptic filtration throughput / cm³. 2 This reduces the amount of filter membrane used and saves production costs.

[0019] (2) The enzymatic hydrolysate uses 1% citric acid as a solvent, which can not only increase the solubility of collagen, but also neutralize it to neutral. The decellularized matrix gel solution is exactly an isotonic buffer solution, so there is no need to add salt solutes such as phosphate and sodium chloride to the aqueous phase, which simplifies the production steps and saves production costs.

[0020] (3) The above technical solution first uses a sterilized strong acid cation exchange resin to remove urea, and then uses a sterilized molecular sieve to remove pepsin, effectively removing the residue of the adjuvant, so it will not have potential cytotoxicity and has high biosafety.

[0021] (4) The above technical solution connects the stainless steel mesh filter head directly to the filling pipeline. The purpose is to disperse the neutralized liquid into a low-particle-size uniform solution, which ensures the stability of the dry matter content during filling and also increases the solubility of the decellularized matrix freeze-dried fiber after freeze-drying.

[0022] (5) The above technical solution involves gelling the prepared decellularized matrix gel solution at 37°C before freeze-drying to form a large-pore hydrogel, and then pre-freezing and freeze-drying it. This not only preserves its triple helix structure and biological activity, but also forms large-pore decellularized matrix freeze-dried fibers, allowing it to quickly fuse with the aqueous phase into a homogeneous sol solution.

[0023] (6) The thermosensitive gel scaffold prepared by the above technical solution can freely load various cell or drug compositions with different properties when it is in the sol state. After being injected into the area to be treated in the form of a solution, it can undergo a sol-gel phase transition at the drug application site under the stimulation of body temperature. The resulting gel scaffold is conducive to cell growth, so that the gel can maintain the therapeutic agent in the body area that needs this treatment and promote tissue wound repair.

[0024] (7) The temperature-sensitive gel scaffold prepared by the above technical solution has a very low moisture content after freeze-drying, which improves the stability of the product, reduces the chance of contamination, and facilitates transportation and storage. Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 It is a freeze-dried thermosensitive gel scaffold material.

[0027] Figure 2 The states of the enzymatic hydrolysate for each embodiment and comparative example are shown.

[0028] Figure 3 The standard curve for urea content in test example 4.

[0029] Figure 4 The morphology of the thermosensitive gel formed by gelation in a water bath at 37°C for 30 minutes in Example 1 is shown.

[0030] Figure 5 The sample from Example 1 gelled within 30 minutes in a water bath at 37°C and then returned to a sol-like state at 4°C.

[0031] Figure 6 This is a scanning electron microscope image of the freeze-dried thermosensitive gel scaffold material.

[0032] Figure 7 The temperature-sensitive gel prepared in Example 1 was used to coat 96-well cell culture plates. After culturing for 48 hours, cell proliferation was observed under a microscope. Group A was the gel-coated test group, and Group B was the non-gel-coated test group.

[0033] Figure 8 Cell viability was detected after coating a 96-well cell culture plate with the thermosensitive gel prepared in Example 1 and culturing for 48 hours.

[0034] Figure 9 Twelve days after the model group was repaired, it was stained with HE at 100X.

[0035] Figure 10 Twelve days after the model group was repaired, it was stained with HE at 400X.

[0036] Figure 11 Twelve days after gel repair, the gel repair group was stained with hematoxylin and eosin (HE) at 100X.

[0037] Figure 12 Twelve days after gel repair, the gel repair group was stained with hematoxylin and eosin (HE) at 400X. Detailed Implementation

[0038] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0039] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for the purpose of describing specific embodiments and not for limiting the scope of protection of the present invention.

[0040] In a typical embodiment of the present invention, a sterile decellularized matrix thermosensitive gel scaffold is provided, which includes at least sterile decellularized matrix lyophilized fibers dissolved in an aqueous phase.

[0041] In another specific embodiment of the present invention, the weight percentage of the sterile decellularized matrix freeze-dried fiber is 0.2-2%, further 0.5-1.5%, and even further 1%.

[0042] The decellularized matrix freeze-dried fiber is obtained by enzymatic hydrolysis of non-human mammalian extracellular matrix with 0.5-2% (preferably 1%) citric acid-pepsin-urea solution, sterile filtration, removal of pepsin and urea, neutralization, and freeze-drying.

[0043] Specifically, the extracellular matrix of the non-human mammals includes, but is not limited to, pigs, cattle, dogs, sheep, rabbits, and mice; more preferably, (small) pigs and (small) cattle.

[0044] The extracellular matrix includes, but is not limited to, any one or more of the following non-human mammalian peritoneum, tendons, cancellous bone, submucosa of the small intestine, submucosa of the bladder, submucosa of the stomach, dermal matrix, pericardium, meninges, amnion, and organ membranes; more preferably, peritoneum.

[0045] The mass ratio of the extracellular matrix of the non-human mammal to the 0.5-2% citrate-pepsin-urea solution is 0.2-2:100, more preferably 1:100;

[0046] The mass ratio of extracellular matrix to pepsin was 50–10:1; the mass concentration of urea solution was 0.5%–2%.

[0047] The specific method of enzymatic hydrolysis includes: placing the above-mentioned enzymatic hydrolysate in an enzymatic hydrolysis container (such as a reaction vessel) for room temperature shearing and stirring enzymatic hydrolysis; more specifically, the shearing can be intermittent shearing, with shearing parameters of 30s / time, an interval of 2h between each shearing, and a stirring speed of 80-150r / min in the reaction vessel; the enzymatic hydrolysis time is controlled to be 12-48h.

[0048] The specific method for aseptic filtration includes: passing the filter through a 200-mesh → 0.45μm → 0.22μm → 0.1μm filter membrane sequentially to achieve rapid aseptic filtration and obtain a sterile filtrate.

[0049] The filter membrane material is polyethersulfone, polysulfone, or polyvinylidene fluoride; polysulfone is more preferred. The diameter is 47mm-300mm, and the pore size is 0.1μm-0.45μm.

[0050] The specific method for removing pepsin and urea includes: removing urea from the above-mentioned sterile filtered enzymatic hydrolysate using a sterilized strong acidic cation exchange resin (such as styrene-divinylbenzene copolymer resin), and then removing pepsin using a sterilized molecular sieve (such as mesoporous SBA-15) to obtain an acidic decellularized matrix gel solution.

[0051] All of the above-mentioned ion exchange resins or molecular sieves are sterilized by irradiation.

[0052] The neutralization method specifically involves adding sterile alkali solution to the above-mentioned acidic decellularized matrix gel solution to adjust the pH to 6.5-7.5.

[0053] In another specific embodiment of the present invention, the alkaline solution can be a 5-15 mol / L (preferably 10 mol / L) sodium hydroxide solution.

[0054] The freeze-drying process may also include filling and shaping processes. Specifically, the neutralized decellularized matrix gel solution is aseptically filled directly into containers (such as vials) after passing through a 200-mesh filter and placed in a freeze dryer. The freeze dryer temperature is set to 33-38℃ and maintained for 10 min to 1 h to allow the sample solution to gel into a solid state. Then, a pre-freezing process is started at -30℃ for 3 h, followed by the freeze-drying program (pre-freezing: -20℃ for 3.5 h; main drying: 0℃ for 12 h; 10℃ for 2 h; 20℃ for 1 h; 30℃ for 1 h). After freeze-drying, sterile decellularized matrix freeze-dried fibers with large pore size (pore size of 20-100 μm) are obtained.

[0055] It should be noted that the filter head can be a stainless steel mesh filter head, which is directly connected to the filling pipeline, thereby dispersing the neutralized liquid into a low-particle-size uniform solution, ensuring the stability of the dry matter content during filling, and also increasing the solubility of the decellularized matrix freeze-dried fibers after freeze-drying.

[0056] The decellularized matrix freeze-dried fibers obtained by the above method are sterile, with large pore size, low residue, and high solubility.

[0057] Meanwhile, in this invention, the aqueous phase can be sterile water for injection.

[0058] In another specific embodiment of the present invention, the sterile decellularized matrix thermosensitive gel scaffold material obtained above is a thermosensitive hydrogel with a sol-gel phase transition critical point of 33-38°C, so that it can be injected into the area to be treated in solution form, and a sol-gel phase transition can occur at the drug application site under the stimulation of body temperature.

[0059] Therefore, the aforementioned sterile decellularized matrix thermosensitive gel scaffold can also load cells and / or drugs, wherein the cells can be traditional somatic cells, immune cells, and various stem cells, such as hepatocytes, pancreatic islet cells, chondrocytes, dendritic cells, cytokine-induced killer cells, lymphokine-activated killer cells, in vitro processed bone marrow or hematopoietic stem cells, and in vitro processed tumor cells. The in vitro cell loading concentration is 1×10⁻⁶. 3 -1×10 5 cells / mL; preferably 1×10 4 -1×10 5 cells / mL; in vivo cell concentration was 1×10⁻⁶. 8 -1×10 10 cells / mL; preferably 1×10 8 -×10 9 cells / mL.

[0060] The drugs include nucleic acids, antibiotics, anti-inflammatory agents, antibodies or antibody fragments thereof, growth factors, cytokines, enzymes, proteins, peptides, fusion proteins, synthetic molecules, organic molecules, metals, carbohydrates or analogs, lipids, hormones, microsomes, their derivatives or variants, and any combination thereof.

[0061] Generally, the cells and drugs mentioned above can be considered as (pharmaceutical) active ingredients, thereby further exerting their role in preventing and / or treating related diseases. Obviously, non-pharmaceutical active ingredients, including suitable excipients such as thickeners (selected from cyclodextrin, HPMC, MC, and HPC, etc.), can also be added to sterile decellularized matrix thermosensitive gel scaffold materials. Of course, the amount of excipients used can be determined based on pharmaceutically acceptable amounts, and no specific limitations are required here.

[0062] In another specific embodiment of the present invention, a method for preparing the above-mentioned sterile decellularized matrix thermosensitive gel scaffold is provided, the method comprising: adding an aqueous phase to a sterile decellularized matrix lyophilized fiber, and mixing well to obtain the scaffold.

[0063] The aqueous phase can be sterile water for injection. In actual production, a sterile syringe can be used to inject the aqueous phase, i.e., sterile water for injection, into the sterile decellularized matrix freeze-dried fiber.

[0064] In another specific embodiment of the present invention, the preparation method further includes adding at least one of cells, drugs, and non-drug active ingredients thereto.

[0065] The cells, drugs, and non-drug active ingredients have already been clearly described in the first aspect above, and will not be repeated here.

[0066] In another specific embodiment of the present invention, the above-mentioned sterile decellularized matrix thermosensitive gel scaffold is provided for use in at least one of the following:

[0067] (a) Drug release or preparation of drug-releasing products;

[0068] (b) As a tissue cell scaffold or for the preparation of tissue cell scaffolds;

[0069] (c) Tissue repair or preparation of tissue repair products.

[0070] Clearly, the above products can be classified as (external) medical products.

[0071] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0072] Example 1

[0073] For the preparation of decellularized matrix lyophilized fibers:

[0074] 10g of bovine peritoneal extracellular matrix, 333mg of pepsin and 10g of urea were added to 1000g of 1% citric acid solution and enzymatically hydrolyzed by room temperature shearing and stirring in a reactor for 36h to obtain the enzymatic hydrolysate. The shearing parameters were 30s / cycle, with an interval of 2h, and the stirring speed of the reactor was 150r / min.

[0075] The enzymatic hydrolysate needs to be filtered rapidly and aseptically through a polysulfone filter membrane in sequence: 200 mesh → 0.45 μm → 0.22 μm → 0.1 μm, to obtain a sterile filtrate. The polysulfone filter membrane has a diameter of 47 mm.

[0076] The sterile filtrate was first passed through an irradiated, strongly acidic cation exchange resin (with a styrene-divinylbenzene copolymer as the backbone, a water content of 42-48%, and a wet apparent density of 0.73-0.88 g / mL) to remove urea, and then through an irradiated, mesoporous SBA-15 molecular sieve to remove pepsin, resulting in an acidic decellularized matrix gel solution.

[0077] Add 14.2 mL of 10 mol / L sterile sodium hydroxide solution to the acidic decellularized matrix gel solution, stir rapidly and adjust the pH to 6.5-7.5 to obtain the decellularized matrix gel solution.

[0078] The decellularized matrix gel solution was filtered through a 200-mesh filter and aseptically filled directly into vials. The vials were then placed in a freeze dryer, and the freeze dryer temperature was set to 37℃ and maintained for 30 minutes to obtain a gelled solid product of the decellularized matrix gel solution. After pre-freezing at -30℃ for 3 hours, the freeze-drying program was started (pre-freezing: -20℃ 3.5 hours; main drying: 0℃ 12 hours; 10℃ 2 hours; 20℃ 1 hour; 30℃ 1 hour). After the program was completed, sterile decellularized matrix freeze-dried fibers with large pore sizes of 20–100 μm were obtained.

[0079] For the preparation of temperature-sensitive gel scaffolds:

[0080] According to the previously aseptically filled volume, inject the corresponding volume of sterile water for injection into the large-pore sterile decellularized matrix lyophilized fiber using a sterile syringe. After rapid mixing, an isotonic thermosensitive gel scaffold is obtained.

[0081] Comparative Example 1

[0082] Used for the preparation of decellularized matrix lyophilized fibers and for the preparation of temperature-sensitive gel scaffolds:

[0083] Compared to Example 1, the only difference is that urea is not added.

[0084] Comparative Example 2

[0085] Used for the preparation of decellularized matrix lyophilized fibers and for the preparation of temperature-sensitive gel scaffolds:

[0086] Compared to Example 1, the only difference is that shearing is not performed in the reactor.

[0087] Comparative Example 3

[0088] For the preparation of decellularized matrix lyophilized fibers:

[0089] 10g of bovine peritoneal extracellular matrix, 333mg of pepsin and 10g of urea were added to 1000g of 0.01mol / L hydrochloric acid and subjected to room temperature shearing and stirring enzymatic hydrolysis in a reaction vessel for 36h to obtain the enzymatic hydrolysate. The shearing parameters were 30s / cycle, with an interval of 2h, and the stirring speed of the reaction vessel was 150r / min.

[0090] The enzymatic hydrolysate needs to be filtered rapidly and aseptically through a polysulfone filter membrane in sequence: 200 mesh → 0.45 μm → 0.22 μm → 0.1 μm, to obtain a sterile filtrate. The polysulfone filter membrane has a diameter of 47 mm.

[0091] The sterile filtrate was first passed through an irradiated, sterilized, strongly acidic cation exchange resin (with a styrene-divinylbenzene copolymer as the backbone, a water content of 42-48%, and a wet apparent density of 0.73-0.88 g / mL) to remove urea, and then through an irradiated, sterilized, mesoporous SBA-15 molecular sieve to remove pepsin, resulting in an acidic decellularized matrix gel solution.

[0092] Add 1 mL of 10 mol / L sterile sodium hydroxide solution to the acidic decellularized matrix gel solution, stir rapidly and adjust the pH to 6.5-7.5 to obtain the decellularized matrix gel solution.

[0093] The decellularized matrix gel solution was filtered through a 200-mesh filter and then aseptically filled into vials. The vials were then placed in a freeze dryer, and the freeze dryer temperature was set to 37°C and maintained for 30 minutes to obtain a gelled solid product of the decellularized matrix gel solution. After pre-freezing at -30°C for 3 hours, the freeze-drying program was started. After the program was completed, sterile decellularized matrix freeze-dried fibers with large pore size were obtained.

[0094] For the preparation of temperature-sensitive gel scaffolds:

[0095] According to the previously aseptically filled volume, inject the corresponding volume of sterile water for injection into the large-pore sterile decellularized matrix freeze-dried fiber using a sterile syringe, and quickly mix to obtain a gel scaffold.

[0096] Comparative Example 4

[0097] Used for the preparation of decellularized matrix lyophilized fibers and for the preparation of temperature-sensitive gel scaffolds:

[0098] Compared with Example 1, the only difference is that the sterile filtrate is not subjected to molecular sieve purification.

[0099] Test Example 1: Observation of the state of the enzyme hydrolysate

[0100] By visually observing the appearance of the enzymatic hydrolysate and comparing the amount of visible solids in each group of hydrolysates, the enzymatic hydrolysis status of each group can be determined. (Table 1) Figure 2 )

[0101] Table 1. Appearance of each group of enzyme hydrolysates

[0102] Example 1 No visible solids Comparative Example 1 A small amount of visible solid matter was found. Comparative Example 2 There was a small amount of visible solid matter, more than in Comparative Example 1. Comparative Example 3 There was a small amount of visible solid matter, slightly less than in Comparative Example 1. Comparative Example 4 No visible solids

[0103] As shown in Table 1, Example 1 and Comparative Example 4 exhibited the best enzymatic hydrolysis results. All groups used the same bovine peritoneal extracellular matrix and pepsin, and the hydrolysis time in the reaction vessel was consistent. The differences in hydrolysis were due to variations in key steps of the preparation methods. The differences between Comparative Examples 1, 2, 3, and 4 and Example 1 were respectively: no urea was used; no shearing hydrolysis was performed; hydrochloric acid solution was used instead of citric acid solution; and molecular sieve purification was not performed. Analysis of the data reveals the following: Comparing Example 1, Comparative Example 4, and Comparative Example 1 shows that adding urea during the preparation of sterile decellularized matrix thermosensitive gel can improve the efficiency of raw material hydrolysis; comparing Example 1 and Comparative Example 2 shows that whether or not the raw material is sheared during the hydrolysis of sterile decellularized matrix thermosensitive gel has a significant impact on the hydrolysis results; comparing Example 1 and Comparative Example 3 shows that the hydrolysis reaction environment provided by 1% citric acid solution is more suitable than that provided by hydrochloric acid solution during the preparation of sterile decellularized matrix thermosensitive gel.

[0104] Test Example 2: Filtration Flux Measurement

[0105] By comparing the time and filtration flux consumed by each group to achieve rapid aseptic filtration by passing through polysulfone membranes of 200 mesh → 0.45μm → 0.22μm → 0.1μm in the same order, the enzymatic hydrolysis status of each group can be determined. The polysulfone membrane diameter is 47mm.

[0106] Table 2. Time and filtration flux consumed for each group of sterile filtration.

[0107] Example 1 93 960 Comparative Example 1 144 796 Comparative Example 2 196 477 Comparative Example 3 129 813 Comparative Example 4 102 955

[0108] As can be seen from the results in Table 2, Example 1 had the highest filtration throughput and the shortest time. All groups used the same bovine peritoneal extracellular matrix and pepsin, and the enzymatic hydrolysis time in the reaction vessel was consistent. The reason for the difference in enzymatic hydrolysis was the difference in the key steps of the preparation method. The differences between Comparative Examples 1, 2, 3, and 4 and Example 1 are respectively: no urea was used, no shearing enzymatic hydrolysis was performed, hydrochloric acid solution was used instead of citric acid solution, and no impurity removal was performed using molecular sieves. Analysis of the above data reveals the following: Comparison of Example 1, Comparative Example 4, and Comparative Example 1 shows that adding urea during the preparation of sterile decellularized matrix thermosensitive gel can improve the efficiency of raw material enzymatic hydrolysis, thereby increasing the volume of hydrolysate that can pass through the filter membrane. Comparison of Example 1 and Comparative Example 2 shows that whether or not the raw material is sheared during the enzymatic hydrolysis of sterile decellularized matrix thermosensitive gel has a significant impact on the hydrolysis process. Shearing helps reduce the occurrence of visible solidification (incompletely hydrolyzed raw material) clogging the filter membrane, thus increasing the filtration throughput. Comparison of Example 1 and Comparative Example 3 shows that the enzymatic reaction environment provided by 1% citric acid solution during the preparation of sterile decellularized matrix thermosensitive gel is more suitable than that provided by hydrochloric acid solution, thereby increasing the volume of hydrolysate that can pass through the filter membrane.

[0109] Test Example 3: Collagen Content Determination

[0110] The collagen content of the enzymatic hydrolysates in the examples and comparative examples was determined according to Appendix A of YY / T 1511-2017 Collagen Sponge Protein Content Determination by Kjeldahl Method after aseptic filtration. Sample digestion: Approximately 1g of the decellularized matrix gel solution sample before lyophilization (equivalent to approximately 1.0mg–2.0mg of nitrogen-containing collagen sponge) was accurately weighed and labeled m1. It was placed in a digestion tube, 0.3g of digestive agent was added, and 2.0mL of concentrated sulfuric acid was added. The tube was placed on an electric heating digestion stove and digested in a fume hood until clear and blue-green, continuing digestion for 60 minutes. A blank digestion control was performed simultaneously. Digestion of non-protein nitrogen: Approximately 8g of the decellularized matrix gel solution sample before lyophilization was accurately weighed and labeled m2. It was soaked in 8mL of water for 30 minutes and then filtered. Take 2 mL of the solution, add 14 mL of water, 2 mL of 10% sodium tungstate solution, and 2 mL of sulfuric acid solution (1.86→100), shake well, let stand for 30 min, filter, accurately measure 5 mL of the filtrate, place it in a digestion tube, and start digestion from the time the digesting agent was added during the sample digestion process.

[0111] The determination method is as follows: Take 10 mL of 2% boric acid absorption solution and place it in a 150 mL Erlenmeyer flask. Immerse the end of the condenser tube of the nitrogen analyzer in the boric acid absorption solution. Transfer the digested sample (m1) into the nitrogen analyzer tube, wash the digestion tube 3-4 times with a small amount of distilled water, transfer the washing solution into the nitrogen analyzer tube, add 10 mL of 50% sodium hydroxide, and then distill. When the total volume of the receiving liquid is about 35 mL-50 mL, remove the end of the condenser tube from the liquid surface, allow the steam to continue rinsing for about 1 minute, rinse the end of the condenser tube with a small amount of distilled water, and then stop distillation. Titrate the receiving liquid with 0.005 mol / L sulfuric acid titrant until the solution changes from blue-green to gray-purple, and record the volume of sulfuric acid titrant consumed, V1. Transfer the digested sample (m2) into the nitrogen analyzer tube, repeat the above distillation and titration steps, and record the volume of sulfuric acid titrant consumed, V2. Transfer the blank digestion control to a nitrogen determination tube, repeat the distillation and titration steps above, and record the volume of sulfuric acid titrant consumed, V0. Correct the result using a blank test. Calculate the total nitrogen content in the sample using the following formula:

[0112]

[0113] Note: In the formula, W1 represents the total nitrogen content of the sample, %;

[0114] V1 represents the volume of sulfuric acid titrant consumed in titrating the sample (m1), in milliliters (mL);

[0115] V0 represents the volume of sulfuric acid titrant consumed in the blank, in milliliters (mL);

[0116] c represents the concentration of the sulfuric acid titrant, expressed in moles per liter (mol / L).

[0117] m1 represents the sample mass, in milligrams (mg);

[0118] m0 represents loss on drying, %.

[0119] The non-protein nitrogen content in the sample is calculated using the following formula:

[0120]

[0121] Note: In the formula, W2 represents the non-protein nitrogen content of the sample, %;

[0122] V2 represents the volume of sulfuric acid titrant consumed in titrating the sample (m2), in milliliters (mL);

[0123] V0 represents the volume of sulfuric acid titrant consumed in the blank, in milliliters (mL);

[0124] c represents the concentration of the sulfuric acid titrant, expressed in moles per liter (mol / L).

[0125] m2 represents the sample mass, expressed in milligrams (mg).

[0126] m0 represents loss on drying, %.

[0127] The protein content in the sample is calculated using the following formula:

[0128] W = (W1 - W2) × F × 100

[0129] Note: In the formula, W represents the protein content of the sample, %;

[0130] F represents 5.55, the conversion factor.

[0131] Table 3 Collagen content of each group

[0132]

[0133]

[0134] As shown in Table 3, the protein content of Examples 1 and Comparative Example 4 was higher. The same bovine peritoneal extracellular matrix was used in all groups, and the difference in protein content was due to differences in key steps of the preparation method. The differences between Comparative Examples 1, 2, 3, and 4 and Example 1 were respectively: no urea was used, no shearing enzymatic hydrolysis was performed, hydrochloric acid solution was used instead of citric acid solution, and no molecular sieve removal was performed. Analysis of the above data reveals that: comparing Example 1 and Comparative Example 1 shows that adding urea during the preparation of sterile decellularized matrix thermosensitive gel can improve enzymatic hydrolysis efficiency, increase the effective filtration capacity of the membrane, and thus increase the collagen content in the decellularized matrix gel solution; comparing Example 1 and Comparative Example 2 shows that whether or not the raw material is sheared during the enzymatic hydrolysis of the sterile decellularized matrix thermosensitive gel has a significant impact on the hydrolysis results. That is, if the hydrolysis time is the same, the amount of matrix dissolved in Example 1 is significantly higher than that in Comparative Example 2. Compared to Comparative Example 2, the effective filtration capacity of the membrane in Example 1 was significantly higher than that in Comparative Example 2, which in turn affected the collagen content in the decellularized matrix gel solution. By comparing Example 1 and Comparative Example 3, it can be seen that the enzymatic reaction environment provided by 1% citric acid solution during the preparation of sterile decellularized matrix thermosensitive gel is more conducive to shortening the enzymatic hydrolysis time and increasing the effective filtration area of ​​the membrane than that provided by hydrochloric acid solution: because for the same enzymatic hydrolysis time, the enzymatic hydrolysis capacity of citric acid-enzymatic hydrolysate is stronger than that of hydrochloric acid-enzymatic hydrolysate. The protein content of Comparative Example 4 was not much different from that of Example 1: because Comparative Example 4 did not remove pepsin and urea, and due to the large solvent volume, it had little effect on the protein content in the decellularized matrix gel before freeze-drying.

[0135] Test Example 4: Urea Residue

[0136] The urea residue was determined according to GB / T 36859-2018, "Determination of Urea Content in Feed". Samples were prepared according to GB / T 20195, pulverized through a 0.45 mm sieve, mixed thoroughly, and stored in a sealed container. Sample solution preparation: Two parallel samples were prepared. Approximately 1 g of sample (accurate to 0.0001 g) was weighed and placed in a 100 mL volumetric flask. 1 g of activated carbon was added, followed by approximately 70 mL of water. The mixture was shaken well and allowed to stand for 10 min. Then, 5 mL of zinc acetate solution and 5 mL of potassium ferrocyanide solution were added separately, and the mixture was shaken and extracted for 30 min. The solution was diluted to the mark with water, shaken well, and allowed to stand for 10 min. The solution was filtered through medium-speed filter paper, and the filtrate was collected. A reagent blank for the sample extraction solution was prepared simultaneously. Standard curve preparation: Accurately pipette 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, 2.0 mL, and 5.0 mL of 1.0 mg / mL standard working solution (containing 0 mg, 0.20 mg, 0.40 mg, 0.60 mg, 0.80 mg, 1.00 mg, 2.00 mg, and 5.00 mg urea respectively) into 25 mL colorimetric tubes. Accurately add 5 mL of phosphate buffer, immediately add 5.0 mL of p-dimethylaminobenzaldehyde (DMAB) solution, dilute to the mark with water, shake well, and let stand for 20 min. Using a 30 mm path length cuvette, with the 0 mL standard working solution (reagent blank) as a reference, measure the absorbance at a wavelength of 420 nm. Plot a standard curve with urea content as the x-axis and absorbance as the y-axis.

[0137] Sample determination: Accurately pipette 5-10 mL of the sample solution into a 25 mL colorimetric tube. Add phosphate buffer and DMAB reagent solution along with the standard working solution for color development. After 20 min, using the reagent blank as a reference, perform colorimetric determination on the reagent blank of the sample solution and the sample extract solution. Measure the absorbance of the sample and the reagent blank, find the urea content on the standard curve, and calculate the urea content of the sample. The urea content w in the sample is expressed as a mass fraction (%), calculated using the formula:

[0138]

[0139] Note: In the formula, w represents the urea content in the sample, %;

[0140] m1 represents the urea content of the sample obtained from the standard curve, in milligrams (mg);

[0141] m2 represents the urea content of the sample extraction solution reagent blank obtained from the standard curve, in milligrams (mg).

[0142] m represents the mass of the sample weighed, in grams (g);

[0143] V represents the final volume of the sample extract, in milliliters (mL);

[0144] V1 represents the volume of sample extract taken during the determination, in milliliters (mL).

[0145] Table 4. Urea residue levels in each group

[0146] Example 1 Not detected Comparative Example 1 Not detected Comparative Example 2 Not detected Comparative Example 3 Not detected Comparative Example 4 1.5％

[0147] As shown in Table 4, except for Comparative Example 1 which did not use urea and Comparative Example 4 which did not undergo molecular sieve removal, the residual amount of urea in each group was not detected. This indicates that urea can be basically removed after irradiation sterilization and molecular sieve removal with specific strong acid cation exchange resin.

[0148] Analysis of the above data reveals that, based on Tables 2 and 3, the addition of urea can increase the filtration flux, indicating that the presence of urea promotes the enzymatic hydrolysis reaction, reduces visible solids (incompletely hydrolyzed raw materials), and thus increases the collagen content. Although the addition of urea has a positive effect on the reaction, failure to remove impurities from the urea after enzymatic hydrolysis can lead to a high residual urea content in the sample, posing a potential risk.

[0149] Test Example 5: Residual Pepsin

[0150] (1) The pepsin potency in the preparation was determined according to the pepsin potency determination method in Part II of the Pharmacopoeia of the People's Republic of China. Hydrochloric acid solution: Take 65 mL of 1 mol / L hydrochloric acid solution and add water to 1000 mL. Test solution: Take an appropriate amount of this product, accurately weigh it, dissolve it in hydrochloric acid solution and quantitatively dilute it to prepare a solution containing about 0.2 to 0.4 units per mL. Reference solution: Take an appropriate amount of tyrosine reference standard, accurately weigh it, dissolve it in hydrochloric acid solution and quantitatively dilute it to prepare a solution containing 0.5 mg per mL.

[0151] Assay: Take 6 test tubes. Accurately add 1 mL of reference solution to 3 tubes and 1 mL of test solution to 3 tubes. Incubate in a 37℃±0.5℃ water bath for 5 minutes. Accurately add 5 mL of hemoglobin test solution preheated to 37℃±0.5℃, shake well, and time accurately. React in a 37℃±0.5℃ water bath for 10 minutes. Immediately add 5 mL of 5% trichloroacetic acid solution, shake well, filter, and collect the filtrate. Take another 2 test tubes and accurately add 5 mL of hemoglobin test solution to each. Incubate in a 37℃±0.5℃ water bath for 10 minutes. Accurately add 5 mL of 5% trichloroacetic acid solution, adding 1 mL of test solution to one tube and 1 mL of hydrochloric acid solution to the other. Shake well, filter, and collect the filtrate. Use these as blank controls for the test sample and reference sample, respectively. Measure the absorbance at 275 nm and calculate the average value. and Calculate using the following formula.

[0152]

[0153] Note: in the formula The average absorbance of the reference standard;

[0154] The average absorbance of the test sample;

[0155] W s The amount of tyrosine contained in 1 mL of the reference solution, in μg;

[0156] W represents the sample size of the test sample, in grams;

[0157] n is the dilution factor of the test sample.

[0158] Under the above conditions, the amount of enzyme that can catalyze the hydrolysis of hemoglobin to produce 1 μmol of tyrosine per minute is defined as one unit of protease activity.

[0159] Table 5. Residual pepsin levels in each group

[0160]

[0161]

[0162] As shown in Table 5, except for Comparative Example 4, which had a higher residual pepsin level due to the lack of molecular sieve purification, all other groups showed no detectable residues. This indicates that pepsin can be largely removed after irradiation sterilization with specific casein molecular sieve packing. Analyzing the data further, Tables 2 and 3 show that Comparative Example 2, due to the lack of shearing, had a significant amount of pepsin failing to penetrate the raw material for enzymatic hydrolysis, resulting in a higher visible solid content and lower filtration throughput of the hydrolysate. However, even so, a large amount of pepsin was largely removed after irradiation sterilization with specific casein molecular sieve packing, indicating that residual pepsin can be controlled through filtration with specific casein molecular sieve packing after irradiation sterilization. Tables 3 and 5 also show that some of the protein content in the sample measured in Comparative Example 4 was due to inactivated enzymes formed from unremoved residues.

[0163] Test Example 6: Gel formation of the enzymatic hydrolysate before lyophilization

[0164] Visually observe whether the enzymatic hydrolysates of each group gelled before freeze-drying under the condition of incubation at 37°C for 30 min. (Table 6) Figure 4 , 5 )

[0165] Table 6. Gel formation of each group after incubation at 37℃

[0166] Example 1 yes Comparative Example 1 yes Comparative Example 2 yes Comparative Example 3 no Comparative Example 4 no

[0167] Table 6 shows that all enzymatic hydrolysates before freeze-drying gelled into solids at 37°C for 30 minutes. Analysis of the data reveals that in Comparative Example 4, urea was not removed, preventing gelation. In Comparative Example 3, enzymatic hydrolysis with hydrochloric acid-enzyme solution resulted in a non-isotonic solution with a low salt concentration after neutralization, hindering gelation and requiring improvement. The differences between Comparative Examples 1 and 2 and Example 1 do not affect the gelation conditions of the decellularized matrix gel solution after aseptic filling, nor do they affect the acquisition of large-pore, sterile decellularized matrix freeze-dried fibers after subsequent freeze-drying.

[0168] Test Example 7: Aseptic Test

[0169] According to the direct inoculation method in the Aseptic Test Method 1101 of the Pharmacopoeia of the People's Republic of China, Staphylococcus aureus was selected as the positive control for aseptic testing. Sample preparation: Using the direct inoculation method, each sample was reconstituted with 3 mL of the corresponding sterile culture medium (temperature not exceeding 45℃). Two tubes of each sample were prepared by adding 30 mL of thioglycolate fluid medium and tryptic soy liquid medium. Negative control: 1 mL of sterile sodium chloride-peptone buffer (pH 7.0) was inoculated into 30 mL of thioglycolate fluid medium. Positive control: Another tube containing the test sample was prepared by adding 1 mL of Staphylococcus aureus bacterial suspension to the thioglycolate fluid medium. The tubes were incubated for 14 days, with the thioglycolate fluid medium incubated at 30–35℃, the tryptic soy liquid medium at 20–25℃, and the positive control tube incubated for 5 days.

[0170] Table 7. Results of aseptic experiments for each group

[0171] Example 1 yes Comparative Example 1 yes Comparative Example 2 yes Comparative Example 3 yes Comparative Example 4 yes

[0172] As shown in Table 7, the sterility test results of the sterile decellularized matrix freeze-dried fibers obtained by aseptic filling and freeze-drying in each group were all qualified. Analysis of the above data indicates that the aseptic filling system can guarantee the sterility of the final product, and the differences between Comparative Examples 1, 2, 3, and 4 and Example 1 do not affect the sterility of the final product—the large-pore sterile decellularized matrix freeze-dried fibers.

[0173] Test Example 8: Aperture Size

[0174] The sample prepared in Example 1 was fixed on the sample stage and vacuum gold-plated for 2 min. Then, the sample was placed under a scanning electron microscope and observed under an accelerating voltage of 1 kV. Based on the aperture size, three magnifications of the scanning electron microscope images were selected: one showing the overall view (50×), one showing the aperture distribution morphology (100×), and one showing the aperture details (200×). The aperture size was measured at an appropriate magnification, and 20 apertures were selected. The dimensions were marked in the images.

[0175] from Figure 6 The results show that the pore size of the samples in Example 1 is basically in the range of 20-100 μm. This proves that the prepared sterile decellularized matrix freeze-dried fibers have a large pore size.

[0176] Test Example 9: Cell Proliferation Experiment

[0177] Thermosensitive gel prepared in Example 1 was used to coat 96-well cell culture plates, with uncoated cell culture plates serving as a control. Primary cultured rat uterine basement membrane cells were seeded at a density of approximately 1000 cells / well, with 5 parallel wells. Cell-free DMEM medium was used as a blank control. The plates were incubated at 37°C, 5% CO2 for 48 hours. 10 μL of CCK-8 solution was added to each well, and the plates were incubated at 37°C for 3 hours. Then, 80 μL of the solution was added to a new 96-well plate, and the absorbance was measured at 450 nm using a microplate reader to calculate cell viability.

[0178] from Figure 7 The results showed that, under a microscope, the number and state of cells in the gel-treated group were significantly better than those in the non-gel-treated group. Figure 8 The results showed that the absorbance of cells in the gel-treated group was 1.004±0.002, while that in the gel-free group was 0.757±0.013, indicating a significant statistical difference (P<0.001). This demonstrates that the gel prepared using the process in Example 1 can effectively promote cell proliferation. The experimental results also showed a significant difference in the growth performance of primary cultured rat uterine basement membrane cells between the two groups, indicating that the in vitro cell proliferation performance of the gel-treated group was better than that of the gel-free group. Furthermore, the cell proliferation performance reflects the lack of cytotoxicity and good biocompatibility of this sterile gel.

[0179] Test Example 10: Endometrial Repair Performance Test in Rats

[0180] Female SD rats aged 8-10 weeks (200-250g) were selected and acclimatized for one week before a mechanical injury model was established. Since the rats have bilateral uteruses, one uterus was used as a blank control and the other as the model.

[0181] Modeling group: The right uterus was exposed, and a curettage instrument (No. 1 long spoon) was used to scrape 12 times from the ovary to the uterus. The wound was then sutured to complete the modeling.

[0182] Gel repair group: The right uterus was exposed, and a curettage instrument (No. 1 long spoon) was used to scrape from the ovary to the uterus 12 times. The wound was sutured and 0.2 mL of the gel prepared in Example 1 was injected.

[0183] Twelve days later, the right uterus of rats in the observation model group and the gel repair group was taken. No obvious gel residue was visible in the right uterus of the gel repair group, which supports the biodegradability of the material. Histopathological sections of the damaged uterine area were prepared and stained with hematoxylin and eosin (HE) to observe the microscopic uterine wall repair.

[0184] Results of the model group: The three layers of the uterine wall in the model group were all relatively thin. Figure 7 The diameter is approximately 0.26 mm. Under high magnification (…). Figure 8 The endometrial epithelium is relatively flat (black arrow), with a thickness of approximately 0.146 mm. Some epithelial cells contain cytoplasmic vacuoles, and a small number of neutrophil infiltrations are observed in the lamina propria, myometrium, and adventitia.

[0185] Results of the gel repair group: In Example 1, the histological layer structure of the uterine wall after repair with the thermosensitive gel was intact, with no obvious abnormalities. The endometrial thickness was thicker than that in the model group, approximately 0.275 mm, and the uterine thickness was approximately 0.6 mm, indicating that the gel prepared by this process can effectively repair damaged endometrium.

[0186] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A sterile decellularized matrix thermosensitive gel scaffold, characterized in that, It comprises at least sterile decellularized matrix lyophilized fibers dissolved in an aqueous phase; the weight percentage of the sterile decellularized matrix lyophilized fibers is 0.2-2%; the decellularized matrix lyophilized fibers are obtained by enzymatic hydrolysis of non-human mammalian extracellular matrix with 0.5-2% citric acid-pepsin-urea solution, sterile filtration, removal of pepsin and urea, neutralization, and lyophilization; the mass ratio of the non-human mammalian extracellular matrix to the 0.5-2% citric acid-pepsin-urea solution is 0.2-2:100; the mass ratio of the extracellular matrix to pepsin is 50-10:1; The mass concentration of the urea solution is 0.5%~2%; The specific method of enzymatic hydrolysis includes: placing the above-mentioned 0.5-2% citric acid-pepsin-urea solution in an enzymatic hydrolysis container for room temperature shearing and stirring enzymatic hydrolysis; the shearing is intermittent shearing, with shearing parameters of 30s / time, an interval of 2h between each shearing, and a stirring speed of 80~150r / min; the enzymatic hydrolysis time is controlled to be 12-48h. The specific method for removing pepsin and urea includes: using a sterilized strong acidic cation exchange resin to remove urea from a sterile filtered 0.5-2% citric acid-pepsin-urea solution, and then removing pepsin through a sterilized molecular sieve to obtain an acidic decellularized matrix gel solution. The freeze-drying process also includes filling and shaping. Specifically, the neutralized decellularized matrix gel solution is aseptically filled into a container after passing through a 200-mesh filter and placed in a freeze dryer. The freeze dryer temperature is set to 33-38℃ and maintained for 10 minutes to 1 hour to allow the sample solution to gel into a solid state. Then, a pre-freezing process is started at -30℃ for 3 hours. After freeze-drying, a large-pore size sterile decellularized matrix freeze-dried fiber is obtained. The filter is a stainless steel mesh filter, which is directly connected to the filling pipeline.

2. The sterile decellularized matrix thermosensitive gel scaffold as described in claim 1, characterized in that, The extracellular matrix of the non-human mammal includes pigs, cattle, dogs, sheep, rabbits, or mice.

3. The sterile decellularized matrix thermosensitive gel scaffold as described in claim 2, characterized in that, The extracellular matrix of the non-human mammal is a pig or a cow.

4. The sterile decellularized matrix thermosensitive gel scaffold as described in claim 3, characterized in that, The extracellular matrix of the non-human mammal is a pig or calf.

5. The sterile decellularized matrix thermosensitive gel scaffold as described in claim 1, characterized in that, The extracellular matrix includes any one or more of the following non-human mammalian peritoneum, tendons, cancellous bone, submucosa of the small intestine, submucosa of the bladder, submucosa of the stomach, dermal matrix, pericardium, meninges, and amnion.

6. The sterile decellularized matrix thermosensitive gel scaffold as described in claim 5, characterized in that, The extracellular matrix is ​​the peritoneum.

7. The sterile decellularized matrix thermosensitive gel scaffold as described in claim 1, characterized in that, The mass ratio of the extracellular matrix of the non-human mammal to the 0.5-2% citric acid-pepsin-urea solution is 1:

100.

8. The sterile decellularized matrix thermosensitive gel scaffold as described in claim 1, characterized in that, The specific method for aseptic filtration includes: passing the solution sequentially through 200 mesh → 0.45 μm → 0.22 μm → 0.1 μm filter membranes to achieve aseptic filtration and obtain aseptic filtrate.

9. The sterile decellularized matrix thermosensitive gel scaffold as described in claim 8, characterized in that, The filter membrane material is polyethersulfone, polysulfone, or polyvinylidene fluoride.

10. The sterile decellularized matrix thermosensitive gel scaffold as described in claim 9, characterized in that, The filter membrane is made of polysulfone.

11. The sterile decellularized matrix thermosensitive gel scaffold as described in claim 1, characterized in that, The neutralization method specifically involves adding sterile alkali solution to the above-mentioned acidic decellularized matrix gel solution to adjust the pH to 6.5-7.

5.

12. The sterile decellularized matrix thermosensitive gel scaffold as described in claim 11, characterized in that, The alkaline solution is a 5-15 mol / L sodium hydroxide solution.

13. The sterile decellularized matrix thermosensitive gel scaffold according to any one of claims 1-12, characterized in that, The sterile decellularized matrix thermosensitive gel scaffold also loads cells and drugs.

14. A method for preparing the sterile decellularized matrix thermosensitive gel scaffold according to any one of claims 1-13, characterized in that, The preparation method includes: adding an aqueous phase to a sterile decellularized matrix freeze-dried fiber, and mixing well to obtain the final product.

15. The method for preparing the sterile decellularized matrix thermosensitive gel scaffold as described in claim 14, characterized in that, The aqueous phase is sterile water for injection.

16. The method for preparing the sterile decellularized matrix thermosensitive gel scaffold as described in claim 14, characterized in that, The preparation method further includes adding at least one of cells and drugs.

17. The use of the sterile decellularized matrix thermosensitive gel scaffold according to any one of claims 1-13 in at least one of the following: (a) Preparation of drug-releasing products; (b) Preparation of tissue cell scaffolds; (c) Prepare tissue repair products.

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