Endometrial repair stent and preparation method thereof

Abstract

The invention relates to the technical field of biomedicine, and provides an endometrial repair stent and a preparation method thereof.The endometrial repair stent is prepared by taking a flaky nanofiber composite membrane as a substrate, combining the flaky nanofiber composite membrane with modified hyaluronic acid, then conducting dynamic crosslinking with carboxymethyl chitosan and then conducting compounding with poly N-isopropylacrylamide; the mass ratio of the modified hyaluronic acid to the carboxymethyl chitosan to the poly (N-isopropylacrylamide) is (1-9): (1-9): (1-90); the thickness of the nanofiber composite membrane is 0.1-0.4 mm, and the nanofiber composite membrane can be applied to treatment of intrauterine adhesion. According to the endometrial repair stent, the nanofiber composite membrane and the temperature-sensitive hydrogel are combined, a uterine cavity is fully paved by utilizing the fluidity of the hydrogel, and the three-dimensional structure of the nanofiber can be used for orderly inducing a wound surface, so that the adhesion and proliferation of cells are facilitated, scar formation is reduced, the repair effect is ensured, and regeneration is promoted. The two parts are combined, so that the plasticity of the whole structure is enhanced, the supporting performance is improved, the indwelling time in the uterine cavity is long, and the double effects of repairing and isolating are fully achieved.

Description

Technical Field

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

[0002] Intrauterine adhesion is a common intrauterine disease in gynecology. Due to the trauma of pregnant or non-pregnant uterus, the basal layer of the endometrium is damaged, resulting in partial or complete occlusion of the uterine cavity, thus leading to menstrual disorders, infertility or recurrent miscarriages, etc. Currently, hysteroscopic resection of intrauterine adhesions is the standard surgical procedure for the treatment of intrauterine adhesions. However, it is difficult for the endometrial wound surface to regenerate after electrocautery separation during the operation, and the pathological repair of the thermally damaged tissue wound surface forms inflammatory granulation tissue and fibrous scars, causing the bare anterior and posterior walls of the uterine cavity without endometrial coverage to adhere again, resulting in infertility. Therefore, measures such as promoting endometrial repair after surgery and placing a barrier to prevent adhesion are particularly important.

[0003] On the one hand, placing an intrauterine device, an intrauterine support balloon, and sodium hyaluronate gel in the uterus after surgery are the main clinical means used at home and abroad to solve the adhesion of damaged uterus. Its principle is mainly to prevent adhesion formation by relying on a simple physical barrier effect. On the other hand, with the development of tissue engineering materials, people have begun to use medical materials for endometrial tissue repair to prevent the occurrence of re-adhesion. The two treatment methods have single functions, and currently there is no dual-function product that can both promote repair and prevent adhesion on the market.

[0004] CN114652892A provides a medical membrane material and a preparation method thereof, an intrauterine adhesion prevention device, and a medical device. The prepared medical membrane material includes a substrate and chemotactic factors grafted on the surface of the substrate. The substrate includes poly(lactic-co-glycolic acid) and a reactive polymer, and the reactive polymer includes gelatin and / or dextran. Although this medical membrane material can promote endometrial repair, its shape does not completely match the shape of the uterine cavity. After being placed in the uterine cavity, it cannot fill every corner of the uterine cavity, and cannot accurately isolate both sides of the uterine cavity and the uterine horn, so the effect of preventing intrauterine re-adhesion is not good.

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

[0006] Therefore, there is a lack of an ideal intrauterine adhesion prevention product in clinical practice that has both tissue repair and sufficient isolation effect to solve the functional defects of traditional hydrogels and simple biological membranes. Summary of the Invention

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

[0008] To achieve the above object, the technical solution adopted by the present invention is: In a first aspect of the present invention, there is provided an endometrial repair stent, which is prepared by using a sheet-like nanofiber composite membrane as a substrate, combining it with dopamine-grafted aldehyde-modified hyaluronic acid, and then dynamically cross-linking with carboxymethyl chitosan based on Schiff base reaction to form a dynamic cross-linked hydrogel, and then compounding with poly(N-isopropylacrylamide); wherein, the mass ratio of the dopamine-grafted aldehyde-modified hyaluronic acid, the carboxymethyl chitosan and the poly(N-isopropylacrylamide) is (1~9):(1~9):(1~90); The thickness of the nanofiber composite membrane is 0.1~0.4 mm.

[0009] 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 in the core-shell structure and the intermediate nanofiber layer are both PLGA, and the mass ratio of the PLGA to the gelatin is 1:1.

[0010] Furthermore, the preparation method of the nanofiber composite membrane includes the following steps: S1: Dissolve PLGA in a first solvent to prepare a first spinning dope with a concentration of 15~30 wt%, and electrospun to obtain the intermediate nanofiber layer; S2: Dissolve gelatin in a second solvent and mix it with a cross-linking agent solution in real time to prepare a second spinning dope; S3: Take another portion of the first spinning dope in step S1 and perform coaxial electrospinning with the second spinning dope in step S2 to respectively obtain the upper nanofiber layer and the lower nanofiber layer; S4: Perform gradient temperature cross-linking on the three-layer nanofiber layer prepared above to obtain the nanofiber composite membrane.

[0011] Further, 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 water, acetic acid and ethyl acetate is (1-9):(1-9):(1-9); the concentration of the gelatin solution is 15-30 wt%; the crosslinking agent is a mixture composed of one or more selected from formaldehyde, glyoxal, glutaraldehyde, genipin and tannic acid, and the concentration of the crosslinking agent solution is 1-5 wt%.

[0012] Further, 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 crosslinking temperature is 80-170 °C, and the crosslinking degree is 50-70%.

[0013] The second aspect of the present invention provides a preparation method of an endometrial repair stent, comprising the following steps: (1) Dissolve hyaluronic acid and add an oxidant for oxidation, then add a reducing agent to terminate the oxidation, and freeze-dry to obtain aldehyde group-modified hyaluronic acid; (2) Dissolve the aldehyde group-modified hyaluronic acid and add dopamine for grafting reaction, and freeze-dry to obtain the dopamine-grafted aldehyde group-modified hyaluronic acid; (3) Dissolve the dopamine-grafted aldehyde group-modified hyaluronic acid, and then sequentially add the film of the nanofiber composite membrane, carboxymethyl chitosan and poly(N-isopropylacrylamide) and mix evenly to prepare the endometrial repair stent.

[0014] Further, in step (1), the concentration of the hyaluronic acid is 0.5-5 wt%; the oxidant is a mixture composed of one or more selected from sodium periodate, basic copper carbonate, chromium anhydride pyridine hydrochloride and lead tetraacetate; the reducing agent is ethylene glycol; the oxidation reaction temperature is 20-40 °C, and the reaction duration is 3-6 h.

[0015] Further, in step (2), the concentration of the aldehyde group-modified hyaluronic acid is 0.5-3 wt%; the molar ratio of the aldehyde group-modified hyaluronic acid to the dopamine is 1:1; the grafting reaction temperature is room temperature, and the reaction duration is 10 h.

[0016] Further, in step (3), the concentration of the dopamine-grafted aldehyde group-modified hyaluronic acid is 0.5-3 wt%.

[0017] Due to the application of the above technical solutions, the present invention has the following advantages compared with the prior art: In the present invention, a nanofiber composite membrane is combined with a thermosensitive hydrogel. The fluidity of the hydrogel is utilized to cover every corner of the uterine cavity. The three-dimensional structure of the nanofibers can orderly induce the wound surface, facilitate cell adhesion and proliferation, reduce scar formation, ensure the repair effect, and promote regeneration. The combination of the two can enhance the plasticity and increase the support of the overall structure, with a long retention time in the uterine cavity, fully achieving the dual effects of repair and isolation.

[0018] In the composite membrane, a hydrophobic material is selected for the middle layer to reduce its swelling in the hydrogel and ensure the integrity of the membrane structure; the upper and lower layer fibers are of a core-shell structure. The PLGA in the core layer serves as the basic framework, providing support and reducing fiber swelling; the shell layer is gelatin, which is crosslinked in real time with a crosslinking agent and the degree of crosslinking is controlled. Moreover, the structures on both sides of the composite membrane are the same, without distinguishing between the front and back, ensuring the same repair effect on both sides of the material.

[0019] Furthermore, the amino groups of the remaining gelatin after crosslinking with the crosslinking agent are grafted with aldehyde-modified hyaluronic acid modified with dopamine. Through chemical crosslinking and physical crosslinking under the covalent binding of dopamine and gelatin, both the binding firmness between the gelatin fibers and the hyaluronic acid gel is ensured, and the swelling degree of the gelatin fiber shell layer is reduced, enhancing the firmness.

[0020] Furthermore, the nanofiber composite membrane exists in the form of flakes in the gel. To ensure the injectability of the hydrogel, the density of the material is relatively loose. Poly(N-isopropylacrylamide) (PNIPAM) shrinks and loses water at body temperature, which can help the nanofiber composite membrane aggregate into a denser structure, forming a "potential repair membrane", enabling the repair membrane to reshape in vivo according to the shape of different people's uteruses. An interpenetrating gel network is formed between the self-healing hydrogel and PNIPAM. PNIPAM serves as a reinforcing rib to ensure the overall firmness of the stent, and the network structure is excreted from the body during menstruation.

[0021] Furthermore, the hydrogel in the present invention is a thermosensitive self-healing hydrogel, with Schiff base as a reversible covalent bond, triggering the healing mechanism without any external stimulation. The hydrogel is in a crosslinked state in the syringe. During injection, under the action of external force, a shear thinning effect occurs, and the gel state spontaneously transforms into a liquid state, and returns to the gel state after the external force disappears, ensuring the injectability of the product. Description of the Drawings

[0022] Some specific embodiments of the present invention will be described in detail hereinafter with reference to the drawings in an exemplary but non-limiting manner. The same reference numerals in the drawings denote the same or similar components or parts. Those skilled in the art should understand that these drawings are not necessarily drawn to scale. In the drawings: Figure 1 is a schematic structural diagram of a thermosensitive endometrial repair stent provided by the present invention; Figure 2 It is a schematic structural diagram of poly N - isopropylacrylamide; Figure 3 It is a schematic structural diagram of the nanofiber composite membrane; Figure 4 It is a schematic structural diagram of the formation of a covalent bond between aldehyde - modified hyaluronic acid and carboxymethyl chitosan; Figure 5 It is the endometrial repair effect diagram of each group of samples at the 2W dissection time point; Figure 6 It is the endometrial repair effect diagram of each group of samples at the 4W dissection time point; Figure 7 It is the result diagram of the ratio of endometrial thickness at the 2W and 4W time points for different samplings; Figure 8 It is the result diagram of the ratio of the number of glands at the 2W and 4W time points for different samplings; Figure 9 It is the result diagram of the ratio of the number of angiogenesis at the 2W and 4W time points for different samplings; Figure 10 It is the electron micrograph of the thermosensitive endometrial repair stent in Example 2; Figure 11 It is the magnified electron micrograph of the fibers of the thermosensitive endometrial repair stent in Example 2; Figure 12 It is the electron micrograph of the endometrial repair stent in Comparative Example 3; Figure 13 It is the magnified electron micrograph of the fibers of the endometrial repair stent in Comparative Example 3. Detailed implementation manners

[0023] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention. Example 1 This example provides a thermosensitive endometrial repair stent and its preparation method, including the following steps:

[0024] (1) Preparation of the nanofiber composite membrane a. Weigh 15 g of PLGA and dissolve it in 85 g of hexafluoroisopropanol to obtain a first spinning dope with a PLGA concentration of 15 wt% (two portions of the first spinning dope are prepared); b. Weigh 15 g of gelatin and dissolve it in a mixed solvent of water, acetic acid and ethyl acetate with a mass ratio of water - acetic acid - ethyl acetate of 1:3:7 to obtain a gelatin solution with a concentration of 15 wt%. Synchronously, weigh 1 g of glyoxal and dissolve it in 99 g of pure water to obtain a cross - linker solution with a glyoxal concentration of 1 wt%. Mix the gelatin solution and the glyoxal solution in real - time to obtain the second spinning dope. c. Take one portion of the first spinning dope and perform electrospinning to obtain the intermediate layer. Among them, the electrospinning voltage is 40 - 60 V, the electrospinning distance is 10 cm, and the electrospinning time is 2 h. Synchronously, take another portion of the first spinning dope and perform coaxial electrospinning with the above - mentioned second spinning dope. The electrospinning voltage is 40 - 60 V, the electrospinning distance is 10 cm, and the electrospinning time is 3 h. Subsequently, heat - treat the electrospun membrane at 80 °C for 2 h, then raise the temperature to 170 °C and keep it for another 3 h to obtain the nanofiber composite membrane (for the specific structure, see Figure 3 ). The thickness of the overall spinning material is 0.1 mm, and the cross - linking degree is 70%. (2) Preparation of dopamine - grafted aldehyde - modified hyaluronic acid 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 concentration of 0.5 wt%. Synchronously, weigh 3 g of sodium periodate and dissolve it in 30 mL of water, and gradually add it drop - by - drop to the hyaluronic acid solution. Stir and react under dark conditions at 35 °C for 4 h. b. Add 200 mL of ethylene glycol to the above - mentioned mixed system and react for 1 h. Then, remove the excessive sodium periodate by dialysis through a dialysis bag, and obtain aldehyde - modified hyaluronic acid after conventional freeze - drying. c. Weigh 0.5 g of the above - mentioned aldehyde - modified hyaluronic acid and dissolve it in 99.5 g of water. Under an N₂ atmosphere and in the dark, add an equimolar amount of dopamine, stir at room temperature for 10 h, then treat it through a dialysis bag for 3 days, and obtain dopamine - grafted aldehyde - modified hyaluronic acid after conventional freeze - drying. (3)Preparation of endometrial repair stent a. Immerse the nanofiber composite membrane prepared in step (1) in liquid nitrogen, and then mechanically crush it, controlling the membrane area to be 1 - 10 mm 2 ; b. Weigh 1 g of dopamine - grafted aldehyde - modified hyaluronic acid and dissolve it in 98 g of water. Add the membrane pieces from step a, and then add 1 g of carboxymethyl chitosan and mix evenly (for the structure, see Figure 4 ); c. Weigh 10 g of poly(N - isopropylacrylamide) (for the specific structure, see Figure 2 ), dissolve it in 90 g of water, and then mix it evenly with the mixed system in step b to obtain the endometrial repair stent (for the specific structure, see Figure 1 ). Example 2 This embodiment provides a temperature-sensitive endometrial repair stent and a preparation method thereof, including the following steps:

[0025] (1) Preparation of nanofiber composite membrane a. Weigh 21.25 g of PLGA and dissolve it in 85 g of hexafluoroisopropanol to obtain a first spinning dope with a PLGA concentration of 20 wt% (two portions of the first spinning dope are prepared); b. Weigh 20 g of gelatin and dissolve it in a mixed solvent of water, acetic acid and ethyl acetate with a mass ratio of water - acetic acid - ethyl acetate of 1:5:9 to obtain a gelatin solution with a gelatin 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%; mix the gelatin solution and the glyoxal solution in real time to obtain a second spinning dope; c. Take one portion of the first spinning dope for electrospinning to obtain an intermediate layer. Among them, the electrospinning voltage is 40 - 60 V, the electrospinning distance is 13 cm, and the electrospinning duration is 3 h; simultaneously take the other portion of the first spinning dope and perform coaxial electrospinning with the above-mentioned second spinning dope, the electrospinning voltage is 40 - 60 V, the electrospinning distance is 13 cm, and the electrospinning duration is 7 h; then heat-treat the electrospun membrane at 80 °C for 2 h, raise the temperature to 170 °C and keep it for another 2 h to obtain 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%; (2) Preparation of dopamine-grafted aldehyde-functionalized hyaluronic acid 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, and gradually add it dropwise to the hyaluronic acid solution, and stir and react in the dark at 35 °C for 4 h; b. Add 200 mL of ethylene glycol to the above mixed system and react for 1 h, then dialyze through a dialysis bag to remove the excess sodium periodate, and obtain aldehyde-functionalized hyaluronic acid after conventional freeze-drying; c. Weigh 2 g of the above aldehyde-functionalized hyaluronic acid and dissolve it in 98 g of water. Under a N2 atmosphere and in the dark, add an equimolar amount of dopamine, stir at room temperature for 10 h, then treat it with a dialysis bag for 3 days, and obtain dopamine-grafted aldehyde-functionalized hyaluronic acid after conventional freeze-drying; (3) Preparation of endometrial repair stent a. Immerse the nanofiber composite membrane prepared in step (1) in liquid nitrogen, and then mechanically crush it, controlling the area of the membrane pieces to be 1 - 10 mm 2 ; b. Weigh 3 g of dopamine-grafted aldehyde-functionalized hyaluronic acid and dissolve it in 94 g of water. Add the nanofiber composite membrane pieces from step a, and then add 3 g of carboxymethyl chitosan and mix evenly (for the structure, see Figure 4 ); c. Weigh 7 g of poly(N-isopropylacrylamide) (for the specific structure, see Figure 2 ), dissolve it in 93 g of water, and then mix it evenly with the mixed system in step b to obtain an endometrial repair stent (for the specific structure, see Figure 1 ). Example 3 This example provides a temperature-sensitive endometrial repair stent and its preparation method, including the following steps:

[0026] (1) Preparation of nanofiber composite membrane a. Weigh 36.43 g of PLGA and dissolve it in 85 g of hexafluoroisopropanol to obtain a first spinning dope with a PLGA concentration of 30 wt% (two portions of the first spinning dope are prepared); b. Weigh 30 g of gelatin and dissolve it in a mixed solvent of water, acetic acid and ethyl acetate with a mass ratio of water - acetic acid - ethyl acetate of 1:7:9 to obtain a solution with a gelatin 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%; mix the gelatin solution and the glyoxal solution in real time to obtain a second spinning dope; c. Take one portion of the first spinning dope for electrospinning to obtain a first one, where the electrospinning voltage is 40 - 60 V, the electrospinning distance is 16 cm, and the electrospinning duration is 4 h; simultaneously take the other portion of the first spinning dope and perform coaxial electrospinning with the above-mentioned second spinning dope, with an electrospinning voltage of 40 - 60 V, an electrospinning distance of 16 cm, and an electrospinning duration of 10 h. Heat-treat the electrospun membrane at 80 °C for 1.5 h, then raise the temperature to 170 °C and keep it for 2 h to obtain a nanofiber composite membrane (for the specific structure, see Figure 3 ), the thickness of the overall spinning material is 0.4 mm, and the cross-linking degree is 50%; (2) Preparation of dopamine-grafted aldehyde-functionalized hyaluronic acid 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, and gradually add it dropwise to the hyaluronic acid solution, and stir and react in the dark at 35 °C for 4 h; b. Add 200 mL of ethylene glycol to the above mixed system and react for 1 h, then dialyze through a dialysis bag to remove the excessive sodium periodate, and perform conventional freeze-drying to obtain aldehyde-functionalized hyaluronic acid; c. Weigh 3 g of the above-mentioned aldehyde-functionalized hyaluronic acid and dissolve it in 97 g of water. Under a N2 atmosphere and in the dark, add an equimolar amount of dopamine, stir at room temperature for 10 h, then treat it with a dialysis bag for 3 days, and perform conventional freeze-drying to obtain dopamine-grafted aldehyde-functionalized hyaluronic acid; (3) Preparation of endometrial repair stent a. Immerse the nanofiber composite membrane prepared in step (1) in liquid nitrogen, then mechanically crush it, and control the membrane area to be 1 - 10 mm 2 ; b. Weigh 5 g of dopamine-grafted aldehyde-functionalized hyaluronic acid, dissolve it in 90 g of water, add the nanofiber composite membrane sheet from step a, and then add 5 g of carboxymethyl chitosan and mix evenly (the structure is shown in Figure 4 ); c. Weigh 5 g of poly(N-isopropylacrylamide) (the specific structure is shown in Figure 2 ), dissolve it in 95 g of water, and then mix it evenly with the mixture system from step b to obtain an endometrial repair stent (the specific structure is shown in Figure 1 ).

[0027] Comparative Example 1 This example provides an endometrial repair stent and its preparation method. The difference from Example 2 is only in the post-treatment of the nanofiber composite membrane prepared in step (1). Specifically, it is first cut into small pieces, dispersed in tert-butanol solution, and continuously stirred at 13,000 rpm for 15 min using a stirrer to achieve solution homogenization, preparing single short fibers, and freeze-drying for later use.

[0028] Comparative Example 2 This example provides an endometrial repair stent and its preparation method. The difference from Example 2 is only that poly(N-isopropylacrylamide) is not added in step (3).

[0029] Comparative Example 3 This example provides an endometrial repair stent and its preparation method. The difference from Example 2 is only that gelatin is used as a single component for electrospinning in step (1) to prepare an electrospun membrane.

[0030] In the present invention, the products prepared in the above examples and comparative examples are characterized in terms of performance, as follows: 1. Cell proliferation experiment Take the samples prepared in Examples 1 - 3 and Comparative Examples 1 - 3, and detect the cell proliferation at 37°C. Add the same amount of hydrogel-nanofiber composite stent (i.e., endometrial repair stent) to a 24-well plate in sequence, add human endometrial epithelial cell suspension, place it in culture at 37°C, and change the medium every other day. After culturing for 3 days, use the MTT method to measure cell proliferation.

[0031] The cell proliferation rate is calculated according to the following formula: Increased cell proliferation rate (%) = × 100% Wherein: 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.

[0032] Table 1 Cell proliferation rate of nanofiber-hydrogel composite material (%)

[0033] It can be seen from the test results that Examples 1 to 3 retained the three-dimensional porous structure of the membrane and the biomimetic extracellular matrix structure, which was more conducive to cell adhesion and proliferation, and the cell proliferation effect was better. In Comparative Example 1, there was no potential repair membrane layer, and the fibers were in a single and dispersed state, and the cell adhesion and proliferation states were poor. In Comparative Example 3, gelatin was used as a single component for spinning. In the hydrogel, the fibers swelled and lost the three-dimensional porous structure, and the cell proliferation was relatively poor. In Comparative Example 2, there was no reinforcing rib structure, and the potential repair membrane was relatively loose, and the cell proliferation was poor.

[0034] 2. Rabbit endometrial repair test A standardized animal test model of rabbit endometrial injury combined with intrauterine adhesion was established by mechanical injury combined with lipopolysaccharide induction method: two incisions were made in the middle part of both sides of the rabbit uterus, the incision spacing was 3 cm, the endometrial tissue was scraped off circularly with a curette, and the damage was made to the muscular layer, the damage length was 3 cm. One end of a cotton thread infiltrated with lipopolysaccharide was buried at the mechanical injury site of the animal uterus, and the other end of the cotton thread was left outside the abdominal wall and pulled out after 24 h. Model group: no treatment after modeling; Experimental group: Inject the samples of Examples 1 to 3 and Comparative Examples 1 to 3 to fill the modeled segment. The specimens were dissected and taken at the 2nd week and 4th week respectively, and the uterus of the normal segment and the modeled segment were taken to measure the ratio of the endometrial thickness, the number of glands, and the number of angiogenesis in the modeled segment to the normal segment to evaluate the repair effect.

[0035] Table 2 Statistical table of the repair effect of thermosensitive hydrogel-nanofiber composite material

[0036] Combined Figures 5 to 9 As shown, the ratios of endometrial thickness, gland number, and angiogenesis number of Examples 1 to 3 at 2W and 4W were significantly higher than those of Comparative Examples 1 to 3, and the repair effect was better than that of Comparative Examples 1 to 3. The repair effect of Comparative Example 1 was the worst because there was no three-dimensional porous structure of the electrospun membrane in Comparative Example 1, and the cell adhesion and proliferation effects were poor; there was no reinforcing rib structure of the hydrogel in Comparative Example 2, the mechanical properties of the gel were poor, the overall structure was not firm, and it was easy to disperse, resulting in an unstable structure of the potential repair membrane and a poor repair effect; in Comparative Example 3, a hydrophobic material was not used to prepare the basic skeleton of the electrospun membrane, resulting in easy water absorption and swelling of the fiber structure of the electrospun membrane sheet, poor cell adhesion, and poor proliferation effect.

[0037] 3. Tensile strength test Weigh the hydrogel-nanofiber composite scaffolds prepared in Examples 1 to 3 and Comparative Examples 1 to 3 with the same mass. Take 5 g of each, place them in a water bath at 37°C, take them out after 0.5 h, and use a universal testing machine to test the mechanical properties of the hydrogel-nanofiber composite scaffolds. The sensor is 100 N. Make the composite scaffolds into a dumbbell shape with a total length of 40 mm, a gauge length of 12 mm, a width of 5 mm, a thickness of 1 mm, and a tensile speed of 50 mm / min. There are 3 parallels in each group.

[0038] Calculate the tensile strength according to the following formula: Tensile strength (MPa) =

[0039] Table 3 Tensile strength test results of hydrogel-nanofiber composite scaffolds (MPa)

[0040] It can be seen from the test results that the tensile strengths of Examples 1 to 3 are all good. In Comparative Example 1, there is no potential repair film layer, and the fibers are in a single and dispersed state, so the mechanical properties are relatively poor. In Comparative Example 2, there is no interpenetrating network in the form of a reinforcing rib, and the mechanical properties are poor.

[0041] 4. Determination of water resistance time Hydrogel adhesion strength test: Weigh the hydrogel-nanofiber composite scaffolds prepared in Examples 1 to 3 and Comparative Example 2 with the same mass. Take 5 g of each, evenly coat them between two pieces of porcine skin tissue, and conduct a tensile experiment at a speed of 10 mm / min in an environment at 37°C to characterize the adhesion strength between the porcine skin tissue and the hydrogel. There are 3 parallels in each group.

[0042] Table 4 Average adhesion strength of nanofiber-hydrogel composite scaffolds (kPa)

[0043] It can be seen from the above table that the tissue adhesion of Examples 1 to 3 is good, while in Comparative Example 2, due to the lack of a cross-linked network formed by the reinforcing rib and the self-healing hydrogel, the tissue adhesion is poor.

[0044] 5. In vitro degradation experiment Weigh the freeze-dried samples of Examples 1 to 3 and Comparative Examples 1 to 3, record the weight as W0, and under sterile conditions at 37°C, immerse each sample in PBS buffer solution and let it stand for 12 h to fully swell. Take samples on the 7th, 14th, and 21st days after swelling, freeze-dry them and weigh the weight as W1, and calculate the degradation rate according to the following formula. Degradation rate (%) =

[0045] Table 5 In vitro degradation rate of hydrogel-nanofiber composite scaffolds (%)

[0046] It can be seen from the test results that with the increase of time, the samples of each group began to degrade. The samples of Examples 1-3, Comparative Example 1 and Comparative Example 3 could still maintain the basic intact structure at 21 days. Due to the lack of fixation of PNIPAM (poly(N-isopropylacrylamide)) reinforcing bars in Comparative Example 2, the hydrogel degraded relatively fast.

[0047] 6. Observation of the swelling of the electrospun fiber membrane by electron microscopy Take the samples of Example 2 and Comparative Example 3. After flushing the gel on the surface of the fiber membrane sheet clean, freeze-dry it, and observe the swelling of the fiber surface by electron microscopy.

[0048] It can be seen from the electron microscopy results that the fibers of Example 2 can maintain a complete three-dimensional porous structure in the hydrogel (the electron microscopy structure is as shown in Figure 10 and Figure 11 ), while in Comparative Example 3, since no hydrophobic material was used as the basic skeleton structure of the fiber, the fiber absorbed water and swelled (the electron microscopy structure is as shown in Figure 12 and Figure 13 ).

[0049] In summary, the nanofiber composite membrane provided in the present invention exists in the hydrogel in the form of a sheet, retains the three-dimensional porous extracellular matrix biomimetic structure of the fiber membrane, is beneficial to cell adhesion and proliferation, and ensures the repair effect. The middle sandwich structure of the composite membrane selects a hydrophobic material to reduce the swelling of the material in the hydrogel and ensure the integrity of the membrane structure. The upper and lower layers of the composite membrane are of a core-shell structure. The PLGA in the core layer serves as the basic skeleton, plays a supporting role and reduces the swelling of the fiber; the shell layer is gelatin, which is crosslinked in real time with glyoxal, and the crosslinking degree is controlled at 50% - 70%.

[0050] And the amino group of the remaining gelatin is grafted with dopamine on hyaluronic acid (see Figure 4 ), which not only ensures the binding firmness between the gelatin fiber and the hyaluronic acid gel, but also reduces the swelling degree of the gelatin fiber shell layer and enhances the firmness through the chemical crosslinking of glyoxal and the physical crosslinking formed by the complexation reaction between dopamine and gelatin.

[0051] After the modification of hyaluronic acid, the dopamine structure is increased, which is not only beneficial to tissue adhesion, but also can better adsorb gelatin fibers, ensuring the connection between gelatin fiber membrane sheets and serving as a connection bridge between the tissue and the composite membrane.

[0052] The lower critical solution temperature (LCST) of the PNIPAM hydrogel is approximately 32 °C. When the temperature is higher than the LCST, the hydrophobic interaction in the hydrogel system dominates. The interaction of hydrophobic groups promotes the contraction of the polymer network, and it precipitates from water to form reinforcing ribs, causing the gel volume to shrink, driving the shrinkage and aggregation of the nanofiber composite membrane, adapting to different uterine cavity structures, and remodeling in vivo to form a dense "potential repair membrane".

[0053] The above embodiments are only used to illustrate the technical concept and characteristics of the present invention. The purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it accordingly, and it cannot be used to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. An endometrial repair stent, characterized in that, It is prepared by using a sheet-like nanofiber composite membrane as a substrate, combining it with dopamine-grafted aldehyde-functionalized hyaluronic acid, dynamically crosslinking with carboxymethyl chitosan based on the Schiff base reaction, and then compounding with poly(N-isopropylacrylamide); wherein, the mass ratio of the dopamine-grafted aldehyde-functionalized hyaluronic acid, the carboxymethyl chitosan, and the poly(N-isopropylacrylamide) is (1-9):(1-9):(1-90); the thickness of the nanofiber composite membrane is 0.1-0.4 mm.

2. The endometrial repair stent according to claim 1, wherein, 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 crosslinking agent, and the mass ratio of the gelatin to the crosslinking agent is (15-30):(1-5); the raw material components of the core layer in the core-shell structure and the intermediate nanofiber layer are both PLGA, and 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 preparation method of the nanofiber composite membrane includes the following steps: S1: Dissolve PLGA in a first solvent to obtain a first spinning dope with a concentration of 15-30 wt%, and electrospun to obtain an intermediate nanofiber layer; S2: Dissolve gelatin in a second solvent, and mix it with a crosslinking agent solution in real time to obtain a second spinning dope; S3: Take another portion of the first spinning dope in step S1, and perform coaxial electrospinning with the second spinning dope in step S2 to respectively obtain an upper nanofiber layer and a lower nanofiber layer; S4: Perform gradient temperature crosslinking on the three-layer nanofiber layer prepared above to obtain the 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 15-30 wt%; the crosslinking agent is a mixture composed of one or more selected from formaldehyde, glyoxal, glutaraldehyde, genipin, and tannic acid, and the concentration of the crosslinking agent solution is 1-5 wt%.

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 crosslinking temperature is 80-170 °C, and the crosslinking degree is 50-70%.

6. The preparation method of an endometrial repair stent according to any one of claims 1 to 5, characterized in that, It includes the following steps: (1) Dissolve hyaluronic acid and then add an oxidant for oxidation, and then add a reducing agent to terminate the oxidation, and freeze-dry to obtain aldehyde-functionalized hyaluronic acid; (2) Dissolve the aldehyde-functionalized hyaluronic acid and then add dopamine for grafting reaction, and freeze-dry to obtain the dopamine-grafted aldehyde-functionalized hyaluronic acid; (3) Dissolve the dopamine-grafted aldehyde-modified hyaluronic acid, and then successively add the membrane sheet of the nanofiber composite membrane, carboxymethyl chitosan, and poly(N-isopropylacrylamide) and mix them evenly to prepare the endometrial repair stent.

7. The preparation method of 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 oxidant is a mixture composed of one or more selected from sodium periodate, basic copper carbonate, pyridinium chlorochromate, and lead tetraacetate; the reducing agent is ethylene glycol; the reaction temperature during oxidation is 20-40 °C, and the reaction duration is 3-6 h.

8. The preparation method of 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 duration is 10 h.

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

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