Injectable temperature-sensitive pregel solution and preparation method and application thereof

Abstract

The present invention relates to an injectable thermosensitive pregel solution and a preparation method and application thereof. The raw materials for preparing the injectable thermosensitive pregel solution include a decellularized matrix and a thermosensitive material; the injectable thermosensitive pregel solution is prepared by a method comprising the following steps: freezing and crushing the decellularized matrix to obtain a decellularized matrix powder, mixing the decellularized matrix powder with an acidic solution to obtain a mixed solution, and mixing the mixed solution with a thermosensitive material to obtain the injectable thermosensitive pregel solution. The injectable thermosensitive pregel solution has a simple preparation method, can be quickly gelled after being injected into the body, and the formed hydrogel has high safety and strong applicability, can specifically repair damaged tissues and organs of the human body, and induce tissue regeneration and repair.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to an injectable temperature-sensitive pre-gel solution and a preparation method and application thereof. Background Art

[0002] In daily life, tissue damage caused by trauma or disease seriously affects people's normal life. Repair of tissue damage is a common problem in clinical medicine. During the tissue repair process, cells at the damaged site and cells from other parts of the body are guided to gather in the damaged area for cell division, proliferation and reconstruction of the extracellular matrix. When cells perform these tasks, their living environment plays a vital role. Any factors that affect the living environment of cells may affect tissue healing. For a long time, people have been exploring various treatment methods, biomaterial products and drugs to shorten tissue healing time, especially the repair of chronic and difficult-to-heal tissue damage.

[0003] Hydrogel is a three-dimensional network polymer that absorbs water and swells and can retain a large amount of water without dissolving. The stability of the network structure is maintained by physical bonding or chemical cross-linking. Hydrogels are widely used in clinical practice because of their high water content, physical structure of extracellular matrix, and ability to mimic human tissue structure. Injectable hydrogels have attracted widespread attention due to their advantages such as simple operation, minimally invasive drug delivery, and low injection cost. It can not only fill small and irregular wounds, but also be injected into complex and difficult-to-reach treatment sites, and can gradually undergo sol-gel phase transition under physiological conditions, thereby achieving precise treatment. Injectable hydrogels can effectively encapsulate bioactive substances and can be injected into the lesion site in a solution state, and then converted into gel through endogenous stimulation (temperature, pH, redox and enzyme) or external stimulation. Among the many environmental stimulation factors, temperature is relatively easy to control and obtain. Because the temperature in the human body is very stable, it can be used as a parameter. The use of temperature difference to trigger the sol-gel transition of amphiphilic polymers can achieve precise control of the in situ formation of injectable hydrogels. Injectable hydrogels can be used in combination with endoscopy and minimally invasive surgery. They are suitable for three-dimensional wounds with complex shapes and can adhere well to their surfaces. The special advantage of thermosensitive hydrogels is that they can achieve perfect fit at the site of tissue damage, reduce damage to surrounding healthy tissues, have mild gelation conditions, and can be wrapped with other bioactive materials through simple physical mixing methods. However, traditional hydrogel materials cannot accurately fuse with surrounding tissue wounds during clinical use, and invasive implantation is likely to cause secondary damage.

[0004] Extracellular matrix (ECM) hydrogel bioactive scaffold materials have excellent bioactivity, biocompatibility, biodegradability, and minimally invasive and injectable advantages. They are widely used in the fields of tissue damage repair and tissue engineering. Extracellular matrix (ECM) is a complex structure secreted by cells outside the cells, including interstitial matrix and basement membrane, which occupies more than one-third of the body mass. The interstitial matrix is ​​mainly formed by type I and type III collagen, fibronectin, elastin and various proteoglycans, forming a loose collagen fiber network. The basement membrane is composed of type IV collagen, laminin, entactin and heparan sulfate proteoglycans, forming a dense lamellar protein network. These substances form a complex grid, support and connect tissue structures, regulate tissue occurrence and cell physiological activities, and play an important role in cell migration, differentiation and proliferation. The study of the composition and function of ECM in various tissues and the imitation and construction of biological scaffolds with high biomimetic properties have become important areas in the field of tissue engineering. Current bioactive hydrogels are limited in simulating the various biological functions and mechanical properties of ECM. Decellularized matrices are natural scaffolds prepared from tissues or organs by removing cellular components and retaining the three-dimensional structure of tissues or organs and some natural fiber components. The scaffold is bioactive, biocompatible and non-immunogenic. Decellularized hydrogels retain many growth factors and play an important role in promoting tissue repair and reconstruction.

[0005] Endometrial damage has become the main factor leading to endometrial adhesions and infertility, which is generally caused by surgical damage or inflammation, such as curettage, abortion, endometrial inflammation, etc. The normal endometrium can repair and regenerate itself, but when the endometrium is damaged and the self-repair ability is weakened, it will lead to regeneration disorders of epithelial and stromal cells, obstructed angiogenesis, etc. When there is a lack of endometrial coverage, fibrosis, scarring and adhesions may occur on the anterior and posterior walls of the uterus. The main methods for preventing and treating intrauterine adhesions include drug therapy, balloons, sodium hyaluronate gel, etc. Among them, drug therapy can only prevent adhesions caused by mild injuries. Balloons and hyaluronic acid gels have good anti-adhesion effects after surgery, but they cannot play a role in endometrial repair, and cannot improve the implantation environment of fertilized eggs and increase pregnancy rates. There are many studies on stem cell repair therapy, but it has not yet entered clinical practical application. The key to preventing the recurrence of intrauterine adhesions is to rebuild the endometrium with normal functions. It is urgent to develop a product that can both prevent adhesions and promote endometrial repair. Currently, therapeutic agents for endometrial repair include vaginal suppositories, douches, and oral tablets, but their efficacy is limited. The main problem is that these preparations cannot ensure that the therapeutic drug is in full contact with the damaged site and maintain an effective concentration. In addition, the rapid turnover of endometrial mucus causes the therapeutic drug to be rapidly lost in the damaged uterine cavity, making it impossible to maintain an effective therapeutic concentration. For this reason, loading the drug into the gel can prolong the drug's action time at the lesion site, reduce losses during delivery, and improve efficacy.

[0006] CN116763724A discloses a method for preparing a recombinant collagen hydrogel loaded with curcumin and its application in the treatment of intrauterine adhesions. The method mixes curcumin with recombinant collagen, and combines the synergistic effect of a cross-linking agent to increase the retention time of the recombinant collagen in the uterine cavity, significantly improving the therapeutic effect of the recombinant collagen in vivo. However, curcumin has the disadvantages of low solubility, poor stability, and low absorption rate; at the same time, chemical cross-linking agents are used in the gel preparation process, which has potential cytotoxicity; and the gel is solid before injection and cannot adapt to the complex surface of the uterine wall.

[0007] CN115804750A discloses a preparation method and application of a composite thermosensitive recombinant collagen hydrogel. Recombinant human type III collagen, poloxamer 407 and poloxamer 188 are dissolved in water to prepare a composite thermosensitive recombinant collagen hydrogel. The gel can effectively prevent tissue adhesion and promote endometrial recovery. However, the recombinant collagen used in the gel has a faster degradation rate, lower biological activity and poor repair effect compared to collagen derived from acellular matrix.

[0008] In summary, how to develop a thermosensitive hydrogel that is simple to prepare, highly safe, can quickly gel after injection into the body, and can induce tissue regeneration and repair has become one of the urgent problems to be solved in this field. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides an injectable thermosensitive pregel solution and its preparation method and application. The injectable thermosensitive pregel solution has a simple preparation method, can quickly gel after being injected into the body, and the formed hydrogel has high safety and strong applicability, and can specifically repair damaged tissues and organs of the human body and induce tissue regeneration and repair.

[0010] To achieve this object, the present invention adopts the following technical solutions:

[0011] In a first aspect, the present invention provides an injectable thermosensitive pregel solution, wherein the raw materials for preparing the injectable thermosensitive pregel solution include a decellularized matrix and a thermosensitive material;

[0012] The injectable temperature-sensitive pregel solution is prepared by a method comprising the following steps:

[0013] The decellularized matrix is ​​frozen and crushed to obtain decellularized matrix powder, the decellularized matrix powder is mixed with an acidic solution to obtain a mixed solution, and the mixed solution is mixed with a thermosensitive material to obtain the injectable thermosensitive pregel solution.

[0014] The present invention provides an injectable thermosensitive pre-gel solution, which can form gel under specific temperature conditions by adding thermosensitive materials, without the need for additional organic solvents, copolymerizers or gel initiators with potential cytotoxicity, and can gel at human body temperature. The present invention utilizes the dynamic balance between the hydrophilic and hydrophobic groups of the main chain polymer of the thermosensitive material, and remains soluble in the solution at room temperature, but spontaneously forms micelle balls and self-associates at physiological temperature.

[0015] The present invention uses acellular matrix materials to prepare pre-gel solutions. Compared with recombinant collagen and purified collagen, acellular matrix collagen materials retain various bioactive components, are rich in adhesion proteins, polysaccharides (including hyaluronic acid and chondroitin sulfate, etc.), and active factors that support and regulate cell growth and differentiation, and can promote rapid recovery of the endometrium. The product can be completely degraded and absorbed, and with the release of various active factors and regenerative factors, it can achieve the repair and regeneration of damaged tissues.

[0016] The present invention freezes and grinds the decellularized matrix. On the one hand, the decellularized matrix obtained by pretreatment is in a moist state, and is freeze-ground for the convenience of subsequent processing and use; on the other hand, in order to avoid the temperature rising during grinding and the inactivation of the bioactive substances in the decellularized matrix, grinding needs to be performed at a low temperature.

[0017] The preparation method of the injectable thermosensitive pregel solution provided by the present invention is simple and convenient, and can retain the natural ECM components to the greatest extent, while retaining the biological activity to the greatest extent. The biologically active factors are hindered in the relatively compact structure and released at a slow rate. After the decellularized matrix is ​​digested with acid, the acidic groups on the collagen molecular chains react with the acid, and the collagen swells and gels, which is more conducive to the release of the biologically active factors.

[0018] Preferably, the injectable thermosensitive pregel solution comprises a decellularized matrix, a thermosensitive material and an antioxidant.

[0019] Preferably, the source of the decellularized matrix includes animal tissues or organs.

[0020] Preferably, the animal tissue or organ comprises any one of porcine dermis, porcine small intestine, porcine bladder, bovine tendon, bovine peritoneum, porcine peritoneum, bovine pericardium or porcine pericardium, or a combination of at least two thereof.

[0021] Preferably, the method for preparing the decellularized matrix comprises: decellularizing, defatting and inactivating viruses from animal tissues or organs to obtain the decellularized matrix.

[0022] Preferably, the preparation method of the decellularized matrix specifically comprises: removing excess parts of animal tissues or organs, retaining the matrix layer, using 0.1%~0.4% (for example, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35% or 0.4%, etc.) v / v polyacrylic acid solution for shaking and soaking for 20~120 min (for example, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min or 120 min, etc.), washing with PBS solution and water in sequence, using 0.05%~0.1% (0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1%, etc.) w / v pancreatin and 0.05%~0.1% (for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1%, etc.) w / v The mixed solution of sodium dodecyl sulfate is shaken for washing for 1-3 h (e.g., 1 h, 1.5 h, 2 h, 2.5 h or 3 h, etc.), washed with PBS solution and water in sequence, freeze-dried, and soaked in an organic degreasing agent for degreasing for 4-16 h (e.g., 4 h, 6 h, 8 h, 10 h, 12 h, 14 h or 16 h, etc.) to obtain the decellularized matrix.

[0023] Preferably, the organic degreasing agent comprises any one of chloroform, ether or isopropanol, or a combination of at least two of them.

[0024] Preferably, the temperature of the freeze-crushing is -15 to -5°C (for example, -15°C, -14°C, -13°C, -12°C, -11°C, -10°C, -9°C, -8°C, -7°C, -6°C or -5°C, etc.).

[0025] Preferably, the diameter of the decellularized matrix powder is 50-300 μm (e.g., 50 μm, 100 μm, 150 μm, 200 μm, 250 μm or 300 μm, etc.), preferably 50-200 μm (e.g., 50 μm, 75 μm, 100 μm, 125 μm, 150 μm, 175 μm or 200 μm, etc.).

[0026] In the present invention, after grinding the decellularized matrix using a tissue grinder, the obtained powder is sieved, and the size of the sieve used is 75 meshes, so that the decellularized matrix powder with a diameter of less than 200 μm can be obtained.

[0027] Preferably, the acidic solution comprises hydrochloric acid solution and / or acetic acid solution, preferably hydrochloric acid solution.

[0028] Preferably, the concentration of the acidic solution is 0.01-0.1 mol / L (for example, 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L or 0.1 mol / L, etc.).

[0029] The present invention allows an acid solution of a certain concentration to act on the decellularized matrix powder at a certain temperature, and after a certain period of digestion, an injectable thermosensitive pre-gel solution is obtained. The specific concentration of the acid solution ensures that the collagen in the decellularized matrix is ​​prevented from being over-gelated, the specific digestion temperature prevents the collagen from being thermally denatured, and the specific digestion time ensures that the decellularized matrix is ​​successfully digested without being over-gelated. The acid-digested decellularized matrix not only retains a large number of intact three-dimensional ultrastructures, but also retains a variety of bioactive components.

[0030] Preferably, the decellularized matrix powder and the acidic solution are mixed by homogenization, and the speed of the homogenization is 200-500 rpm (eg, 200 rpm, 250 rpm, 300 rpm, 350 rpm, 400 rpm, 450 rpm or 500 rpm, etc.).

[0031] A specific mixing speed is set in the present invention. Since the viscosity of the mixed liquid is relatively high, a too low speed will lead to uneven stirring, while a too high speed will generate bubbles and affect the acid digestion process. The preferred speed is 300 rpm.

[0032] Preferably, the decellularized matrix powder is mixed with the acidic solution for 4 to 12 h (e.g., 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h or 12 h, etc.).

[0033] The present invention sets a specific acid digestion time. If the digestion time is too short, the digestion effect is poor, the solution is not uniform, a small amount of powder is suspended, the viscosity is low, and the reaction is incomplete. If the digestion time is too long, the collagen is over-hydrolyzed and loses its activity. The decellularized matrix after digestion denatures and lumps appear, and it cannot gel at 37°C. The preferred acid digestion time is 6 h.

[0034] Preferably, the temperature of the decellularized matrix powder and the acidic solution when mixed is 25-35°C (e.g., 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C, etc.).

[0035] The present invention sets a specific acid digestion temperature. If the digestion temperature is too low, a better digestion effect cannot be obtained, the viscosity is low, and the reaction is incomplete; if the digestion temperature is too high, collagen denaturation will occur, and gelation cannot be achieved at 37°C. The preferred acid digestion temperature is 32°C.

[0036] Preferably, the thermosensitive material comprises any one or a combination of at least two of chitosan / β-sodium glycerophosphate, poly N-isopropylacrylamide, a block copolymer of polyoxyethylene and polyoxypropylene, gelatin or a copolymer of polylactic acid-hydroxybutyl ester and polyethylene glycol, and the block copolymer of polyoxyethylene and polyoxypropylene comprises poloxamer 407 and / or poloxamer 188.

[0037] Preferably, the temperature-sensitive material is poloxamer 407 and poloxamer 188, and the mass ratio of poloxamer 407 to poloxamer 188 is (5-24):1.

[0038] The specific point values ​​of 5 to 24 mentioned above can be selected from 5, 8, 10, 12, 14, 16, 18, 20, 22 or 24, etc.

[0039] Preferably, the method of mixing with the temperature-sensitive material specifically comprises: immersing the temperature-sensitive material in a solution, and leaving it to stand at 0-8°C (for example, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C, etc.) for 12-24 h (for example, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h or 24 h, etc.), until the temperature-sensitive material to be added is completely dissolved.

[0040] Preferably, the mass percentage of the thermosensitive material in the injectable thermosensitive pregel solution is 15% to 30% (eg, 15%, 17.5%, 20%, 22.5%, 25%, 27.5% or 30%, etc.).

[0041] Preferably, after mixing with the acidic solution, the method further comprises the step of using an alkaline solution to adjust the pH of the solution.

[0042] Preferably, after mixing with the temperature-sensitive material, the method further comprises the step of using a buffer solution to adjust the osmotic pressure of the solution.

[0043] The present invention uses a buffer solution to adjust the osmotic pressure of the solution to the normal human osmotic pressure range. When the pre-gel solution is injected into the human body, it helps the gel to quickly adapt to the human body environment and improve the adaptability of the gel. The pH of the pre-gel solution is stabilized between 6 and 8, which is conducive to the rapid formation of the gel.

[0044] Preferably, the buffer solution comprises any one of PBS buffer solution, Tris-HCl buffer solution or glycine-Tris buffer solution, or a combination of at least two thereof.

[0045] Preferably, the osmotic pressure of the injectable thermosensitive pregel solution is 200-400 mOsmol / kg (e.g., 200 mOsmol / kg, 225 mOsmol / kg, 250 mOsmol / kg, 275 mOsmol / kg, 300 mOsmol / kg, 325 mOsmol / kg, 350 mOsmol / kg, 375 mOsmol / kg or 400 mOsmol / kg, etc.).

[0046] Preferably, after mixing with the acidic solution, the method further comprises the step of mixing with an antioxidant.

[0047] The invention helps to prevent the oxidative denaturation of collagen by adding an antioxidant, maintains the activity of collagen molecules, and makes it difficult for the gel to separate into phases.

[0048] Preferably, the antioxidant comprises any one of brown algae oligosaccharide, carrageenan oligosaccharide, agar oligosaccharide, tea polyphenols, sodium citrate, ascorbic acid, rutin, epicatechin, catechin, mannitol or glycerol, or a combination of at least two thereof.

[0049] Glycerol is a radiation protection agent. By adding an appropriate amount of glycerol to the pre-gel solution provided by the present invention, some important components can be protected from being affected or having a small impact after radiation.

[0050] Preferably, the mass ratio of the antioxidant to the mixed solution is 1:(5-100), more preferably 1:(20-50).

[0051] The specific point values ​​of 5~100 mentioned above can be selected as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 etc.

[0052] The specific point values ​​of 20~50 mentioned above can be selected as 20, 25, 30, 35, 40, 45 or 50, etc.

[0053] In a second aspect, the present invention provides a method for preparing the injectable temperature-sensitive pregel solution as described in the first aspect, the preparation method comprising the following steps:

[0054] (1) Obtaining the decellularized matrix: decellularizing, defatting and inactivating viruses from animal tissues or organs including any one or a combination of at least two of porcine dermis, porcine small intestine, porcine bladder, bovine tendon, bovine peritoneum, porcine peritoneum, bovine pericardium or porcine pericardium to obtain the decellularized matrix;

[0055] The preparation method of the decellularized matrix specifically comprises: removing excess parts of animal tissues or organs, retaining the matrix layer, using 0.1%-0.4% v / v polyacrylic acid solution for shaking and soaking for 20-120 min, washing with PBS solution and water in sequence, using a mixed solution containing 0.05%-0.1% w / v pancreatic enzyme and 0.05%-0.1% w / v sodium dodecyl sulfate for shaking and washing for 1-3 h, washing with PBS solution and water in sequence, freeze-drying, soaking and defatting with an organic degreasing agent for 4-16 h, and obtaining the decellularized matrix;

[0056] (2) Obtaining decellularized matrix powder: freezing and crushing the decellularized matrix described in step (1) at -15 to -5°C to obtain decellularized matrix powder with a diameter of 50 to 300 μm;

[0057] (3) Digestion of decellularized matrix powder: the decellularized matrix powder in step (2) is homogenously mixed with a 0.01-0.1 mol / L acidic solution at a temperature of 25-35° C. and a rotation speed of 200-500 rpm for 4-12 h; the acidic solution includes a hydrochloric acid solution and / or an acetic acid solution;

[0058] (4) Solution pH adjustment: using an alkaline solution to adjust the pH of the solution obtained in step (3) to 6-8;

[0059] (5) Mixing with a temperature-sensitive material: mixing the solution obtained in step (4) with a temperature-sensitive material;

[0060] The thermosensitive material comprises any one or a combination of at least two of chitosan / β-sodium glycerophosphate, poly-N-isopropylacrylamide, a block copolymer of polyoxyethylene and polyoxypropylene, gelatin or a copolymer of polylactic acid-hydroxybutyl ester and polyethylene glycol; the block copolymer of polyoxyethylene and polyoxypropylene comprises poloxamer 407 and / or poloxamer 188; the mass percentage of the thermosensitive material in the injectable thermosensitive pregel solution is 15% to 30%;

[0061] (6) Adjusting the osmotic pressure of the solution: adjusting the osmotic pressure of the solution obtained in step (5) using a buffer solution; the buffer solution comprises any one of a PBS buffer solution, a Tris-HCl buffer solution or a glycine-Tris buffer solution, or a combination of at least two thereof;

[0062] (7) Mixing with an antioxidant: mixing the solution obtained in step (6) with an antioxidant; the antioxidant comprises any one of brown algae oligosaccharides, carrageenan oligosaccharides, agar oligosaccharides, tea polyphenols, sodium citrate, ascorbic acid, rutin, epicatechin, catechin, mannitol or glycerol, or a combination of at least two thereof; the mass ratio of the antioxidant to the mixed solution is 1:(5-100).

[0063] In a third aspect, the present invention provides an injectable thermosensitive gel, wherein the injectable thermosensitive gel is obtained by gelling the injectable thermosensitive pregel solution described in the first aspect.

[0064] The gel prepared by the method provided by the present invention has the characteristics of high wettability and strong applicability. It is a flowable liquid at room temperature before injection, and can quickly gel into a solid at body temperature after being injected into the human body. When injected into a wound defect or cavity at room temperature, it can achieve full coverage and filling of irregular defect positions, and at the same time, it can quickly change from a flowable state to a non-flowable solid under physiological temperature conditions.

[0065] The gel prepared by the method provided by the present invention has the characteristics of high safety and high activity, and also has many ideal properties, including thermosensitivity, injectability, biodegradability, good biocompatibility and continuous release of biological factors on the wound surface, retains adhesion proteins, polysaccharides and active factors that support and regulate cell growth and differentiation, can effectively promote cell aggregation and growth at the material interface, can inhibit scars caused by excessive proliferation and deposition of collagen, make the repaired wound surface smooth and flat, can specifically repair damaged tissues and organs of the human body, and induce tissue regeneration and repair.

[0066] Preferably, the bubbles in the pre-gel solution are removed by centrifugation before gelation, so as to form a densely structured gel after gelation.

[0067] Preferably, the gelation temperature is 25-40°C (for example, 25°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, etc.).

[0068] Preferably, the gelation time is 1-40 min (eg, 1 min, 3 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min or 40 min, etc.).

[0069] The present invention can adjust the gelation temperature and time according to the concentration and volume of the pre-gel solution.

[0070] In a fourth aspect, the present invention provides a use of the injectable thermosensitive pregel solution as described in the first aspect or the injectable thermosensitive gel as described in the third aspect in the preparation of a product for preventing endometrial adhesion and / or promoting endometrial repair.

[0071] The present invention forms gel at physiological temperature by in situ injection into the uterine cavity, and the formed gel has high viscosity, which can avoid outflow after injection, prolong the retention time of the gel in the uterine cavity, and form a protective film on the endometrium, thereby preventing endometrial adhesion. The amount of thermosensitive material added affects the viscosity of the obtained gel. At 37°C, the viscosity of the gel containing 16% thermosensitive material is 28.25 pa·s, the viscosity of the gel containing 20% ​​thermosensitive material is 39.42 pa·s, and the viscosity of the gel containing 25% thermosensitive material is 52.04 pa·s. The gel is prepared using acellular matrix material and contains a variety of bioactive ingredients, which can promote rapid recovery of the endometrium and achieve repair and regeneration of damaged tissues.

[0072] In a fifth aspect, the present invention provides a drug for preventing endometrial adhesions and / or promoting endometrial repair, the drug comprising the injectable temperature-sensitive pre-gel solution described in the first aspect or the injectable temperature-sensitive gel described in the third aspect.

[0073] The drug provided by the present invention, which contains an injectable temperature-sensitive pregel solution, can also be loaded with other drugs that help prevent endometrial adhesion and / or promote endometrial repair. The pregel solution can carry the drug to quickly reach the diseased site and cooperate with the acellular matrix gel to play a therapeutic role.

[0074] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0075] Compared with the prior art, the present invention has the following beneficial effects:

[0076] (1) The present invention provides an injectable thermosensitive pre-gel solution, which is prepared using a decellularized matrix and a thermosensitive material. It remains soluble at room temperature, spontaneously gels at physiological temperature, has good fluidity, can achieve perfect filling when injected into the body, and can solidify in situ within 2 minutes at body temperature;

[0077] (2) The present invention provides a thermosensitive gel obtained by gelling an injectable thermosensitive pregel solution. The gel has a certain viscosity and a certain volume, and can play a role in isolating the wound surface, thereby preventing endometrial adhesion. The bioactive substances contained in the gel can also promote the rapid recovery of the endometrium and achieve the repair and regeneration of damaged tissues.

[0078] (3) The decellularized matrix of the present invention still effectively maintains the natural micro-nano structure of the original tissue or organ after being crushed and digested. This nanofiber structure highly mimics the natural scaffold of the endometrial ECM, providing a support environment for cells and promoting tissue regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 The microstructure of the decellularized matrix after digestion

[0080] Figure 2 is the phase transition result diagram of the pre-gel solution;

[0081] Figure 3 This is the gelation result diagram of the pre-gel solution;

[0082] Figure 4 This is a scan of the left endometrial cells (H&E) of rats in the treatment group;

[0083] Figure 5 This is a scan of the left endometrial cells (H&E) of rats in the model group;

[0084] Figure 6 The graph shows the endometrial thickness and gland number results of rats in the model group and the treatment group. DETAILED DESCRIPTION

[0085] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0086] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0087] Example 1

[0088] This embodiment provides a method for preparing an injectable temperature-sensitive pregel solution, the preparation method comprising the following steps:

[0089] (1) The excess part of the porcine bladder was removed, and the matrix layer was retained. The porcine bladder was soaked in 0.25% v / v polyacrylic acid solution for 70 min, washed with PBS solution and water in turn, washed with a mixed solution containing 0.08% w / v pancreatic enzyme and 0.08% w / v sodium dodecyl sulfate for 2 h, washed with PBS solution and water in turn, freeze-dried, and soaked in ether for degreasing for 10 h to obtain porcine bladder decellularized matrix (UBM);

[0090] (2) Freeze and crush the UBM at -10°C, and sieve to obtain UBM powder with a diameter of 50-200 μm;

[0091] (3) The UBM powder was homogenously mixed with 0.05 mol / L hydrochloric acid solution at 30 °C and 350 rpm for 8 h;

[0092] (4) Use alkaline solution to adjust the pH of the solution to 6-8. The microstructure is as follows: Figure 1 As shown;

[0093] (5) Immerse the thermosensitive material poloxamer 407 in the mixed solution, stir it slightly every 2 hours, and let it stand for 24 hours. The mass percentage of the thermosensitive material in the pre-gel solution is 20%;

[0094] (6) Use PBS buffer solution to adjust the osmotic pressure of the solution to ensure that the osmotic pressure of the final pre-gel solution is in the range of 280~400 mOsmol / kg;

[0095] (7) Adding antioxidant glycerol, wherein the mass ratio of the antioxidant to the mixed solution is 1:25.

[0096] The gelling effect of the pre-gel solution is as follows Figure 2 and Figure 3 As shown, Figure 2 As shown, it is in liquid state at room temperature and in gel state after treatment at 37℃. Figure 3 As shown, the pre-gel solution is loaded into a syringe, and the pre-gel solution can quickly gel in a 37°C environment.

[0097] Example 2

[0098] This embodiment provides a method for preparing an injectable temperature-sensitive pregel solution, the preparation method comprising the following steps:

[0099] (1) Remove the excess part of the porcine small intestine, retain the matrix layer, and use 0.4% v / v polyacrylic acid solution to shake and soak for 20 min, wash with PBS solution and water in turn, shake and wash with a mixed solution containing 0.1% w / v pancreatic enzyme and 0.05% w / v sodium dodecyl sulfate for 3 h, wash with PBS solution and water in turn, freeze-dry, and soak in chloroform for defatting for 4 h to obtain porcine small intestine decellularized matrix (SIS);

[0100] (2) Freeze and crush SIS at -15°C, and sieve to obtain SIS powder with a diameter of 50-200 μm;

[0101] (3) SIS powder was homogenously mixed with 0.1 mol / L acetic acid solution at 35 °C and 200 rpm for 12 h;

[0102] (4) Use alkaline solution to adjust the pH of the solution to 6-8;

[0103] (5) Immerse the thermosensitive materials poloxamer 407 and poloxamer 188 in the mixed solution, stir slightly every 2 hours, and let stand for 24 hours. The mass percentage of the thermosensitive material poloxamer 407 in the pre-gel solution is 20%, and the mass percentage of the thermosensitive material poloxamer 188 is 0.5%;

[0104] (6) Using Tris-HCl buffer solution to adjust the osmotic pressure of the solution to ensure that the osmotic pressure of the final pregel solution is in the range of 280-400 mOsmol / kg;

[0105] (7) Adding antioxidant glycerol, wherein the mass ratio of the antioxidant to the mixed solution is 1:5.

[0106] Example 3

[0107] This embodiment provides a method for preparing an injectable temperature-sensitive pregel solution, the preparation method comprising the following steps:

[0108] (1) Remove the excess part of the porcine bladder, retain the matrix layer, and use 0.1% v / v polyacrylic acid solution to shake and soak for 120 min, wash with PBS solution and water in turn, shake and wash with a mixed solution containing 0.05% w / v pancreatic enzyme and 0.1% w / v sodium dodecyl sulfate for 1 h, wash with PBS solution and water in turn, freeze-dry, and soak in isopropanol for degreasing for 16 h to obtain porcine bladder decellularized matrix (UBM);

[0109] (2) The UBM was frozen and crushed at -5°C, and then sieved to obtain UBM powder with a diameter of 50-200 μm;

[0110] (3) The UBM powder was homogenously mixed with 0.01 mol / L hydrochloric acid solution at 25 °C and 500 rpm for 4 h;

[0111] (4) Use alkaline solution to adjust the pH of the solution to 6-8;

[0112] (5) Immerse the thermosensitive material poly (N-isopropylacrylamide) in the mixed solution, stir it slightly every 2 hours, and let it stand for 24 hours. The mass percentage of the thermosensitive material in the pre-gel solution is 15%;

[0113] (6) Using glycine-Tris buffer solution to adjust the osmotic pressure of the solution to ensure that the osmotic pressure of the final pre-gel solution is in the range of 280-400 mOsmol / kg;

[0114] (7) Adding antioxidant glycerol, wherein the mass ratio of the antioxidant to the mixed solution is 1:100.

[0115] Example 4

[0116] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from that of Embodiment 1 only in that the homogenizing and mixing time in step (3) is 3 h.

[0117] Example 5

[0118] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from the method of embodiment 1 only in that the homogenizing and mixing time in step (3) is 4 h.

[0119] Example 6

[0120] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from the method of embodiment 1 only in that the homogenizing and mixing time in step (3) is 12 h.

[0121] Example 7

[0122] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from the method of embodiment 1 only in that the homogenizing and mixing time in step (3) is 13 h.

[0123] Example 8

[0124] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 1 only in that the temperature of the homogenous mixing in step (3) is 24°C.

[0125] Example 9

[0126] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from the method of embodiment 1 only in that the temperature of the homogenous mixing in step (3) is 25°C.

[0127] Example 10

[0128] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from the method of embodiment 1 only in that the temperature of the homogenous mixing in step (3) is 35°C.

[0129] Embodiment 11

[0130] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 1 only in that the temperature of the homogenous mixing in step (3) is 36°C.

[0131] Example 12

[0132] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 1 only in that the mass percentage of the thermosensitive material in the pre-gel solution in step (5) is 14%.

[0133] Example 13

[0134] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 1 only in that the mass percentage of the thermosensitive material in the pre-gel solution in step (5) is 15%.

[0135] Embodiment 14

[0136] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 1 only in that the mass percentage of the thermosensitive material in the pre-gel solution in step (5) is 30%.

[0137] Embodiment 15

[0138] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 1 only in that the mass percentage of the thermosensitive material in the pre-gel solution in step (5) is 31%.

[0139] Example 16

[0140] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 1 only in that the mass percentage of the thermosensitive material in the pre-gel solution in step (5) is 17%.

[0141] Embodiment 17

[0142] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 1 only in that the mass percentage of the thermosensitive material in the pre-gel solution in step (5) is 18%.

[0143] Embodiment 18

[0144] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 1 only in that the mass percentage of the thermosensitive material in the pre-gel solution in step (5) is 19%.

[0145] Embodiment 19

[0146] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 1 only in that step (5) is: immersing the thermosensitive materials poloxamer 407 and poloxamer 188 in a mixed solution, stirring slightly every 2 hours, and standing for 24 hours. The mass percentage of the thermosensitive material poloxamer 407 in the pre-gel solution is 20%, and the mass percentage of the thermosensitive material poloxamer 188 is 0.5%.

[0147] Embodiment 20

[0148] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 19 only in that the mass percentages of the thermosensitive materials poloxamer 407 and poloxamer 188 in the pre-gel solution in step (5) are 21% and 4%, respectively.

[0149] Embodiment 21

[0150] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 19 only in that the mass percentages of the thermosensitive materials poloxamer 407 and poloxamer 188 in the pre-gel solution in step (5) are 22% and 3%, respectively.

[0151] Embodiment 22

[0152] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 19 only in that the mass percentages of the thermosensitive materials poloxamer 407 and poloxamer 188 in the pre-gel solution in step (5) are 23% and 2%, respectively.

[0153] Embodiment 23

[0154] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 19 only in that the mass percentages of the thermosensitive materials poloxamer 407 and poloxamer 188 in the pre-gel solution in step (5) are 24% and 1%, respectively.

[0155] Embodiment 24

[0156] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 19 only in that the mass percentages of the thermosensitive materials poloxamer 407 and poloxamer 188 in the pre-gel solution in step (5) are 20% and 1%, respectively.

[0157] Embodiment 25

[0158] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 19 only in that the mass percentages of the thermosensitive materials poloxamer 407 and poloxamer 188 in the pre-gel solution in step (5) are 20% and 1.5%, respectively.

[0159] Embodiment 26

[0160] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 19 only in that the mass percentages of the thermosensitive materials poloxamer 407 and poloxamer 188 in the pre-gel solution in step (5) are 20% and 2% respectively.

[0161] Embodiment 27

[0162] This embodiment provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Embodiment 19 only in that the mass percentages of the thermosensitive materials poloxamer 407 and poloxamer 188 in the pre-gel solution in step (5) are 20% and 2.5%, respectively.

[0163] Comparative Example 1

[0164] This comparative example provides a method for preparing an injectable thermosensitive pregel solution, which differs from Example 1 only in that step (3) is to mix the UBM powder with an aqueous solution containing 3 mg / mL pepsin and 0.05 mol / L hydrochloric acid, and homogenize and mix them at a temperature of 30° C. and a rotation speed of 350 rpm for 48 h.

[0165] Comparative Example 2

[0166] This comparative example provides a method for preparing an injectable thermosensitive pre-gel solution, which is different from Example 1 only in that step (5) is omitted, that is, no thermosensitive material is added to the pre-gel solution.

[0167] Test Example 1

[0168] In this test example, a pre-gel solution was prepared by the method of Examples 1-11, and the gelation of the pre-gel solution and the acid digestion result were tested. The results are shown in Table 1.

[0169] Table 1

[0170]

[0171] From Table 1, we can conclude that:

[0172] (1) Comparing Example 1 with Example 4, it can be seen that if the digestion time is shorter than 4 h, the digestion time is too short, the digestion effect is poor, the solution viscosity is low, the reaction is incomplete, and only a suspension of UBM powder is obtained, and the decellularized matrix pregel cannot be expected to be obtained;

[0173] (2) Comparing Example 1 with Example 7, it can be seen that if the digestion time is longer than 12 h, the digestion time is too long, the collagen is over-hydrolyzed and loses its activity, and the final decellularized matrix pregel is agglomerated, indicating that the collagen in the decellularized matrix has been denatured. At the same time, long-term acid immersion may cause the bioactive factors in the decellularized matrix to be inactivated;

[0174] (3) Comparing Example 1 with Example 8, it can be seen that when the digestion temperature is lower than 25°C, the digestion temperature is too low to obtain a better digestion effect, the reaction is incomplete, and the solution viscosity is low; from Examples 1, 5-6, it can be seen that when other conditions are within the set range, when the homogenization mixing time is between 4 h and 8 h, the digestion effect is good and a uniform matrix solution can be obtained;

[0175] (4) By comparing Example 1 with Example 11, it can be seen that when the digestion temperature is higher than 35°C, the collagen in the decellularized matrix will undergo a large amount of denaturation, causing the decellularized matrix solution after digestion to agglomerate and fail to gel. At the same time, the bioactive factors in the decellularized matrix will be destroyed and lose their activity. It can be seen from Examples 1, 9-10 that when other conditions are within the set range, when the temperature of the homogenized mixing is between 25-35°C, the digestion effect is good and a uniform matrix solution can be obtained.

[0176] Test Example 2

[0177] In this test example, the pre-gel solution was prepared by the method provided in Examples 1-3, 12-27 and Comparative Examples 1-2, and the gelation and gelation time of the pre-gel solution were tested. The gelation time of the injectable thermosensitive gel was determined by the vial pouring method. When the vial was turned over, the time required for the sample in the vial to stop flowing was the gelation time of the sample under the corresponding conditions. The results are shown in Table 2.

[0178] Table 2

[0179]

[0180] From Table 2, we can conclude that:

[0181] (1) Comparing Example 1 with Example 12, it can be seen that when the proportion of the thermosensitive material is less than 15%, for example, 14% as provided in Example 12, the proportion of the thermosensitive material in the pre-gel solution is too low and gelation cannot be achieved;

[0182] (2) Comparing Example 1 with Example 15, it can be seen that when the mass percentage of the thermosensitive material in the pre-gel solution exceeds 30%, for example, when it is 31% provided in Example 15, the proportion of the thermosensitive material in the pre-gel solution is too high and gelation can be achieved. However, due to the high concentration, gelation has already occurred at room temperature and subsequent operations cannot be performed.

[0183] (3) Comparing Example 1 with Examples 13-14, 16-18, it can be seen that as the mass percentage of the thermosensitive material in the pre-gel solution gradually decreases, the gelation time gradually increases. That is, the higher the concentration of poloxamer 407, the shorter the gelation time;

[0184] (4) Comparing Example 1 with Example 19 and Examples 24-27, it can be seen that the addition of poloxamer 188 can adjust the gelation time of the gel. As the amount of poloxamer 188 in the thermosensitive material gradually increases, the gelation time gradually increases.

[0185] (5) It can be seen from Examples 20-23 that when the total mass fraction of the added thermosensitive material remains unchanged, the gelation time of the pre-gel solution can be precisely regulated by changing the mass ratio of poloxamer 407 to poloxamer 188, and the gelation time can be precisely controlled within 2 min.

[0186] Test Example 3

[0187] In this test example, the pre-gel solution was prepared using the method provided in Example 1. According to the requirements of GB / T 16886.5-2017 Biological Evaluation of Medical Devices, the in vitro cytotoxicity of the sample was evaluated. The results showed that the sample had no potential toxic effect on L929 cells, indicating that the injectable thermosensitive gel has good biocompatibility. The cytotoxicity results of Example 1 are shown in Table 3, where the negative control group was high-density polyethylene, extracted with MEM culture medium containing 10% fetal bovine serum, the positive control group was zinc diethyldithiocarbamate, extracted with MEM culture medium containing 10% fetal bovine serum, and the blank group only contained MEM culture medium containing 10% fetal bovine serum.

[0188] Table 3

[0189]

[0190] Test Example 4

[0191] This test example performs physiological tests on the pregel solution obtained by the method provided in Example 1. A rat intrauterine adhesion uterine injury model is constructed, and then the temperature-sensitive pregel solution is injected into the injured uterus to observe the repair of the rat endometrium.

[0192] (1) Experimental methods:

[0193] Experimental design: This experiment was divided into a model group and a treatment group. The rat endometrial injury model was established by injecting 95% ethanol into the endometrium, and then the thermosensitive hydrogel was injected into the damaged uterus to observe the repair of the rat endometrium.

[0194] Establishment of animal model: Female SD rats (200-220 g, 8 weeks old) with normal estrus cycle were randomly divided into two groups: model group and treatment group. Rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (0.3 mL / 100 g), and the uterine horns were exposed by incision. The skin and each layer of the abdominal wall were cut in turn; the uterus was separated, and the proximal and distal ends of one side of the uterus were gently clamped with vascular clamps respectively, and the uterus was gently straightened with toothless forceps. Sterile saline gauze was used to protect the surrounding tissues to prevent 95% anhydrous ethanol from overflowing and damaging. The syringe needle entered the uterine cavity and kept it parallel to the longitudinal axis of the uterus. 95% anhydrous ethanol was slowly injected into one end of the uterus to keep the uterine cavity filled. The needle stayed for 4-5 min and was slowly removed; the residual anhydrous ethanol was gently squeezed out, and then rinsed with saline twice, and the vascular clamp was loosened; the other side of the uterus was treated in the same way. To prevent adhesion, the abdominal cavity was rinsed with saline and dried with sterile gauze. The uterus was restored to its normal position and the abdomen was closed layer by layer. After the model was established, the wound was disinfected, and the rat was placed on a warming pad and put into a feeding cage after waking up.

[0195] (2) Experimental steps:

[0196] Based on the established intrauterine adhesion model, the treatment group injected thermosensitive pregel solution into both uteri. In the model group, the intrauterine adhesion model was established by chemical injury, and then 100 μL of normal saline was injected into both uteri.

[0197] Results: One week after surgery, the rats were killed and the uterus was stained with hematoxylin-eosin (H&E) to evaluate the healing effect of the uterus in each group. The tissue morphology, H&E, endometrial thickness and gland number were detected.

[0198] (3) Experimental conclusion:

[0199] like Figure 4 and Figure 5 The results of the experiment showed that compared with the model group, the 7-day treatment group formed a physical space in the animal's uterine cavity, separating the mucosal surface that had not yet been fully epithelialized, and better preventing the occurrence of new adhesions. Figure 6As shown, the thickness of the endometrium and the number of glands in the treatment group were also better than those in the model group, proving that the bioactive substances contained in the gel can promote rapid recovery of the endometrium and achieve repair and regeneration of damaged tissues.

[0200] In summary, the present invention provides an injectable thermosensitive pre-gel solution, which is prepared by a cell-free matrix and a thermosensitive material. The preparation method is simple and the solution can quickly gel after being injected into the body. The formed hydrogel has high safety and strong applicability. It can specifically repair damaged tissues and organs of the human body and induce tissue regeneration and repair. It can be used to prevent endometrial adhesion and / or promote endometrial repair.

[0201] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. An injectable temperature-sensitive pre-gel solution, characterized in that: The raw materials for preparing the injectable thermosensitive pregel solution include a decellularized matrix and a thermosensitive material; The injectable temperature-sensitive pregel solution is prepared by a method comprising the following steps: Freezing and crushing the decellularized matrix to obtain decellularized matrix powder, mixing the decellularized matrix powder with an acidic solution to obtain a mixed solution, and mixing the mixed solution with a temperature-sensitive material to obtain the injectable temperature-sensitive pregel solution; The acidic solution is a hydrochloric acid solution, the diameter of the decellularized matrix powder is 50-300 μm, the concentration of the acidic solution is 0.01-0.1 mol / L, the time for mixing the decellularized matrix powder and the acidic solution is 4-12 h, and the temperature when the decellularized matrix powder and the acidic solution are mixed is 25-35° C.; The mass percentage of the thermosensitive material in the injectable thermosensitive pregel solution is 15% to 30%, the thermosensitive material is poloxamer 407 and poloxamer 188, and the mass ratio of poloxamer 407 to poloxamer 188 is (5 to 24): 1; The osmotic pressure of the injectable thermosensitive pregel solution is 200-400 mOsmol / kg; The decellularized matrix is ​​derived from animal tissues or organs, and the animal tissues or organs are any one of porcine dermis, porcine small intestine, porcine bladder, bovine tendon, bovine peritoneum, porcine peritoneum, bovine pericardium or porcine pericardium, or a combination of at least two thereof.

2. The injectable temperature-sensitive pregel solution according to claim 1, characterized in that: The method for preparing the decellularized matrix comprises: performing decellularization, defatting and virus inactivation on animal tissues or organs to obtain the decellularized matrix.

3. The injectable temperature-sensitive pregel solution according to claim 1, characterized in that: The temperature of the freezing and crushing is -15~-5°C; The method for mixing the decellularized matrix powder and the acidic solution is homogenization, and the rotation speed of the homogenization is 200-500 rpm.

4. The injectable temperature-sensitive pregel solution according to claim 1, characterized in that: After mixing with the acidic solution, the step of adjusting the pH of the solution using an alkaline solution is also included; After the mixture is mixed with the temperature-sensitive material, the step of using a buffer solution to adjust the osmotic pressure of the solution is also included; The buffer solution includes any one of PBS buffer solution, Tris-HCl buffer solution or glycine-Tris buffer solution or a combination of at least two thereof.

5. The injectable temperature-sensitive pregel solution according to claim 1, characterized in that: After the mixing with the acidic solution, the step of mixing with an antioxidant is also included; The antioxidant includes any one of brown algae oligosaccharide, carrageenan oligosaccharide, agar oligosaccharide, tea polyphenols, sodium citrate, ascorbic acid, rutin, epicatechin, catechin, mannitol or glycerol, or a combination of at least two thereof; The mass ratio of the antioxidant to the mixed solution is 1:(5-100).

6. An injectable temperature-sensitive gel, characterized in that: The injectable thermosensitive gel is obtained by gelling the injectable thermosensitive pregel solution according to any one of claims 1 to 5.

7. The injectable thermosensitive gel according to claim 6, characterized in that: The gelation temperature is 25-40° C., and the gelation time is 1-40 min.

8. Use of the injectable thermosensitive pregel solution according to any one of claims 1 to 5 or the injectable thermosensitive gel according to claim 6 or 7 in the preparation of a product for preventing endometrial adhesions and / or promoting endometrial repair.

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