Endometrial repair stent and preparation method thereof

Abstract

The present invention relates to the field of biomedical technology, and a kind of endometrial repair stent and preparation method thereof is provided, with sheet-like nanofiber composite membrane as substrate, first combined with modified hyaluronic acid, then after being dynamically cross-linked with carboxymethyl chitosan, and then composited with poly N-isopropyl acrylamide to obtain; the mass ratio of modified hyaluronic acid, carboxymethyl chitosan and poly N-isopropyl acrylamide is (1~9): (1~9): (1~90); the thickness of nanofiber composite membrane is 0.1~0.4mm, which can be applied to treat intrauterine adhesions. Endometrial repair stent combines nanofiber composite membrane with thermosensitive hydrogel, utilizes the fluidity of hydrogel to cover the uterine cavity, and the three-dimensional structure of nanofiber can orderly induce the wound surface, is beneficial to the adhesion and proliferation of cells, reduces scar formation, ensures repair effect, and promotes regeneration. The two are combined, and the plasticity of the overall structure can be enhanced, and supportiveness is increased. The retention time in the uterine cavity is long, and the dual effect of repair and isolation is fully achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an endometrial repair stent and a preparation method thereof. Background Art

[0002] Intrauterine adhesions are a common gynecological intrauterine disease. They are caused by trauma to the pregnant or non-pregnant uterus, leading to damage to the basal layer of the endometrium, partial or complete occlusion of the uterine cavity, and consequently, menstrual abnormalities, infertility, or recurrent miscarriage. Currently, hysteroscopic intrauterine adhesion resection is the standard surgical procedure for treating intrauterine adhesions. However, the endometrial wound surface after electrocautery separation during surgery is difficult to regenerate. Pathological repair of the thermally damaged tissue surface forms inflammatory granulation tissue and fibrous scars, causing the exposed anterior and posterior walls of the uterine cavity, which are not covered by the endometrium, to adhere again, leading to infertility. Therefore, postoperative measures such as promoting endometrial repair and placing a barrier to prevent adhesions are particularly important.

[0003] On the one hand, postoperative intrauterine contraceptive devices, intrauterine support balloons, and sodium hyaluronate gel are the main clinical treatments for damaged uterine adhesions both domestically and internationally. These methods rely primarily on a simple physical barrier to prevent adhesion formation. On the other hand, with the development of tissue engineering materials, medical materials are being used to repair endometrial tissue and prevent further adhesions. Both treatments are limited in function, and currently, no dual-functional product is available on the market that can both promote repair and prevent adhesions.

[0004] CN114652892A discloses a medical membrane material, a preparation method thereof, an anti-uterine adhesion device, and a medical apparatus. The prepared medical membrane material comprises a substrate comprising poly(lactic-glycolic acid) and a reactive polymer comprising gelatin and / or dextran, and a chemokine grafted onto the surface of the substrate. While this medical membrane material can promote endometrial repair, its shape does not fully match that of the uterine cavity. After insertion, it cannot fill every corner of the uterine cavity and cannot accurately isolate the uterine cavity and the uterine horns, resulting in poor effectiveness in preventing re-adhesion.

[0005] Hydrogel is a three-dimensional material with a three-dimensional structure similar to the extracellular matrix. It can provide a three-dimensional growth environment for cells, but it cannot directly induce orderly cell growth. It lacks support in the uterine cavity, has poor continuous spreading effect, and is difficult to retain. Therefore, the isolation effect is poor and it cannot be widely used in tissue repair.

[0006] Therefore, the clinic lacks an ideal anti-uterine adhesion product that has both tissue repair and sufficient isolation effects to address the functional defects of traditional hydrogels and simple biofilms. Summary of the Invention

[0007] The purpose of the present invention is to provide an endometrial repair stent and a preparation method thereof, and to provide a temperature-sensitive endometrial repair stent with the dual functions of uterine cavity tissue repair and isolation.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides an endometrial repair stent, which is prepared by combining a sheet-like nanofiber composite membrane with dopamine-grafted aldehyde-modified hyaluronic acid, dynamically cross-linking the membrane with carboxymethyl chitosan based on a Schiff base reaction to form a dynamic cross-linked hydrogel, and then compounding the membrane with poly (N-isopropylacrylamide).

[0010] Wherein, the mass ratio of the dopamine-grafted aldehyde-hydrated hyaluronic acid, the carboxymethyl chitosan and the poly N-isopropylacrylamide is (1~9):(1~9):(1~90);

[0011] The thickness of the nanofiber composite membrane is 0.1-0.4 mm.

[0012] Furthermore, the nanofiber composite membrane includes an upper nanofiber layer, a lower nanofiber layer, and an intermediate nanofiber layer formed between the upper nanofiber layer and the lower nanofiber layer; the nanofibers in the upper nanofiber layer and the lower nanofiber layer are both core-shell structures, and the shell layer raw material components in the core-shell structure include gelatin and a cross-linking agent, and the mass ratio of the gelatin to the cross-linking agent is (15~30): (1~5); the raw material components of the core layer and the intermediate nanofiber layer in the core-shell structure are both PLGA, and the mass ratio of the PLGA to the gelatin is 1:1.

[0013] Furthermore, the method for preparing the nanofiber composite membrane comprises the following steps:

[0014] S1: dissolving PLGA in a first solvent to prepare a first spinning solution with a concentration of 15-30 wt%, and electrospinning to prepare an intermediate nanofiber layer;

[0015] S2: dissolving gelatin in a second solvent and mixing it with a cross-linking agent solution in real time to prepare a second spinning solution;

[0016] S3: taking the first spinning solution in step S1 and coaxially electrospinning it with the second spinning solution in step S2 to prepare an upper nanofiber layer and a lower nanofiber layer respectively;

[0017] S4: performing gradient temperature-raising cross-linking on the three nanofiber layers prepared above, thereby preparing a nanofiber composite membrane.

[0018] Furthermore, in step S1, the first solvent is hexafluoroisopropanol; in step S2, the second solvent is a mixture of water, acetic acid and ethyl acetate, and the mass ratio of the water, the acetic acid and the ethyl acetate is (1~9):(1~9):(1~9); the concentration of the gelatin solution is 15~30wt%; the cross-linking agent is a mixture of one or more selected from formaldehyde, glyoxal, glutaraldehyde, genipin and tannic acid, and the concentration of the cross-linking agent solution is 1~5wt%.

[0019] Furthermore, in step S1, the electrospinning voltage is 40~60V, the spinning distance is 10~16cm, and the spinning time is 2~6h; in step S3, the coaxial electrospinning voltage is 40~60V, the spinning distance is 10~16cm, and the spinning time is 3~10h; in step S4, the cross-linking temperature is 80~170℃, and the cross-linking degree is 50~70%.

[0020] A second aspect of the present invention provides a method for preparing an endometrial repair stent, comprising the following steps:

[0021] (1) After dissolving hyaluronic acid, an oxidant is added to oxidize it, and then a reducing agent is added to terminate the oxidation, and the aldehyde-modified hyaluronic acid is obtained by freeze-drying;

[0022] (2) dissolving the aldehyde-modified hyaluronic acid, adding dopamine to carry out a grafting reaction, and freeze-drying to obtain the dopamine-grafted aldehyde-modified hyaluronic acid;

[0023] (3) The dopamine-grafted aldehyde-modified hyaluronic acid is dissolved, and then the nanofiber composite membrane sheet, carboxymethyl chitosan and poly (N-isopropylacrylamide) are added in sequence and mixed evenly to prepare the endometrial repair scaffold.

[0024] Furthermore, in step (1), the concentration of the hyaluronic acid is 0.5-5 wt %; the oxidizing agent is a mixture of one or more selected from sodium periodate, basic copper carbonate, chromic anhydride pyridinium hydrochloride and lead tetraacetate; the reducing agent is ethylene glycol; the oxidation reaction temperature is 20-40° C., and the reaction time is 3-6 h.

[0025] Furthermore, in step (2), the concentration of the aldehyde-modified hyaluronic acid is 0.5-3 wt %; the molar ratio of the aldehyde-modified hyaluronic acid to the dopamine is 1:1; the grafting reaction temperature is room temperature, and the reaction time is 10 h.

[0026] Furthermore, in step (3), the concentration of the dopamine-grafted aldehyde-hydrated hyaluronic acid is 0.5-3 wt %.

[0027] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0028] This invention combines a nanofiber composite membrane with a thermosensitive hydrogel, leveraging the hydrogel's fluidity to cover every corner of the uterine cavity. The three-dimensional nanofiber structure systematically guides the wound surface, promoting cell adhesion and proliferation, minimizing scarring, ensuring repair, and promoting regeneration. The combination of these two enhances the overall structural plasticity and support, allowing for extended retention in the uterine cavity, fully achieving the dual functions of repair and isolation.

[0029] The composite membrane features a hydrophobic middle layer to minimize swelling in the hydrogel and maintain structural integrity. The upper and lower fiber layers are constructed with a core-shell structure, with the PLGA core layer serving as a basic skeleton, providing support and minimizing fiber swelling. The shell layer is constructed of gelatin, which is crosslinked in real time using a crosslinking agent, with the degree of crosslinking controlled. The composite membrane's top and bottom sides share the same structure, ensuring consistent repair results on both sides.

[0030] Furthermore, the amino groups of the remaining gelatin after cross-linking by the cross-linking agent are grafted with the aldehyde-modified hyaluronic acid modified by dopamine, respectively, through chemical cross-linking and physical cross-linking under the covalent bonding of dopamine and gelatin, which not only ensures the bonding strength between the gelatin fiber and the hyaluronic acid gel, but also reduces the swelling degree of the gelatin fiber shell, thereby enhancing the firmness.

[0031] Furthermore, the nanofiber composite membrane exists in sheet form within the gel. To ensure the injectability of the hydrogel, the material density is relatively loose. However, the shrinkage of poly (N-isopropylacrylamide) (PNIPAM) at body temperature helps the nanofiber composite membrane aggregate into a denser structure, forming a "potential repair membrane" that can reshape within the body to adapt to the shape of the individual uterus. An interpenetrating gel network is formed between the self-healing hydrogel and PNIPAM, with the PNIPAM acting as a reinforcement to ensure the overall robustness of the scaffold. This network is then excreted from the body during menstruation.

[0032] Furthermore, the hydrogel in this invention is a thermosensitive, self-healing hydrogel, using Schiff bases as reversible covalent bonds, triggering the healing mechanism without any external stimulation. The hydrogel is cross-linked within the syringe. During injection, external force causes a shear-thinning effect, causing the gel to spontaneously transform into a liquid state. Upon removal of the external force, the gel returns to its original state, ensuring the product's injectability. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Hereinafter, some specific embodiments of the present invention will be described in detail in an exemplary and non-limiting manner with reference to the accompanying drawings. The same reference numerals in the accompanying drawings indicate the same or similar components or parts. It should be understood by those skilled in the art that these drawings are not necessarily drawn to scale. In the accompanying drawings:

[0034] Figure 1A schematic structural diagram of a temperature-sensitive endometrial repair stent provided by the present invention;

[0035] Figure 2 Schematic diagram of the structure of poly (N-isopropylacrylamide);

[0036] Figure 3 Schematic diagram of the structure of the nanofiber composite membrane;

[0037] Figure 4 Schematic diagram of the structure of covalent bond between aldehyde-modified hyaluronic acid and carboxymethyl chitosan;

[0038] Figure 5 The endometrial repair effect diagram of each group of samples at the 2W dissection time point;

[0039] Figure 6 This is the endometrial repair effect diagram of each group of samples at the 4W dissection time point;

[0040] Figure 7 The graph shows the ratio of endometrial thickness at 2 weeks and 4 weeks after different samples were taken.

[0041] Figure 8 The results of the ratio of gland numbers at 2 weeks and 4 weeks after different samples were taken are shown in the figure;

[0042] Figure 9 The results of the ratio of angiogenesis number at 2 weeks and 4 weeks after different samples were taken are shown;

[0043] Figure 10 This is an electron micrograph of the temperature-sensitive endometrial repair stent of Example 2;

[0044] Figure 11 This is a magnified electron microscopic image of the temperature-sensitive endometrial repair scaffold fiber in Example 2;

[0045] Figure 12 This is an electron microscope image of the endometrial repair stent of comparative example 3;

[0046] Figure 13 This is a magnified electron microscope image of the endometrial repair scaffold fiber in comparative example 3. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be described clearly and completely below. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Example 1

[0048] This embodiment provides a temperature-sensitive endometrial repair stent and a preparation method thereof, comprising the following steps:

[0049] (1) Preparation of nanofiber composite membrane

[0050] a. Weigh 15 g of PLGA and dissolve it in 85 g of hexafluoroisopropanol to obtain a first spinning solution with a PLGA concentration of 15 wt% (two portions of the first spinning solution are prepared);

[0051] b. Weigh 15 g of gelatin and dissolve it in a mixed solvent of water, acetic acid, and ethyl acetate, with the mass ratio of water-acetic acid-ethyl acetate being 1:3:7, to obtain a gelatin solution with a concentration of 15 wt%; simultaneously weigh 1 g of glyoxal and dissolve it in 99 g of pure water to obtain a cross-linking agent solution with a glyoxal concentration of 1 wt%; the gelatin solution and the glyoxal solution are mixed in real time to obtain a second spinning solution;

[0052] c. Take one of the first spinning solutions and perform electrospinning to obtain an intermediate layer, wherein the spinning voltage is 40-60V, the spinning distance is 10cm, and the spinning time is 2h; simultaneously take another first spinning solution and perform coaxial electrospinning with the second spinning solution, wherein the spinning voltage is 40-60V, the spinning distance is 10cm, and the spinning time is 3h; then heat-treat the spun membrane at 80℃ for 2h, raise the temperature to 170℃ and maintain for 3h, thereby obtaining a nanofiber composite membrane (for the specific structure, see Figure 3 ), the thickness of the overall spinning material is 0.1mm and the cross-linking degree is 70%;

[0053] (2) Preparation of dopamine-grafted aldehyde-modified hyaluronic acid

[0054] a. Weigh 0.5 g of hyaluronic acid and dissolve it in 99.5 g of water to obtain a hyaluronic acid solution with a hyaluronic acid concentration of 0.5 wt%. Simultaneously weigh 3 g of sodium periodate and dissolve it in 30 mL of water. Add the solution dropwise to the hyaluronic acid solution and stir at 35°C in the dark for 4 h.

[0055] b. Add 200 mL of ethylene glycol to the above mixture and react for 1 hour, then dialyze through a dialysis bag to remove excess sodium periodate, and then freeze-dry to obtain aldehyde-modified hyaluronic acid;

[0056] c. Weigh 0.5 g of the above-mentioned aldehyde-modified hyaluronic acid and dissolve it in 99.5 g of water. Add an equal mole of dopamine under a nitrogen atmosphere and in the dark. Stir at room temperature for 10 h, then dialysis bag for 3 days and freeze-dry to obtain dopamine-grafted aldehyde-modified hyaluronic acid.

[0057] (3) Preparation of endometrial repair scaffold

[0058] a. Soak the nanofiber composite membrane prepared in step (1) in liquid nitrogen, and then mechanically crush it to control the membrane area to 1~10mm 2 ;

[0059] b. Weigh 1g dopamine grafted aldehyded hyaluronic acid, dissolve it in 98g water, add the membrane from step a, and then add 1g carboxymethyl chitosan and mix well (see the structure). Figure 4 );

[0060] c. Weigh 10 g of poly (N-isopropylacrylamide) (see Figure 2 ), dissolved in 90g water, and then mixed evenly with the mixed system of step b to prepare the endometrial repair stent (for specific structure, see Figure 1 ). Example 2

[0061] This embodiment provides a temperature-sensitive endometrial repair stent and a preparation method thereof, comprising the following steps:

[0062] (1) Preparation of nanofiber composite membrane

[0063] a. Weigh 21.25 g of PLGA and dissolve it in 85 g of hexafluoroisopropanol to obtain a first spinning solution with a PLGA concentration of 20 wt% (the first spinning solution is prepared in two portions);

[0064] b. Weigh 20 g of gelatin and dissolve it in a mixed solvent of water, acetic acid, and ethyl acetate, with the mass ratio of water-acetic acid-ethyl acetate being 1:5:9, to obtain a gelatin solution with a concentration of 20 wt%; simultaneously weigh 3 g of glyoxal and dissolve it in 97 g of pure water to obtain a cross-linking agent solution with a glyoxal concentration of 3 wt%; the gelatin solution and the glyoxal solution are mixed in real time to obtain a second spinning solution;

[0065] c. Take one of the first spinning solutions and perform electrospinning to obtain the middle layer, wherein the spinning voltage is 40-60V, the spinning distance is 13cm, and the spinning time is 3h; simultaneously take another first spinning solution and perform coaxial electrospinning with the second spinning solution, wherein the spinning voltage is 40-60V, the spinning distance is 13cm, and the spinning time is 7h; then heat-treat the spun membrane at 80℃ for 2h, raise the temperature to 170℃ and maintain for 2h, thereby obtaining the nanofiber composite membrane (for the specific structure, see Figure 3 ), the thickness of the overall spinning material is 0.2 mm and the cross-linking degree is 60%;

[0066] (2) Preparation of dopamine-grafted aldehyde-modified hyaluronic acid

[0067] a. Weigh 3 g of hyaluronic acid and dissolve it in 97 g of water to obtain a hyaluronic acid solution with a hyaluronic acid concentration of 3 wt%. Simultaneously weigh 5 g of sodium periodate and dissolve it in 50 mL of water. Add the solution dropwise to the hyaluronic acid solution and stir at 35°C in the dark for 4 h.

[0068] b. Add 200 mL of ethylene glycol to the above mixture and react for 1 hour, then dialyze through a dialysis bag to remove excess sodium periodate, and then freeze-dry to obtain aldehyde-modified hyaluronic acid;

[0069] c. Weigh 2 g of the above-mentioned aldehyde-modified hyaluronic acid and dissolve it in 98 g of water. Add an equal mole of dopamine under a nitrogen atmosphere and in the dark. Stir at room temperature for 10 h, then dialysis bag for 3 days and freeze-dry to obtain dopamine-grafted aldehyde-modified hyaluronic acid.

[0070] (3) Preparation of endometrial repair scaffold

[0071] a. Soak the nanofiber composite membrane prepared in step (1) in liquid nitrogen, and then mechanically crush it to control the membrane area to 1~10mm 2 ;

[0072] b. Weigh 3g of dopamine-grafted aldehyde-modified hyaluronic acid, dissolve it in 94g of water, add the nanofiber composite membrane prepared in step a, and then add 3g of carboxymethyl chitosan and mix well (see the structure for details). Figure 4 );

[0073] c. Weigh 7 g of poly (N-isopropylacrylamide) (see Figure 2 ), dissolved in 93g water, and then mixed evenly with the mixed system of step b to obtain the endometrial repair stent (for the specific structure, see Figure 1 ). Example 3

[0074] This embodiment provides a temperature-sensitive endometrial repair stent and a preparation method thereof, comprising the following steps:

[0075] (1) Preparation of nanofiber composite membrane

[0076] a. Weigh 36.43 g of PLGA and dissolve it in 85 g of hexafluoroisopropanol to obtain a first spinning solution with a PLGA concentration of 30 wt% (the first spinning solution is prepared in two portions);

[0077] b. Weigh 30 g of gelatin and dissolve it in a mixed solvent of water, acetic acid, and ethyl acetate, with the mass ratio of water-acetic acid-ethyl acetate being 1:7:9, to obtain a gelatin solution with a concentration of 30 wt%; simultaneously weigh 5 g of glyoxal and dissolve it in 95 g of pure water to obtain a cross-linking agent solution with a glyoxal concentration of 5 wt%; the gelatin solution and the glyoxal solution are mixed in real time to obtain a second spinning solution;

[0078] c. Take one of the first spinning solutions and perform electrospinning to obtain the first, wherein the spinning voltage is 40-60V, the spinning distance is 16cm, and the spinning time is 4h; simultaneously take another first spinning solution and perform coaxial electrospinning with the second spinning solution, wherein the spinning voltage is 40-60V, the spinning distance is 16cm, and the spinning time is 10h. The spun membrane is heat-treated at 80°C for 1.5h, and then heated to 170°C and maintained for 2h to obtain a nanofiber composite membrane (for specific structure, see Figure 3 ), the thickness of the overall spinning material is 0.4 mm and the cross-linking degree is 50%;

[0079] (2) Preparation of dopamine-grafted aldehyde-modified hyaluronic acid

[0080] a. Weigh 5 g of hyaluronic acid and dissolve it in 95 g of water to obtain a hyaluronic acid solution with a hyaluronic acid concentration of 5 wt%. Simultaneously weigh 7 g of sodium periodate and dissolve it in 70 mL of water. Add the solution dropwise to the hyaluronic acid solution and stir at 35°C in the dark for 4 h.

[0081] b. Add 200 mL of ethylene glycol to the above mixture and react for 1 hour, then dialyze through a dialysis bag to remove excess sodium periodate, and then freeze-dry to obtain aldehyde-modified hyaluronic acid;

[0082] c. Weigh 3 g of the above-mentioned aldehyde-modified hyaluronic acid and dissolve it in 97 g of water. Add an equal mole of dopamine under a nitrogen atmosphere and in the dark. Stir at room temperature for 10 h, then dialysis bag for 3 days and freeze-dry to obtain dopamine-grafted aldehyde-modified hyaluronic acid.

[0083] (3) Preparation of endometrial repair scaffold

[0084] a. Soak the nanofiber composite membrane prepared in step (1) in liquid nitrogen, and then mechanically crush it to control the membrane area to 1~10mm 2 ;

[0085] b. Weigh 5g of dopamine-grafted aldehyde-modified hyaluronic acid, dissolve it in 90g of water, add the nanofiber composite membrane prepared in step a, and then add 5g of carboxymethyl chitosan and mix well (see the structure for details). Figure 4 );

[0086] c. Weigh 5 g of poly (N-isopropylacrylamide) (see Figure 2 ), dissolved in 95g water, and then mixed evenly with the mixed system of step b to obtain the endometrial repair stent (for the specific structure, see Figure 1 ).

[0087] Comparative Example 1

[0088] This embodiment provides an endometrial repair stent and a preparation method thereof. The only difference between the embodiment 2 and the embodiment 2 is that the post-treatment of the nanofiber composite membrane prepared in step (1) is different. Specifically, the nanofiber composite membrane is first cut into small pieces, dispersed in a tert-butanol solution, and stirred continuously at 13,000 rpm for 15 minutes to achieve solution homogenization, prepare single short fibers, and freeze-dry for later use.

[0089] Comparative Example 2

[0090] This embodiment provides an endometrial repair stent and a preparation method thereof, which differs from Example 2 only in that poly (N-isopropylacrylamide) is not added in step (3).

[0091] Comparative Example 3

[0092] This embodiment provides an endometrial repair stent and a preparation method thereof, which differs from Example 2 only in that gelatin is used as a single component for electrospinning in step (1) to prepare an electrospun membrane.

[0093] The performance of the products prepared in the above examples and comparative examples is characterized as follows:

[0094] 1. Cell proliferation assay

[0095] Samples prepared in Examples 1-3 and Comparative Examples 1-3 were tested for cell proliferation at 37°C. Equal amounts of hydrogel-nanofiber composite scaffolds (i.e., endometrial repair scaffolds) were added to a 24-well plate, followed by a human endometrial epithelial cell suspension. The plates were incubated at 37°C, with the medium replaced every other day. After three days of incubation, cell proliferation was measured using the MTT assay.

[0096] The cell proliferation rate was calculated according to the following formula:

[0097] Proliferation rate of feed cells (%) Proliferation rate (%) = ×100%

[0098] OD0: the average absorbance of each well in the blank control group at 570 nm; OD1: the average absorbance of each well in the test group at 570 nm.

[0099] Table 1 Cell proliferation rate of nanofiber-hydrogel composite materials (%)

[0100]

[0101] The test results show that Examples 1-3 retain the three-dimensional porous structure of the membrane and the biomimetic extracellular matrix structure, which is more conducive to cell adhesion and proliferation, and the cell proliferation effect is good. In Comparative Example 1, there is no potential repair membrane layer, the fibers are in a single dispersed state, and the cell adhesion and proliferation are poor. In Comparative Example 3, gelatin is spun as a single component. In the hydrogel, the fibers swell and lose the three-dimensional porous structure, resulting in relatively poor cell proliferation. In Comparative Example 2, there is no reinforcing rib structure, the potential repair membrane is loose, and cell proliferation is poor.

[0102] 2. Rabbit endometrial repair test

[0103] A standardized rabbit model of endometrial injury and intrauterine adhesions was established using mechanical injury combined with lipopolysaccharide induction. Two incisions were made bilaterally in the mid-uterine segment, 3 cm apart. Endometrial tissue was scraped in a circular pattern using a spatula, damaging the myometrium to a depth of 3 cm. A cotton thread soaked in lipopolysaccharide was placed at the site of mechanical injury in the uterus, with the other end remaining outside the abdominal wall and removed 24 hours later. The model group received no treatment after modeling. The experimental group received injections of the samples from Examples 1-3 and Comparative Examples 1-3, filling the model segment. Uterine sections were dissected at two and four weeks, respectively, and the ratio of endometrial thickness, glandular count, and angiogenesis between the model and normal segments was measured to evaluate the repair efficacy.

[0104] Table 2 Statistics of repair effects of thermosensitive hydrogel-nanofiber composite materials

[0105]

[0106] Combine Figures 5 to 9 As shown, the endometrial thickness ratio, gland number ratio, and angiogenesis number ratio of 2W and 4W Examples 1 to 3 are significantly higher than those of Comparative Examples 1 to 3, and the repair effect is better than that of Comparative Examples 1 to 3. The repair effect of Comparative Example 1 is the worst because the three-dimensional porous structure of the electrospun membrane is not present in Comparative Example 1, resulting in poor cell adhesion and proliferation effects; the rib structure of the hydrogel is not present in Comparative Example 2, resulting in poor mechanical properties of the gel, a weak overall structure, and easy dispersion, resulting in a weak potential repair membrane structure and poor repair effect; and the fact that the hydrophobic material is not used to prepare the basic skeleton of the electrospun membrane in Comparative Example 3 results in the fiber structure of the electrospun membrane being easily swollen by water, poor cell adhesion, and poor proliferation effect.

[0107] 3. Tensile strength test

[0108] Weigh 5g of each hydrogel-nanofiber composite scaffold prepared in Examples 1-3 and Comparative Examples 1-3. Place in a water bath at 37°C for 0.5 hours and remove after 0.5 hours. Mechanical properties of the hydrogel-nanofiber composite scaffolds were tested using a universal testing machine with a 100N sensor. The composite scaffolds were fabricated into a dumbbell shape with a total length of 40mm, a gauge length of 12mm, a width of 5mm, and a thickness of 1mm. Three parallel sets were prepared at a tensile speed of 50mm / min.

[0109] Calculate the tensile strength according to the following formula:

[0110] Tensile strength (MPa) =

[0111] Table 3 Experimental results of tensile strength of hydrogel-nanofiber composite scaffolds (MPa)

[0112]

[0113] The test results show that the tensile strength of Examples 1 to 3 is relatively good. In Comparative Example 1, there is no potential repair membrane layer, the fibers are in a single dispersed state, and the mechanical properties are relatively poor. In Comparative Example 2, there is no interpenetrating network in the form of reinforcing ribs, and the mechanical properties are poor.

[0114] 4. Water blocking time determination

[0115] Hydrogel adhesion strength test: Weigh 5 g of the hydrogel-nanofiber composite scaffolds prepared in Examples 1 to 3 and Comparative Example 2, and evenly apply them between two pieces of pigskin tissue. A tensile test was performed at a speed of 10 mm / min at 37°C to characterize the adhesion strength between the pigskin tissue and the hydrogel. Three parallel groups were tested in each group.

[0116] Table 4 Average adhesion strength of nanofiber-hydrogel composite scaffolds (kPa)

[0117]

[0118] As can be seen from the above table, Examples 1 to 3 have good tissue adhesion, while Comparative Example 2 has poor tissue adhesion due to the lack of reinforcing ribs to form a cross-linked network with the self-healing hydrogel.

[0119] 5. In vitro degradation experiment

[0120] The freeze-dried samples of Examples 1-3 and Comparative Examples 1-3 were weighed and recorded as W0. Each sample was immersed in a PBS buffer solution at 37°C under aseptic conditions and allowed to stand for 12 hours to fully swell. Samples were taken 7, 14, and 21 days after swelling, and the weight after freeze-drying was W1. The degradation rate was calculated according to the following formula: Degradation rate (%) =

[0121] Table 5 In vitro degradation rate of hydrogel-nanofiber composite scaffolds (%)

[0122]

[0123] The test results show that all samples began to degrade over time. Examples 1-3 and Comparative Examples 1 and 3 maintained a largely intact structure after 21 days. However, the hydrogel in Comparative Example 2 degraded more rapidly due to the lack of PNIPAM (poly (N-isopropylacrylamide)) reinforcement.

[0124] 6. Electron microscopy observation of the swelling of electrospun fiber membrane

[0125] Samples from Example 2 and Comparative Example 3 were taken, the gel on the surface of the fiber membrane was washed clean, and then freeze-dried, and the swelling of the fiber surface was observed under an electron microscope.

[0126] The electron microscopy results show that the fibers of Example 2 can maintain a complete three-dimensional porous structure in the hydrogel (electron microscopy structure as shown in FIG. Figure 10 and Figure 11 As shown), while Comparative Example 3 is better than the one that does not use hydrophobic material as the basic skeleton structure of the fiber, the fiber swells after absorbing water (the electron microscope structure is shown in Figure 12 and Figure 13 shown).

[0127] In summary, the nanofiber composite membrane provided in the present invention is present in the hydrogel in sheet form, retaining the three-dimensional porous extracellular matrix biomimetic structure of the fiber membrane, which is beneficial to the adhesion and proliferation of cells and ensures the repair effect. The middle sandwich structure of the composite membrane is made of hydrophobic material, the purpose of which is to reduce the swelling of the material in the hydrogel and ensure the integrity of the membrane structure. The upper and lower fibers of the composite membrane are a core-shell structure. The PLGA of the core layer serves as the basic skeleton, plays a supporting role and reduces fiber swelling; the shell layer is gelatin, which is cross-linked in real time using glyoxal to control the cross-linking degree to 50%~70%.

[0128] The remaining gelatin amino groups were grafted with dopamine on hyaluronic acid (see Figure 4 ), which not only ensures the bonding strength between gelatin fiber and hyaluronic acid gel, but also forms physical crosslinks through chemical crosslinking of glyoxal and complexation reaction of dopamine and gelatin. The double crosslinking reduces the swelling of gelatin fiber shell and enhances its firmness.

[0129] After hyaluronic acid is modified, a dopamine structure is added, which is beneficial for tissue adhesion and can better adsorb gelatin fibers, ensuring the connection between gelatin fiber membranes and serving as a connecting bridge between tissues and composite membranes.

[0130] The lower critical solution temperature (LCST) of PNIPAM hydrogel is about 32°C. When the temperature is higher than the LCST, the hydrophobic effect of the hydrogel system dominates. The interaction between hydrophobic groups drives the polymer network to shrink and precipitate from the water to form reinforcing ribs, which reduces the volume of the gel and drives the nanofiber composite membrane to shrink and aggregate, adapting to different uterine cavity structures and reshaping in the body to form a dense "potential repair membrane."

[0131] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. An endometrial repair stent, characterized in that: The nanofiber composite membrane is prepared by combining a sheet-like nanofiber composite membrane with dopamine-grafted aldehyde-modified hyaluronic acid, dynamically cross-linking the membrane with carboxymethyl chitosan based on a Schiff base reaction, and then compounding the membrane with poly (N-isopropyl acrylamide). The mass ratio of the dopamine-grafted aldehyde-modified hyaluronic acid, the carboxymethyl chitosan, and the poly (N-isopropyl acrylamide) is (1-9): (1-9): (1-90). The thickness of the nanofiber composite membrane is 0.1-0.4 mm. The nanofiber composite membrane includes an upper nanofiber layer, a lower nanofiber layer and an intermediate nanofiber layer formed between the upper nanofiber layer and the lower nanofiber layer; the nanofibers in the upper nanofiber layer and the lower nanofiber layer are both core-shell structures, and the shell layer raw material components in the core-shell structure include gelatin and a cross-linking agent; the raw material components of the core layer and the intermediate nanofiber layer in the core-shell structure are both PLGA.

2. The endometrial repair stent according to claim 1, characterized in that: The mass ratio of the gelatin to the cross-linking agent is (15-30): (1-5); the mass ratio of the PLGA to the gelatin is 1:

1.

3. The endometrial repair stent according to claim 2, characterized in that: The method for preparing the nanofiber composite membrane comprises the following steps: S1: dissolving PLGA in a first solvent to prepare a first spinning solution with a concentration of 15-30 wt%, and electrospinning to prepare an intermediate nanofiber layer; S2: dissolving gelatin in a second solvent and mixing it with a cross-linking agent solution in real time to prepare a second spinning solution; S3: taking the first spinning solution in step S1 and coaxially electrospinning it with the second spinning solution in step S2 to prepare an upper nanofiber layer and a lower nanofiber layer respectively; S4: performing gradient temperature-raising cross-linking on the three nanofiber layers prepared above, thereby preparing a nanofiber composite membrane.

4. The endometrial repair stent according to claim 3, characterized in that: In step S1, the first solvent is hexafluoroisopropanol; in step S2, the second solvent is a mixture of water, acetic acid and ethyl acetate, and the mass ratio of the water, the acetic acid and the ethyl acetate is (1-9): (1-9): (1-9); the concentration of the gelatin solution is 15-30wt%; the cross-linking agent is a mixture of one or more selected from formaldehyde, glyoxal, glutaraldehyde, genipin and tannic acid, and the concentration of the cross-linking agent solution is 1-5wt%.

5. The endometrial repair stent according to claim 3, characterized in that: In step S1, the electrospinning voltage is 40-60 V, the spinning distance is 10-16 cm, and the spinning time is 2-6 h; in step S3, the coaxial electrospinning voltage is 40-60 V, the spinning distance is 10-16 cm, and the spinning time is 3-10 h; in step S4, the cross-linking temperature is 80-170 ° C, and the cross-linking degree is 50-70%.

6. The method for preparing an endometrial repair stent according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) After dissolving hyaluronic acid, an oxidant is added to oxidize it, and then a reducing agent is added to terminate the oxidation, and the aldehyde-modified hyaluronic acid is obtained by freeze-drying; (2) dissolving the aldehyde-modified hyaluronic acid, adding dopamine to carry out a grafting reaction, and freeze-drying to obtain the dopamine-grafted aldehyde-modified hyaluronic acid; (3) The dopamine-grafted aldehyde-modified hyaluronic acid is dissolved, and then the nanofiber composite membrane sheet, carboxymethyl chitosan and poly (N-isopropylacrylamide) are added in sequence and mixed evenly to prepare the endometrial repair scaffold.

7. The method for preparing an endometrial repair stent according to claim 6, characterized in that: In step (1), the concentration of the hyaluronic acid is 0.5-5 wt %; the oxidizing agent is a mixture of one or more selected from sodium periodate, basic copper carbonate, chromic anhydride pyridine hydrochloride and lead tetraacetate; the reducing agent is ethylene glycol; the reaction temperature during oxidation is 20-40° C., and the reaction time is 3-6 h.

8. The method for preparing an endometrial repair stent according to claim 6, characterized in that: In step (2), the concentration of the aldehyde-modified hyaluronic acid is 0.5-3 wt %; the molar ratio of the aldehyde-modified hyaluronic acid to the dopamine is 1:1; the grafting reaction temperature is room temperature, and the reaction time is 10 h.

9. The method for preparing an endometrial repair stent according to claim 6, characterized in that: In step (3), the concentration of the dopamine-grafted aldehyde-modified hyaluronic acid is 0.5-3 wt %.

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