Injectable thermosensitive pre-gel solution, and preparation method therefor and use thereof

Abstract

The present application relates to an injectable thermosensitive pre-gel solution, and a preparation method therefor and a use thereof. Raw materials for preparing the injectable thermosensitive pre-gel solution comprise a decellularized matrix and a thermosensitive material. The injectable thermosensitive pre-gel solution is prepared by a method comprising the following steps: freezing and pulverizing 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 the thermosensitive material to obtain the injectable thermosensitive pre-gel solution. The preparation method for the injectable thermosensitive pre-gel solution is simple; when injected into the body, the injectable thermosensitive pre-gel solution can quickly form a gel; the formed hydrogel has high safety and strong applicability, and can specifically repair damaged tissues and organs in the human body and induce tissue regeneration and repair.

Description

An injectable thermosensitive pregel solution, its preparation method and application Technical Field

[0001] This application relates to the field of biomedicine, and in particular to an injectable thermosensitive pregel solution, its preparation method, and its application. Background Technology

[0002] In daily life, tissue damage caused by trauma or disease severely impacts people's normal lives. Tissue repair is a common problem in clinical medicine. During tissue repair, cells from the injured site and other parts of the body are guided to converge on the damaged area to undergo cell division, proliferation, and extracellular matrix reconstruction. The cell's living environment plays a crucial role in performing these tasks, and any factor affecting the cellular environment can influence tissue healing. For a long time, researchers have been exploring various treatment methods, biomaterials, and drugs to shorten tissue healing time, especially for the repair of chronic, difficult-to-heal tissue injuries.

[0003] Hydrogels are three-dimensional network polymers that absorb and swell while retaining a large amount of water without dissolving. The stability of the network structure is maintained through physical bonding or chemical cross-linking. Due to their high water content, extracellular matrix-like physical structure, and ability to mimic human tissue structure, hydrogels are widely used clinically. Injectable hydrogels have attracted widespread attention due to their ease of use, minimally invasive drug delivery, and low injection cost. They can fill small, irregular wounds and be injected into complex, hard-to-reach treatment sites. Under physiological conditions, they can gradually undergo a sol-gel phase transition, enabling precise treatment. Injectable hydrogels can effectively encapsulate bioactive substances and can be injected into lesions in solution form, subsequently transforming into a gel through endogenous stimuli (temperature, pH, redox, and enzymes) or external stimuli. Among many environmental stimuli, temperature is relatively easy to control and obtain. Because the body's internal temperature is very stable, it can be used as a parameter. By utilizing temperature differences to trigger the sol-gel transition of amphiphilic polymers, precise control of in-situ formation of injectable hydrogels can be achieved. Injectable hydrogels can be used in conjunction with endoscopy and minimally invasive surgery, making them suitable for complex three-dimensional wounds while adhering well to their surfaces. Thermosensitive hydrogels offer the unique advantage of achieving perfect adhesion to damaged tissues, minimizing damage to surrounding healthy tissues. Their gelation process is gentle, and they can encapsulate other bioactive materials through simple physical mixing methods. However, traditional hydrogel materials often fail to fuse accurately with surrounding wound tissues in clinical applications, and invasive implantation can potentially cause secondary damage.

[0004] Extracellular matrix (ECM) hydrogel bioactive scaffold materials possess excellent bioactivity, biocompatibility, biodegradability, and minimally invasive and injectable advantages, making them widely used in tissue damage repair and tissue engineering. The extracellular matrix (ECM) is a complex structure secreted by cells outside the cell, including the interstitial matrix and basement membrane, accounting for more than one-third of the body's mass. The interstitial matrix is ​​mainly composed of 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, nestin, and heparan sulfate proteoglycans, forming a dense, sheet-like protein network. These substances form a complex scaffold that supports and connects tissue structures, regulates tissue development and cellular physiological activities, and plays a crucial role in cell migration, differentiation, and proliferation. Researching the composition and function of the ECM in various tissues, as well as the mimicry and construction of biomimetic scaffolds with high biomimetic properties, has become an important area in tissue engineering. Current bioactive hydrogels are limited in their ability to mimic the various biological functions and mechanical properties of the ECM (extracellular matrix). Decellularized matrix, on the other hand, is a natural scaffold prepared from tissues or organs by removing cellular components while retaining the three-dimensional structure and some natural fibrous components of the tissue or organ. This scaffold possesses bioactivity, biocompatibility, and non-immunogenicity. Decellularized hydrogels retain many growth factors, playing an important role in promoting tissue repair and remodeling.

[0005] Endometrial damage has become a major factor leading to endometrial adhesions and infertility, generally caused by surgical injury or inflammation, such as curettage, abortion, and endometritis. Normal endometrium can repair and regenerate itself, but when the endometrium is damaged, its self-repair ability is weakened, leading to impaired regeneration of epithelial and stromal cells, and obstructed angiogenesis. Without endometrial coverage, fibrosis, scarring, and adhesions can occur on the anterior and posterior walls of the uterus. Methods for preventing and treating intrauterine adhesions mainly include drug therapy, balloon angioplasty, and sodium hyaluronate gel. Drug therapy can only prevent adhesions caused by mild damage. Balloon angioplasty and hyaluronic acid gel have good anti-adhesion effects after surgery, but they cannot repair the endometrium, improve the implantation environment of the fertilized egg, or increase pregnancy rates. Stem cell repair therapy has been extensively researched, but it has not yet entered clinical application. The key to preventing recurrence of intrauterine adhesions is to rebuild a normally functioning endometrium; therefore, there is an urgent need to develop a product that can both prevent adhesions and promote endometrial repair. Currently, treatments for endometrial repair include vaginal suppositories, douches, and oral tablets, but their efficacy is limited. The main problem is that these formulations cannot ensure sufficient contact between the medication and the damaged site and maintain an effective concentration. Furthermore, the rapid turnover of endometrial mucus causes the medication to be quickly lost from the damaged uterine cavity, making it impossible to maintain an effective therapeutic concentration. Therefore, encapsulating the medication in a gel can prolong the duration of action at the lesion site, reduce losses during delivery, and improve efficacy.

[0006] CN116763724A discloses a method for preparing a curcumin-loaded recombinant collagen hydrogel and its application in the treatment of intrauterine adhesions. This method mixes curcumin with recombinant collagen, and with the synergistic effect of a cross-linking agent, increases the retention time of the recombinant collagen in the uterine cavity, significantly improving its therapeutic effect in vivo. However, curcumin has drawbacks such as low solubility, poor stability, and low absorption rate; furthermore, the use of a chemical cross-linking agent in the gel preparation process poses potential cytotoxicity; and the gel is solid before injection, making it unable to adapt to the complex surface of the uterine wall.

[0007] CN115804750A discloses a method for preparing a compound thermosensitive recombinant collagen hydrogel and its application. A compound thermosensitive recombinant collagen hydrogel is prepared by dissolving recombinant human type III collagen, poloxamer 407, and poloxamer 188 in water. This gel can effectively prevent tissue adhesion and promote endometrial recovery. However, the recombinant collagen used in this gel degrades faster and has lower bioactivity compared to collagen derived from decellularized matrix, resulting in a less effective repair.

[0008] In summary, developing a thermosensitive hydrogel that is easy to prepare, highly safe, can rapidly gel in vivo, 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] This application provides an injectable thermosensitive pregel solution, its preparation method, and its application. The preparation method of this injectable thermosensitive pregel solution is simple, and it rapidly gels upon injection into the body. The resulting hydrogel is highly safe, has wide applicability, and can specifically repair damaged human tissues and organs, inducing tissue regeneration and repair.

[0010] In a first aspect, this application provides an injectable thermosensitive pregel solution, wherein the raw materials for preparing the injectable thermosensitive pregel solution include decellularized matrix and thermosensitive materials;

[0011] The injectable thermosensitive pregel solution is prepared by a method comprising the following steps:

[0012] The decellularized matrix is ​​cryogenically pulverized to obtain decellularized matrix powder. The decellularized matrix powder is mixed with an acidic solution to obtain a mixed solution. The mixed solution is then mixed with a thermosensitive material to obtain the injectable thermosensitive pregel solution.

[0013] This application provides an injectable thermosensitive pregel solution. By adding a thermosensitive material, the injectable thermosensitive pregel solution can gel under specific temperature conditions without the need for additional potentially cytotoxic organic solvents, copolymers, or gel initiators, and gelation can occur at human body temperature. This application utilizes the dynamic balance between the hydrophilic and hydrophobic groups of the thermosensitive material's main polymer chain, maintaining solubility in solution at room temperature, but spontaneously forming micelles and self-associating at physiological temperatures.

[0014] This application utilizes decellularized matrix materials to prepare a pregel solution. Compared to recombinant collagen and purified collagen, the decellularized matrix gel raw material retains various bioactive components, is rich in adhesion proteins, polysaccharides (including hyaluronic acid and chondroitin sulfate, etc.), and active factors that support and regulate cell growth and differentiation, thus promoting rapid endometrial recovery. This product is completely degradable and absorbable, and with the release of various active and regenerative factors, it achieves the repair and regeneration of damaged tissues.

[0015] This application utilizes cryogenic pulverization of decellularized matrix. On one hand, the decellularized matrix obtained through pretreatment is in a moist state, and it is cryogenically pulverized to facilitate subsequent processing and use. On the other hand, in order to avoid the deactivation of bioactive substances in the decellularized matrix due to temperature rise during grinding, grinding needs to be carried out at a low temperature.

[0016] The method for preparing the injectable, temperature-sensitive pregel solution provided in this application is simple and can retain the natural ECM components to the greatest extent while maximizing bioactivity. Bioactive factors are hindered and released slowly within the relatively compact structure. After acid digestion of the decellularized matrix, the acidic groups on the collagen molecular chains react with the acid, causing the collagen to swell and gel, which further facilitates the release of bioactive factors.

[0017] Preferably, the injectable thermosensitive pregel solution comprises a decellularized matrix, a thermosensitive material, and an antioxidant.

[0018] Preferably, the decellularized matrix is ​​derived from animal tissues or organs.

[0019] Preferably, the animal's tissues or organs include any one or a combination of at least two of the following: pig dermis, pig small intestine, pig bladder, bovine tendon, bovine peritoneum, pig peritoneum, bovine pericardium, or pig pericardium.

[0020] Preferably, the method for preparing the decellularized matrix includes: decellularizing, defatting and inactivating viruses in animal tissues or organs to obtain the decellularized matrix.

[0021] Preferably, the method for preparing the decellularized matrix specifically includes: removing excess portions of animal tissues or organs, retaining the matrix layer, and soaking the tissues or organs in a 0.1%–0.4% (e.g., 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, or 0.4%) v / v polyacrylic acid solution with shaking for 20–120 min (e.g., 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, or 120 min, etc.) solution, followed by sequential washing with PBS solution and water, and then soaking the tissues or organs in a 0.05%–% (v / v) polyacrylic acid solution. The decellularized matrix was obtained by shaking and washing with a mixed solution of 0.1% (0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%, etc.) w / v trypsin and 0.05%–0.1% (e.g., 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%, etc.) w / v sodium dodecyl sulfate for 1–3 h (e.g., 1 h, 1.5 h, 2 h, 2.5 h, or 3 h, etc.), followed by washing with PBS solution and water, freeze-drying, and degreasing with an organic degreasing agent for 4–16 h (e.g., 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, or 16 h, etc.).

[0022] Preferably, the organic degreasing agent includes any one or a combination of at least two of chloroform, diethyl ether, or isopropanol.

[0023] Preferably, the temperature of the cryogenic pulverization is -15 to -5℃ (e.g., -15℃, -14℃, -13℃, -12℃, -11℃, -10℃, -9℃, -8℃, -7℃, -6℃ or -5℃, etc.).

[0024] 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.), and more preferably 50–200 μm (e.g., 50 μm, 75 μm, 100 μm, 125 μm, 150 μm, 175 μm or 200 μm, etc.).

[0025] After grinding the decellularized matrix using a tissue homogenizer, the obtained powder is sieved using a 75-mesh sieve to obtain decellularized matrix powder with a diameter of less than 200 μm.

[0026] Preferably, the acidic solution includes hydrochloric acid solution and / or acetic acid solution, with hydrochloric acid solution being the most preferred.

[0027] Preferably, the concentration of the acidic solution is 0.01 to 0.1 mol / L (e.g., 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.).

[0028] This application describes a process where a specific concentration of acid solution is applied to decellularized matrix powder at a specific temperature and for a specific time to obtain an injectable, temperature-sensitive pre-gel solution. The specific acid concentration prevents excessive gelation of collagen in the decellularized matrix, the specific digestion temperature prevents thermal denaturation of collagen, and the specific digestion time ensures successful digestion of the decellularized matrix without excessive gelation. The acid-digested decellularized matrix not only retains a large number of intact three-dimensional ultrastructures but also retains various bioactive components.

[0029] Preferably, the method for mixing the decellularized matrix powder with the acidic solution is homogenization, and the homogenization speed is 200-500 rpm (e.g., 200 rpm, 250 rpm, 300 rpm, 350 rpm, 400 rpm, 450 rpm or 500 rpm, etc.).

[0030] This application sets a specific mixing speed. Since the viscosity of the mixture is relatively high, too low a speed will result in uneven stirring, while too high a speed will generate bubbles, affecting the acid digestion process. The preferred speed is 300 rpm.

[0031] Preferably, the time for mixing the decellularized matrix powder with the acidic solution is 4 to 12 hours (e.g., 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours).

[0032] This application sets a specific acid digestion time. If the digestion time is too short, the digestion effect will be poor, the solution will be uneven, a small amount of powder will be suspended, the viscosity will be low, and the reaction will be incomplete. If the digestion time is too long, the collagen will be over-hydrolyzed, lose its activity, and the decellularized matrix after digestion will denature and clump together, and it will not be able to gel at 37°C. The preferred acid digestion time is 6 hours.

[0033] Preferably, the temperature at which the decellularized matrix powder is mixed with the acidic solution 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).

[0034] This application sets a specific acid digestion temperature. If the digestion temperature is too low, the digestion effect will not be good, the viscosity will be low, and the reaction will be incomplete. If the digestion temperature is too high, it will cause collagen denaturation, and gelation will not be possible at 37°C. The preferred acid digestion temperature is 32°C.

[0035] Preferably, the temperature-sensitive material comprises any one or a combination of at least two of the following: chitosan / sodium β-glycerophosphate, poly(N-isopropylacrylamide), a block copolymer of polyethylene oxide and polypropylene oxide, gelatin, or a copolymer of polylactic acid-hydroxybutyl ester and polyethylene glycol, wherein the block copolymer of polyethylene oxide and polypropylene oxide comprises poloxamer 407 and / or poloxamer 188.

[0036] 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.

[0037] The specific point values ​​from 5 to 24 mentioned above can be 5, 8, 10, 12, 14, 16, 18, 20, 22, or 24, etc.

[0038] Preferably, the method of mixing with the temperature-sensitive material specifically includes: immersing the temperature-sensitive material in a solution and allowing it to stand at 0-8℃ (e.g., 0℃, 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, or 8℃, etc.) for 12-24 hours (e.g., 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, etc.) until the temperature-sensitive material to be added is completely dissolved.

[0039] Preferably, the thermosensitive material in the injectable thermosensitive pregel solution has a mass percentage content of 15% to 30% (e.g., 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, or 30%).

[0040] Preferably, the step of mixing with an acidic solution further includes adjusting the pH of the solution using an alkaline solution.

[0041] Preferably, after mixing with the temperature-sensitive material, the step further includes adjusting the osmotic pressure of the solution using a buffer solution.

[0042] This application uses a buffer solution to adjust the osmotic pressure of the solution to within the normal range of human osmotic pressure. When the pre-gelling solution is injected into the human body, it helps the gel to quickly adapt to the human environment, improving the gel's adaptability. Maintaining the pH of the pre-gelling solution between 6 and 8 is also beneficial for rapid gel formation.

[0043] Preferably, the buffer solution comprises any one or a combination of at least two of PBS buffer solution, Tris-HCl buffer solution, or glycine-Tris buffer solution.

[0044] Preferably, the osmotic pressure of the injectable temperature-sensitive 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.).

[0045] Preferably, the step of mixing with an antioxidant is further included after mixing with an acidic solution.

[0046] This application incorporates antioxidants, which helps prevent collagen from undergoing oxidative denaturation, maintains the activity of collagen molecules, and makes the gel less prone to phase separation.

[0047] Preferably, the antioxidant comprises any one or a combination of at least two of the following: fucoidan, carrageenan, agar oligosaccharide, tea polyphenols, sodium citrate, ascorbic acid, rutin, epicatechin, catechin, mannitol, or glycerol.

[0048] Glycerin is an irradiant. By adding an appropriate amount of glycerin to the pregel solution provided in this application, some important components can be protected from irradiation or be minimally affected.

[0049] Preferably, the mass ratio of the antioxidant to the mixed solution is 1:(5-100), more preferably 1:(20-50).

[0050] The specific point values ​​from 5 to 100 can be selected as 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, etc.

[0051] The specific point values ​​for the above 20 to 50 can be selected as 20, 25, 30, 35, 40, 45 or 50, etc.

[0052] Secondly, this application provides a method for preparing an injectable thermosensitive pregel solution as described in the first aspect, the method comprising the following steps:

[0053] (1) Obtaining decellularized matrix: Decellularize, defatt, and inactivate viruses any one or at least two combinations of animal tissues or organs including porcine dermis, porcine small intestine, porcine bladder, bovine tendon, bovine peritoneum, porcine peritoneum, bovine pericardium, or porcine pericardium to obtain decellularized matrix;

[0054] The method for preparing the decellularized matrix specifically includes: removing excess parts of animal tissues or organs, retaining the matrix layer, soaking in 0.1%–0.4% v / v polyacrylic acid solution with shaking for 20–120 min, washing with PBS solution and water in sequence, washing with a mixed solution containing 0.05%–0.1% w / v trypsin and 0.05%–0.1% w / v sodium dodecyl sulfate with shaking for 1–3 h, washing with PBS solution and water in sequence, freeze-drying, and soaking in an organic defatting agent for defatting for 4–16 h to obtain the decellularized matrix;

[0055] (2) Obtaining decellularized matrix powder: The decellularized matrix described in step (1) is frozen and pulverized at -15 to -5°C to obtain decellularized matrix powder with a diameter of 50 to 300 μm;

[0056] (3) Digestion treatment of decellularized matrix powder: The decellularized matrix powder described in step (2) is homogenized with a 0.01-0.1 mol / L acidic solution at a temperature of 25-35℃ and a rotation speed of 200-500 rpm for 4-12 h; the acidic solution includes hydrochloric acid solution and / or acetic acid solution;

[0057] (4) Solution pH adjustment: Use an alkaline solution to adjust the pH of the solution obtained in step (3) to 6-8;

[0058] (5) Mixing with temperature-sensitive material: Mix the solution obtained in step (4) with temperature-sensitive material;

[0059] The thermosensitive material comprises any one or a combination of at least two of the following: chitosan / sodium β-glycerophosphate, poly(N-isopropylacrylamide), a block copolymer of polyethylene oxide and polypropylene oxide, gelatin, or a copolymer of polylactic acid-hydroxybutyl ester and polyethylene glycol; the block copolymer of polyethylene oxide and polypropylene oxide comprises poloxamer 407 and / or poloxamer 188; the mass percentage of the thermosensitive material in the injectable thermosensitive pregel solution is 15% to 30%.

[0060] (6) Solution osmotic pressure adjustment: Adjust the osmotic pressure of the solution obtained in step (5) using a buffer solution; the buffer solution includes any one or a combination of at least two of PBS buffer solution, Tris-HCl buffer solution or glycine-Tris buffer solution;

[0061] (7) Mixing with antioxidants: The solution obtained in step (6) is mixed with antioxidants; the antioxidants include any one or a combination of at least two of the following: fucoidan, carrageenan, agar oligosaccharide, tea polyphenols, sodium citrate, ascorbic acid, rutin, epicatechin, catechin, mannitol or glycerol; the mass ratio of the antioxidant to the mixed solution is 1:(5-100).

[0062] Thirdly, this application provides an injectable thermosensitive gel, which is obtained by gelling the injectable thermosensitive pregel solution described in the first aspect.

[0063] The gel prepared using the method provided in this application has the characteristics of high wettability and wide applicability. Before injection, it is a flowable liquid at room temperature, and after injection into the human body, it can quickly gel and become solid at body temperature. When the gel is injected into wound defects or cavities at room temperature, it can achieve full coverage and filling of irregular defect locations. At the same time, under physiological temperature conditions, it rapidly changes from a flowable state to a non-flowable solid state.

[0064] The gel prepared using the method provided in this application is characterized by high safety and high activity, and also possesses many ideal properties, including thermosensitivity, injectability, biodegradability, good biocompatibility, and continuous release of biological factors from the wound. It retains adhesion proteins, polysaccharides, and active factors that support and regulate cell growth and differentiation, effectively promoting cell aggregation and growth at the material interface. It can inhibit excessive collagen proliferation and deposition leading to scarring, resulting in a smooth and flat repaired wound surface. It can specifically repair damaged human tissues and organs and induce tissue regeneration and repair.

[0065] Preferably, the air bubbles in the pregel solution are removed by centrifugation before gelation, which facilitates the formation of a dense gel after gelation.

[0066] Preferably, the gelation temperature is 25–40°C (e.g., 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.).

[0067] Preferably, the gelation time is 1-40 min (e.g., 1 min, 3 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min or 40 min, etc.).

[0068] This application allows for adjustment of the gelation temperature and time based on the concentration and volume of the pregelation solution.

[0069] Fourthly, this application provides the 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 products for preventing endometrial adhesions and / or promoting endometrial repair.

[0070] This application utilizes intrauterine injection to form a gel at physiological temperature. The resulting gel has high viscosity, preventing leakage after injection and prolonging its retention time within the uterine cavity. It forms a protective film on the endometrium, thus preventing endometrial adhesions. The amount of thermosensitive material added affects the viscosity of the obtained gel. At 37°C, the gel viscosity is 28.25 Pa·s for gels containing 16% thermosensitive material, 39.42 Pa·s for gels containing 20% ​​thermosensitive material, and 52.04 Pa·s for gels containing 25% thermosensitive material. The gel is prepared using a decellularized matrix material and contains various bioactive components, which can promote rapid endometrial recovery and achieve the repair and regeneration of damaged tissue.

[0071] Fifthly, this application provides a medicament for preventing endometrial adhesions and / or promoting endometrial repair, the medicament comprising the injectable thermosensitive pregel solution described in the first aspect or the injectable thermosensitive gel described in the third aspect.

[0072] The drug provided in this application, which contains an injectable thermosensitive pregel solution, can also be loaded with other drugs that help prevent endometrial adhesions and / or promote endometrial repair. The pregel solution can carry the drug to the affected area quickly and work synergistically with the decellularized matrix gel to achieve a therapeutic effect.

[0073] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0074] Compared with the prior art, this application has the following beneficial effects:

[0075] (1) This application provides an injectable thermosensitive pregel solution, which is prepared by decellularized matrix and thermosensitive material. It remains soluble at room temperature, spontaneously gels under physiological temperature conditions, has good fluidity, can achieve perfect filling when injected into the body, and can solidify in situ within 2 minutes at body temperature.

[0076] (2) This application provides a thermosensitive gel, which is obtained by gelling an injectable thermosensitive pregel solution. It has a certain viscosity and a certain volume, which can isolate the wound surface and prevent 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 tissue.

[0077] (3) The decellularized matrix of this application 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. Attached Figure Description

[0078] Figure 1 shows the microstructure after decellularized matrix digestion.

[0079] Figure 2 shows the phase transition results of the pregelation solution;

[0080] Figure 3 shows the gelation results of the pregel solution;

[0081] Figure 4 shows a scan of the left endometrial cells (H&E) of rats in the treatment group;

[0082] Figure 5 shows a scan of endometrial cells (H&E) on the left side of the rats in the model group;

[0083] Figure 6 shows the results of endometrial thickness and glandular number in rats in the model group and treatment group. Detailed Implementation

[0084] To further illustrate the technical means and effects adopted in this application, the following description, in conjunction with embodiments and accompanying drawings, will provide further details. It is understood that the specific embodiments described herein are merely for explaining this application and not for limiting it.

[0085] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0086] Example 1

[0087] This embodiment provides a method for preparing an injectable temperature-sensitive pregel solution, the preparation method including the following steps:

[0088] (1) Remove excess parts of the pig bladder, retain the matrix layer, and soak it in 0.25% v / v polyacrylic acid solution with shaking for 70 min. Then wash it with PBS solution and water in sequence. Wash it with a mixed solution containing 0.08% w / v trypsin and 0.08% w / v sodium dodecyl sulfate for 2 h with shaking. Then wash it with PBS solution and water in sequence. Freeze-dry it and soak it in ether for 10 h to defatt it, and obtain the decellularized pig bladder matrix (UBM).

[0089] (2) UBM was frozen and pulverized at -10℃, and then sieved to obtain UBM powder with a diameter of 50 to 200 μm;

[0090] (3) The UBM powder and 0.05 mol / L hydrochloric acid solution were homogenized and mixed for 8 h at a temperature of 30 °C and a rotation speed of 350 rpm.

[0091] (4) The pH of the solution was adjusted to 6-8 using an alkaline solution, and the microstructure is shown in Figure 1.

[0092] (5) Immerse the thermosensitive material poloxamer 407 in the mixed solution, stir slightly every 2 hours, and let stand for 24 hours. The mass percentage of the thermosensitive material in the pregel solution is 20%.

[0093] (6) Use PBS 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 to 400 mOsmol / kg;

[0094] (7) Add the antioxidant glycerin, wherein the mass ratio of the antioxidant to the mixed solution is 1:25.

[0095] The gelation effect of the pregelation solution is shown in Figures 2 and 3. As shown in Figure 2, the solution is in a liquid state at room temperature and becomes a gel after treatment at 37°C. As shown in Figure 3, the pregelation solution is loaded into a syringe and can rapidly gel at 37°C.

[0096] Example 2

[0097] This embodiment provides a method for preparing an injectable temperature-sensitive pregel solution, the preparation method including the following steps:

[0098] (1) Remove excess parts of the pig small intestine, retain the matrix layer, and soak it in 0.4% v / v polyacrylic acid solution with shaking for 20 min. Then wash it with PBS solution and water in sequence. Wash it with a mixed solution containing 0.1% w / v trypsin and 0.05% w / v sodium dodecyl sulfate for 3 h with shaking. Then wash it with PBS solution and water in sequence. Freeze dry and defatt it with chloroform soaking for 4 h to obtain decellularized pig small intestine matrix (SIS);

[0099] (2) The SIS was frozen and pulverized at -15℃ and then sieved to obtain SIS powder with a diameter of 50 to 200 μm;

[0100] (3) The SIS powder and 0.1 mol / L acetic acid solution were homogenized and mixed for 12 h at a temperature of 35 °C and a rotation speed of 200 rpm.

[0101] (4) Adjust the pH of the solution to 6-8 using an alkaline solution;

[0102] (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 thermosensitive material poloxamer 407 in the pregel solution is 20%, and the mass percentage of thermosensitive material poloxamer 188 is 0.5%.

[0103] (6) Use 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 to 400 mOsmol / kg;

[0104] (7) Add the antioxidant glycerin, wherein the mass ratio of the antioxidant to the mixed solution is 1:5.

[0105] Example 3

[0106] This embodiment provides a method for preparing an injectable temperature-sensitive pregel solution, the preparation method including the following steps:

[0107] (1) Remove excess parts of the pig bladder, retain the matrix layer, and soak it in 0.1% v / v polyacrylic acid solution with shaking for 120 min. Then wash it with PBS solution and water in sequence. Wash it with a mixed solution containing 0.05% w / v trypsin and 0.1% w / v sodium dodecyl sulfate for 1 h with shaking. Then wash it with PBS solution and water in sequence. Freeze-dry it and soak it in isopropanol for 16 h to defatt it, and obtain the decellularized pig bladder matrix (UBM).

[0108] (2) UBM was frozen and pulverized at -5℃, and then sieved to obtain UBM powder with a diameter of 50 to 200 μm;

[0109] (3) The UBM powder and 0.01 mol / L hydrochloric acid solution were homogenized and mixed for 4 h at a temperature of 25 °C and a rotation speed of 500 rpm.

[0110] (4) Adjust the pH of the solution to 6-8 using an alkaline solution;

[0111] (5) Immerse the thermosensitive material polyN-isopropylacrylamide in the mixed solution, stir slightly every 2 hours, let stand for 24 hours, and the mass percentage of the thermosensitive material in the pregel solution is 15%.

[0112] (6) Use glycine-Tris 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;

[0113] (7) Add the antioxidant glycerin, wherein the mass ratio of the antioxidant to the mixed solution is 1:100.

[0114] Example 4

[0115] This embodiment provides a method for preparing an injectable temperature-sensitive pregel solution, which differs from Example 1 only in that the homogenization time in step (3) is 3 hours.

[0116] Example 5

[0117] This embodiment provides a method for preparing an injectable temperature-sensitive pregel solution, which differs from Example 1 only in that the homogenization time in step (3) is 4 hours.

[0118] Example 6

[0119] This embodiment provides a method for preparing an injectable temperature-sensitive pregel solution, which differs from Example 1 only in that the homogenization time in step (3) is 12 hours.

[0120] Example 7

[0121] This embodiment provides a method for preparing an injectable temperature-sensitive pregel solution, which differs from Example 1 only in that the homogenization time in step (3) is 13 hours.

[0122] Example 8

[0123] This embodiment provides a method for preparing an injectable temperature-sensitive pregel solution, which differs from Example 1 only in that the homogenization temperature in step (3) is 24°C.

[0124] Example 9

[0125] This embodiment provides a method for preparing an injectable temperature-sensitive pregel solution, which differs from Example 1 only in that the homogenization temperature in step (3) is 25°C.

[0126] Example 10

[0127] This embodiment provides a method for preparing an injectable temperature-sensitive pregel solution, which differs from Example 1 only in that the homogenization temperature in step (3) is 35°C.

[0128] Example 11

[0129] This embodiment provides a method for preparing an injectable temperature-sensitive pregel solution, which differs from Example 1 only in that the homogenization temperature in step (3) is 36°C.

[0130] Example 12

[0131] This embodiment provides a method for preparing an injectable thermosensitive pregel solution, which differs from Example 1 only in that the mass percentage of thermosensitive material in the pregel solution in step (5) is 14%.

[0132] Example 13

[0133] This embodiment provides a method for preparing an injectable thermosensitive pregel solution, which differs from Example 1 only in that the mass percentage of thermosensitive material in the pregel solution in step (5) is 15%.

[0134] Example 14

[0135] This embodiment provides a method for preparing an injectable thermosensitive pregel solution, which differs from Example 1 only in that the mass percentage of thermosensitive material in the pregel solution in step (5) is 30%.

[0136] Example 15

[0137] This embodiment provides a method for preparing an injectable thermosensitive pregel solution, which differs from Example 1 only in that the mass percentage of thermosensitive material in the pregel solution in step (5) is 31%.

[0138] Example 16

[0139] This embodiment provides a method for preparing an injectable thermosensitive pregel solution, which differs from Example 1 only in that the mass percentage of thermosensitive material in the pregel solution in step (5) is 17%.

[0140] Example 17

[0141] This embodiment provides a method for preparing an injectable thermosensitive pregel solution, which differs from Example 1 only in that the mass percentage of thermosensitive material in the pregel solution in step (5) is 18%.

[0142] Example 18

[0143] This embodiment provides a method for preparing an injectable thermosensitive pregel solution, which differs from Example 1 only in that the mass percentage of thermosensitive material in the pregel solution in step (5) is 19%.

[0144] Example 19

[0145] This embodiment provides a method for preparing an injectable thermosensitive pregel solution. The only difference from Embodiment 1 is that step (5) is as follows: the thermosensitive materials poloxamer 407 and poloxamer 188 are immersed in a mixed solution, stirred slightly every 2 hours, and left to stand for 24 hours. The mass percentage of the thermosensitive material poloxamer 407 in the pregel solution is 20%, and the mass percentage of the thermosensitive material poloxamer 188 is 0.5%.

[0146] Example 20

[0147] This embodiment provides a method for preparing an injectable thermosensitive pregel solution. The only difference from Example 19 is that in step (5), the mass percentages of the thermosensitive materials poloxamer 407 and poloxamer 188 in the pregel solution are 21% and 4%, respectively.

[0148] Example 21

[0149] This embodiment provides a method for preparing an injectable thermosensitive pregel solution. The only difference from Example 19 is that in step (5), the mass percentages of the thermosensitive materials poloxamer 407 and poloxamer 188 in the pregel solution are 22% and 3%, respectively.

[0150] Example 22

[0151] This embodiment provides a method for preparing an injectable thermosensitive pregel solution. The only difference from Example 19 is that in step (5), the mass percentages of the thermosensitive materials poloxamer 407 and poloxamer 188 in the pregel solution are 23% and 2%, respectively.

[0152] Example 23

[0153] This embodiment provides a method for preparing an injectable thermosensitive pregel solution. The only difference from Example 19 is that in step (5), the mass percentages of the thermosensitive materials poloxamer 407 and poloxamer 188 in the pregel solution are 24% and 1%, respectively.

[0154] Example 24

[0155] This embodiment provides a method for preparing an injectable thermosensitive pregel solution. The only difference from Example 19 is that in step (5), the mass percentages of the thermosensitive materials poloxamer 407 and poloxamer 188 in the pregel solution are 20% and 1%, respectively.

[0156] Example 25

[0157] This embodiment provides a method for preparing an injectable thermosensitive pregel solution. The only difference from Example 19 is that in step (5), the mass percentages of the thermosensitive materials poloxamer 407 and poloxamer 188 in the pregel solution are 20% and 1.5%, respectively.

[0158] Example 26

[0159] This embodiment provides a method for preparing an injectable thermosensitive pregel solution. The only difference from Example 19 is that in step (5), the mass percentages of the thermosensitive materials poloxamer 407 and poloxamer 188 in the pregel solution are 20% and 2%, respectively.

[0160] Example 27

[0161] This embodiment provides a method for preparing an injectable thermosensitive pregel solution. The only difference from Example 19 is that in step (5), the mass percentages of the thermosensitive materials poloxamer 407 and poloxamer 188 in the pregel solution are 20% and 2.5%, respectively.

[0162] Comparative Example 1

[0163] This comparative example provides a method for preparing an injectable temperature-sensitive pregel solution. The only difference from Example 1 is that step (3) involves mixing UBM powder with an aqueous solution containing 3 mg / mL pepsin and 0.05 mol / L hydrochloric acid, and homogenizing the mixture for 48 h at a temperature of 30°C and a rotation speed of 350 rpm.

[0164] Comparative Example 2

[0165] This comparative example provides a method for preparing an injectable thermosensitive pregel solution, which differs from Example 1 only in that step (5) is omitted, i.e., no thermosensitive material is added to the pregel solution.

[0166] Test Example 1

[0167] This test example prepared a pregel solution using the methods of Examples 1-11. The results of the test were shown in Table 1.

[0168] Table 1

[0169] From Table 1, we can conclude that:

[0170] (1) Comparing Example 1 and Example 4, it can be seen that if the digestion time is less than 4 hours, 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.

[0171] (2) Comparing Example 1 and Example 7, it can be seen that if the digestion time is longer than 12 hours, the collagen will be over-hydrolyzed and lose its activity. Furthermore, the pregel of the decellularized matrix obtained at the end will clump together, indicating that the collagen in the decellularized matrix has been denatured. At the same time, prolonged acid soaking may cause the bioactive factors in the decellularized matrix to become inactive.

[0172] (3) Comparing Example 1 and Example 8, it can be seen that when the digestion temperature is below 25°C, the digestion effect is not good, the reaction is incomplete, and the solution viscosity is low. From Examples 1 and 5-6, it can be seen that when other conditions are within the set range, the digestion effect is good and a uniform matrix solution can be obtained when the homogenization time is between 4 and 8 hours.

[0173] (4) 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 clump and not form a gel. At the same time, the bioactive factors in the decellularized matrix will be destroyed and lose their activity. As can be seen from Examples 1 and 9-10, when other conditions are within the set range, when the homogenization temperature is between 25-35°C, the digestion effect is good and a homogeneous matrix solution can be obtained.

[0174] Test Example 2

[0175] This test example used the methods provided in Examples 1-3, 12-27 and Comparative Examples 1-2 to prepare pregel solutions. The gelation time and whether these pregel solutions formed gels were then tested. The gelation time of the injectable thermosensitive gel was determined using the vial-pouring method. The time required for the sample to stop flowing after the vial was inverted was the gelation time of the sample under the corresponding conditions. The results are shown in Table 2.

[0176] Table 2

[0177] From Table 2, we can conclude that:

[0178] (1) Comparing Example 1 and Example 12, it can be seen that when the proportion of temperature-sensitive material is less than 15%, such as the 14% provided in Example 12, the proportion of temperature-sensitive material in the pregel solution is too low to form a gel.

[0179] (2) Comparing Example 1 and Example 15, it can be seen that when the mass percentage of the thermosensitive material in the pregel solution exceeds 30%, for example, when it is 31% as provided in Example 15, the proportion of the thermosensitive material in the pregel solution is too high. It can form a gel, but because the concentration is too high, it has already formed a gel at room temperature and cannot be operated subsequently.

[0180] (3) Comparing Example 1 with Examples 13-14 and 16-18, it can be seen that as the mass percentage of the thermosensitive material in the pregel solution gradually decreases, the gelation time gradually increases; that is, the higher the concentration of poloxamer 407, the shorter the gelation time.

[0181] (4) Comparing Example 1 with Example 19 and Examples 24-27, it can be seen that adding 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.

[0182] (5) As can be seen from Examples 20-23, when the total mass fraction of the added temperature-sensitive material remains unchanged, the gelation time of the pregel solution can be precisely controlled by changing the mass ratio of poloxamer 407 to poloxamer 188, and the gelation time can be precisely controlled within 2 minutes.

[0183] Test Example 3

[0184] This test example used the method provided in Example 1 to prepare a pre-gel solution. The in vitro cytotoxicity of the sample was evaluated according to the requirements of GB / T 16886.5-2017 Medical Device Biological Evaluation. 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. 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 contained only MEM culture medium containing 10% fetal bovine serum.

[0185] Table 3

[0186] Test Example 4

[0187] This test case utilizes the pregel solution obtained through the method provided in Example 1 for physiological testing. A rat model of intrauterine adhesions and uterine injury was constructed, and then a thermosensitive pregel solution was injected into the injured uterus to observe the endometrial repair in the rats.

[0188] (1) Experimental method:

[0189] Experimental Design: This experiment was divided into a model group and a treatment group. A rat endometrial injury model was established by injecting 95% ethanol into the endometrium. Then, a thermosensitive hydrogel was injected into the injured uterus, and the endometrial repair in rats was observed.

[0190] Establishment of the animal model: Female SD rats (200-220g, 8 weeks old) with normal estrous cycles were randomly divided into two groups: a model group and a treatment group. Rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (0.3mL / 100g), and the uterine horn was exposed through an incision. The skin and abdominal wall layers were sequentially incised; the uterus was separated, and the proximal and distal ends of one side of the uterus were gently clamped with vascular clamps. The uterus was gently straightened with toothless forceps, and the surrounding tissues were protected with sterile saline gauze to prevent leakage and damage from 95% anhydrous ethanol. A syringe needle was inserted into the uterine cavity, kept parallel to the longitudinal axis of the uterus, and 95% anhydrous ethanol was slowly injected into one end of the uterus to keep the cavity full. The needle was left in place for 4-5 minutes and then slowly removed. Residual anhydrous ethanol was gently squeezed out, and the uterus was rinsed twice with physiological saline before releasing the vascular clamps. The other uterus was treated in the same way. To prevent adhesions, the abdominal cavity was rinsed with physiological saline and dried with sterile gauze. The uterus was restored to its normal position, and the abdomen was closed layer by layer. After the modeling was completed, the wound was disinfected, the rat was placed on a warming pad, and after it woke up, it was placed in a feeding cage.

[0191] (2) Experimental steps:

[0192] Based on the established intrauterine adhesion model, the treatment group received a thermosensitive pre-gel solution injected into both uteri. The model group, after establishing an intrauterine adhesion model through chemical injury, received 100 μL of normal saline injected into both uteri.

[0193] Results and processing: One week after surgery, rats were sacrificed, and the uterus was stained with hematoxylin and eosin (H&E) to evaluate the uterine healing effect in each group. Histological morphology, H&E, endometrial thickness, and glandular number were also examined.

[0194] (3) Experimental conclusions:

[0195] The experimental results shown in Figures 4 and 5 indicate that, compared to the model group, the 7-day treatment group formed physical spaces within the animal's uterine cavity, separating the incompletely epithelialized mucosal surface and effectively preventing the occurrence of new adhesions. As shown in Figure 6, the endometrial thickness and glandular quantity in the treatment group were also superior to those in the model group, demonstrating that the bioactive substances contained in the gel can promote rapid endometrial recovery and achieve the repair and regeneration of damaged tissue.

[0196] In summary, this application provides an injectable thermosensitive pregel solution, which is prepared using a decellularized matrix and thermosensitive materials. The preparation method is simple, and it can quickly form a gel when injected into the body. The resulting hydrogel has high safety and strong applicability, and can specifically repair damaged human tissues and organs, induce tissue regeneration and repair, and can be used to prevent endometrial adhesions and / or promote endometrial repair.

[0197] The applicant declares that the above description is only a specific implementation of this application, but the protection scope of this application is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application fall within the protection and disclosure scope of this application.

Claims

1. An injectable thermosensitive pregel solution, the raw materials for which are prepared include decellularized matrix and thermosensitive material; The injectable thermosensitive pregel solution is prepared by a method comprising the following steps: The decellularized matrix is ​​cryogenically pulverized to obtain decellularized matrix powder; the decellularized matrix powder is mixed with an acidic solution to obtain a mixed solution; the mixed solution is mixed with a thermosensitive material to obtain the injectable thermosensitive 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 mixing time of the decellularized matrix powder and the acidic solution is 4-12 h, and the temperature of the decellularized matrix powder and the acidic solution during mixing is 25-35 °C. The temperature-sensitive material is poloxamer 407 and poloxamer 188, and the mass ratio of poloxamer 407 to poloxamer 188 is (5-24):

1.

2. The injectable temperature-sensitive pregel solution according to claim 1, wherein, The decellularized matrix may be derived from animal tissues or organs; The animal's tissues or organs include any one or a combination of at least two of the following: pig dermis, pig small intestine, pig bladder, bovine tendon, bovine peritoneum, pig peritoneum, bovine pericardium, or pig pericardium.

3. The injectable temperature-sensitive pregel solution according to claim 1, wherein, The method for preparing the decellularized matrix includes: decellularizing, defatting and inactivating viruses in animal tissues or organs to obtain the decellularized matrix.

4. The injectable temperature-sensitive pregel solution according to claim 1, wherein, The temperature of the cryogenic pulverization is -15 to -5°C; The method for mixing the decellularized matrix powder with the acidic solution is homogenization, and the homogenization speed is 200-500 rpm.

5. The injectable thermosensitive pregel solution according to claim 1, wherein, The injectable thermosensitive pregel solution contains 15% to 30% by mass of thermosensitive material.

6. The injectable temperature-sensitive pregel solution according to claim 1, wherein, The process of mixing with an acidic solution further includes the step of adjusting the pH of the solution using an alkaline solution. After mixing with the temperature-sensitive material, the method further includes the step of adjusting the osmotic pressure of the solution using a buffer solution; The buffer solution includes any one or a combination of at least two of the following: PBS buffer solution, Tris-HCl buffer solution, or glycine-Tris buffer solution.

7. The injectable thermosensitive pregel solution according to claim 1, wherein, The process of mixing with an acidic solution further includes a step of mixing with an antioxidant. The antioxidants include any one or a combination of at least two of the following: fucoidan, carrageenan, agar oligosaccharide, tea polyphenols, sodium citrate, ascorbic acid, rutin, epicatechin, catechin, mannitol, or glycerol; The mass ratio of the antioxidant to the mixed solution is 1:(5-100).

8. An injectable thermosensitive gel, obtained by gelling the injectable thermosensitive pregel solution according to any one of claims 1 to 7.

9. The injectable thermosensitive gel according to claim 8, wherein, The gelation temperature is 25–40°C, and the gelation time is 1–40 min.

10. The use of an injectable thermosensitive pregel solution as described in any one of claims 1 to 7 or an injectable thermosensitive gel as described in claim 8 or 9 in the preparation of products for preventing endometrial adhesions and / or promoting endometrial repair.

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