A collagen-exosome composite membrane material for uterine cavity repair and preparation method and application thereof

Abstract

The application discloses a collagen-exosome composite film material for uterine cavity repair and a preparation method and application thereof. The collagen-exosome composite film material is prepared by compounding type I collagen and mesenchymal stem cell-derived exosomes. The type I collagen is obtained by cross-linking reaction of animal tissue and a cross-linking agent. The mesenchymal stem cell-derived exosomes express one or more exosome marker proteins of CD9, CD63 and TSG101. The collagen-exosome composite film material disclosed by the application maintains flexibility at body temperature, can be attached to an endometrial wound surface, realizes slow release of the exosomes and uterine cavity physical isolation, has good biological activity, and can promote endometrial regeneration and angiogenesis.

Description

Technical Field

[0001] This application relates to a collagen-exosome composite membrane material for uterine cavity repair, its preparation method, and its application, belonging to the field of uterine cavity repair material preparation technology. Background Technology

[0002] Endometrial damage and intrauterine adhesions are significant causes of female infertility and recurrent miscarriage. Existing treatments include mechanical isolation or pharmacological intervention, but their repair effects are limited. Mesenchymal stem cell-derived exosomes (MSC-Exosomes) can promote endometrial epithelial cell proliferation and angiogenesis, but direct injection of exosomes results in short retention time and low local concentration within the uterine cavity. Patent CN115054678A discloses a thermosensitive collagen exosome hydrogel that can sustainably release exosomes, but the hydrogel morphology is unstable, and its in-situ maintenance effect is limited. Therefore, there is a need to develop a structurally stable, controllable degradable membrane material that combines sustained release and physical support functions. Summary of the Invention

[0003] To address the aforementioned issues, this invention provides a collagen-exosome composite membrane, its preparation method, and its applications. The composite membrane of this invention is used for uterine cavity repair and endometrial regeneration, possessing both exosome sustained-release function and physical isolation effect. The preparation method is simple and the operation is controllable.

[0004] According to a first aspect of this application, a collagen-exosome composite membrane material for uterine cavity repair is provided, the collagen-exosome composite membrane material being prepared by combining type I collagen with mesenchymal stem cell-derived exosomes;

[0005] The type I collagen matrix is ​​obtained by film formation and cross-linking of animal-derived type I collagen, which is obtained by extraction and purification of animal tissue.

[0006] The mesenchymal stem cell-derived exosomes express one or more exosome marker proteins among CD9, CD63, and TSG101.

[0007] Optionally, the crosslinking agent for the crosslinking treatment is 1-ethyl-3-(3-dimethylpropyl)carbodiimide hydrochloride / N-hydroxysuccinimide.

[0008] Optionally, the mass ratio of 1-ethyl-3-(3-dimethylpropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 2:1 to 5:1.

[0009] Optionally, the mass ratio of 1-ethyl-3-(3-dimethylpropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is independently selected from any value of 2:1, 3:1, 4:1, 5:1 or a range between any two of the above.

[0010] Optionally, the particle size of the mesenchymal stem cell-derived exosomes is 30~150 nm.

[0011] Optionally, the particle size of the mesenchymal stem cell-derived exosomes is independently selected from any value of 30nm, 50nm, 70nm, 90nm, 110nm, 130nm, 150nm, or a range between any two of the above.

[0012] According to a second aspect of this application, a method for preparing the collagen-exosome composite membrane material described above is provided, the method comprising the following steps:

[0013] Step S1: Animal tissues are subjected to acid swelling, tissue disruption, pepsin hydrolysis, enzyme inactivation, pH adjustment to acidic, adsorption, ultrafiltration, dialysis, concentration, secondary ultrafiltration dialysis, and freeze drying to obtain type I collagen;

[0014] Step S2: Acid-soluble treatment of type I collagen, pH adjustment, dilution with phosphate buffer, self-assembly, centrifugation, pouring into a mold, freeze-drying, to obtain type I collagen membrane material;

[0015] Step S3: The mixture containing type I collagen membrane material and cross-linking agent is subjected to cross-linking reaction and washed to obtain collagen solution;

[0016] Step S4: Mix the collagen solution and the mesenchymal stem cell-derived exosome solution, freeze-dry, and obtain the collagen-exosome composite membrane material.

[0017] Optionally, the preparation method further includes impregnating the collagen-exosome composite membrane material with an impregnation solution to obtain a porous collagen-exosome composite membrane material.

[0018] Optionally, the impregnation solution includes water, dopamine, and bioactive powder.

[0019] Optionally, the bioactive powder is selected from at least one of bioactive glass, chitosan, and sodium hyaluronate.

[0020] Optionally, the immersion treatment time is 5 to 30 minutes.

[0021] Optionally, the immersion treatment time is independently selected from any value of 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, or a range between any two of the above.

[0022] Optionally, the pore size of the porous collagen-exosome composite membrane material is 50~300μm.

[0023] Optionally, the pore size of the porous collagen-exosome composite membrane material is independently selected from any value of 50μm, 100μm, 150μm, 200μm, 250μm, 300μm or a range between any two of the above.

[0024] Optionally, in step S1, the animal tissue is derived from at least one of pig skin, pig Achilles tendon, cow skin, and cow Achilles tendon.

[0025] Optionally, in step S2, the pH value is adjusted to 6-8.

[0026] Optionally, the phosphate buffer is diluted to a final concentration of type I collagen of 1-10 mg / mL.

[0027] Optionally, the phosphate buffer dilution to achieve a final concentration of type I collagen independently selected from any value of 1 mg / mL, 2 mg / mL, 4 mg / mL, 5 mg / mL, 8 mg / mL, 10 mg / mL, or a range between any two of the above.

[0028] Optionally, the self-assembly temperature is 18~30℃.

[0029] Optionally, the self-assembly temperature is independently selected from any value of 18°C, 20°C, 22°C, 25°C, 28°C, 30°C, or a range between any two of the above.

[0030] Optionally, the thickness of the type I collagen membrane material is 0.1~0.5 mm.

[0031] Optionally, the thickness of the type I collagen membrane material is independently selected from any value of 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, or a range between any two of the above.

[0032] Optionally, in step S3, the crosslinking agent is 1-ethyl-3-(3-dimethylpropyl)carbodiimide hydrochloride / N-hydroxysuccinimide.

[0033] Optionally, the mass ratio of 1-ethyl-3-(3-dimethylpropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 2:1 to 5:1.

[0034] Optionally, the mass ratio of 1-ethyl-3-(3-dimethylpropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is independently selected from any value of 2:1, 3:1, 4:1, 5:1 or a range between any two of the above.

[0035] Optionally, the crosslinking reaction is carried out at a temperature of 20-30°C for 5-24 hours.

[0036] Optionally, the temperature of the crosslinking reaction is independently selected from any value of 20°C, 22°C, 25°C, 28°C, 30°C, or a range between any two of the above.

[0037] Optionally, the time of the crosslinking reaction is independently selected from any value of 5h, 8h, 12h, 15h, 18h, 20h, 24h or a range between any two of the above.

[0038] Optionally, the cleaning includes washing with phosphate buffer 1 to 3 times, followed by washing with water 1 to 3 times.

[0039] Optionally, in step S4, the concentration of the mesenchymal stem cell-derived exosome solution is 10~100 μg / mL.

[0040] Optionally, in step S4, the concentration of the mesenchymal stem cell-derived exosome solution is any value from 10 μg / mL, 20 μg / mL, 30 μg / mL, 50 μg / mL, 80 μg / mL, 100 μg / mL, or a range between any two of the above.

[0041] Optionally, the mesenchymal stem cell-derived exosomes in the mesenchymal stem cell-derived exosome solution express one or more exosome marker proteins among CD9, CD63, and TSG101.

[0042] Optionally, the freeze-drying includes pre-freezing, primary drying, and secondary drying, wherein the pre-freezing temperature is -40℃ to -80℃, the primary drying temperature is -30℃ to -10℃, and the secondary drying temperature is 15℃ to 25℃; the total freeze-drying time is 24 to 36 hours.

[0043] Optionally, in step S4, a freeze-drying protectant is added to the mixture of collagen solution and mesenchymal stem cell-derived exosome solution before freeze-drying.

[0044] Optionally, the freeze-drying protectant is selected from at least one of trehalose, sucrose, mannitol, and sodium trehalose.

[0045] Optionally, the final concentration of the freeze-drying protectant in the mixed system is 2% to 10% (w / v).

[0046] According to a third aspect of this application, a collagen-exosome composite membrane material prepared by the above-described collagen-exosome composite membrane material or the preparation method of the above-described collagen-exosome composite membrane material is provided for uterine cavity repair.

[0047] As an optional implementation, this application is achieved through the following technical solution:

[0048] Step (1) Preparation of collagen solution: Animal tissues rich in type I collagen are selected as the source of collagen extraction, such as pig skin, pig Achilles tendon, cow skin, and cow Achilles tendon, which are easy to obtain. The collected animal tissues are weighed and then subjected to the following steps in sequence: acid swelling, tissue disruption, pepsin hydrolysis, enzyme inactivation, pH adjustment to acidic, diatomaceous earth adsorption, ultrafiltration, dialysis, concentration, secondary ultrafiltration dialysis, and freeze drying to obtain type I collagen.

[0049] The obtained type I collagen was lyophilized for easy storage. The lyophilized type I collagen was then acid-soluble, and the pH was adjusted to 6-8. Phosphate buffer was added to achieve a final collagen concentration of 1-10 mg / mL. The collagen solution was self-assembled at 18-30°C, centrifuged, poured into a mold, and lyophilized to form a type I collagen membrane material with a thickness of 0.1-0.5 mm.

[0050] The lyophilized collagen membrane was added to a cross-linking agent, which was 1-ethyl-3-(3-dimethylpropyl)carbodiimide hydrochloride / N-hydroxysuccinimide (EDC / NHS), with the mass ratio of the EDC / NHS solution ranging from 2:1 to 5:1. Cross-linking was carried out at room temperature for 5 to 24 hours. The membrane was then washed 1 to 3 times with PBS and 1 to 3 times with purified water to remove residual cross-linking agent, thus obtaining a collagen solution.

[0051] Step (2) Exosome extraction and purification: Exosomes derived from mesenchymal stem cells (MSCs) were extracted using ultracentrifugation or SEC column chromatography. The concentration of exosomes was 10-100 μg / mL. Exosome characteristic markers included CD9, CD63, and TSG101. The isolated exosomes were verified by Western blotting and flow cytometry.

[0052] The exosome particle size was confirmed using a nanoparticle size tracking analyzer, and exosomes in the range of 30~150nm were selected.

[0053] Step (3) Preparation of collagen-exosome composite membrane: The collagen solution from step (1) and the exosome solution from step (2) are mixed and shaken at low temperature to ensure that the exosome solution is uniformly attached to the collagen. The mixture is then freeze-dried for 24-36 h to obtain a porous collagen-exosome composite membrane material. The prepared porous collagen-exosome composite membrane material is then sterilized by γ-ray or EO.

[0054] Step (4) Membrane surface modification and performance control:

[0055] To improve the porosity and surface hydrophilicity of porous collagen-exosome composite membrane materials, a secondary solution impregnation treatment can be performed on the surface of the composite membrane: the impregnation solution consists of water, a small amount of dopamine, and optional bioactive powders; the impregnation time is 5-30 minutes, followed by ultrasonic dispersion, removal of excess solution, air drying or oven drying, and freeze drying. After freeze drying, the membrane surface exhibits a porous structure with a pore size of 50-300 μm, accounting for >85%, which is beneficial for the sustained release of exosomes and the adhesion of endometrial cells.

[0056] The beneficial effects that this application can produce include:

[0057] 1) The collagen-exosome composite membrane material for uterine cavity repair prepared in this invention can achieve long-term sustained release of exosomes. It forms a three-dimensional fiber network structure through the self-assembly of type I collagen, and then constructs a stable spatial network structure through EDC / NHS cross-linking, forming a porous membrane with a controllable pore size (50-300 μm). Exosomes are embedded in the interstices of collagen fibers and the microporous structure, achieving fixation through physical embedding and weak interactions. In the body fluid environment, they are gradually released through membrane degradation and diffusion mechanisms, thereby significantly prolonging the retention time of exosomes in the uterine cavity and achieving a continuous, physiologically-level release effect.

[0058] 2) The collagen-exosome composite membrane material for uterine cavity repair prepared in this invention can promote endometrial regeneration and angiogenesis. The collagen used is a natural extracellular matrix component, which can provide a supporting microenvironment for endometrial epithelial cells and stromal cells. At the same time, the porous structure is conducive to cell migration and vascular endothelial cell ingrowth. Exosomes can regulate local cell proliferation and angiogenesis, thereby significantly improving endometrial thickness and functional recovery level.

[0059] 3) The collagen-exosome composite membrane material prepared by this invention has high biodegradability and safety. It uses type I collagen extracted from animals, which is a natural material with good biocompatibility. The degradation products are amino acids or small molecule peptides, which can be metabolized and absorbed by the body. EDC / NHS is a zero-length cross-linking system, which does not introduce permanent toxic residues and has good tissue compatibility and safety.

[0060] 4) The preparation process of this invention is controllable, the membrane morphology is stable, and it is easy to store, transport, and use. The step-by-step process allows for precise control of key parameters (pH, temperature, crosslinking time, pore size range), ensuring batch stability. After freeze-drying, the membrane structure is stable and the thickness is controllable, allowing for long-term storage at low temperatures. When in use, simple humidification restores its flexibility and allows it to adhere to the uterine cavity wound, making operation convenient. Attached Figure Description

[0061] Figure 1 This is a schematic diagram of the collagen-exosome composite membrane of this application.

[0062] Figure 2This is a schematic diagram illustrating the flexible adhesion of the composite film of this application at body temperature.

[0063] Figure 3 This is a statistical chart of the intima thickness in animal experiments for this application.

[0064] Figure 4 This is an HE staining image of the intima from an animal experiment in this application.

[0065] Figure 5 This is a statistical chart showing the number of large blood vessels in the intima during animal experiments for this application. Detailed Implementation

[0066] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0067] Unless otherwise specified, all raw materials used in the embodiments of this application were purchased through commercial channels.

[0068] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0069] 1-Ethyl-3-(3-dimethylpropyl)carbodiimide hydrochloride (EDC·HCl, CAS No.: 25952-53-8) and N-hydroxysuccinimide (NHS, CAS No.: 6066-82-6) are both commercially available reagents. For example, EDC·HCl is available from Sigma-Aldrich, catalog number E6383, or Tokyo Chemical Industry Co., Ltd., catalog number D1601; NHS is available from Sigma-Aldrich, catalog numbers 130672 or 804518, or Tokyo Chemical Industry Co., Ltd., catalog number H0623. The sources of these reagents are merely examples and do not constitute a limitation on the scope of protection of this application.

[0070] Example 1: Preparation of collagen-exosome composite membrane

[0071] This embodiment provides a method for extracting porcine skin collagen, preparing collagen membrane sheets, and further loading human umbilical cord mesenchymal stem cell-derived exosomes to form a composite membrane. The method steps are as follows.

[0072] (1) Extraction of collagen from pig skin

[0073] Select fresh, disease-free pigskin as raw material. Cut the pigskin into 0.5-1 cm pieces. 2Small pieces were placed in a 0.5 mol / L hydrochloric acid solution and soaked at 4 ℃ for 12 h at a material-to-liquid ratio of 1:10 (w / v). The acid solution was changed every 3 h to ensure thorough removal of impurities and expansion of the raw material. After acid soaking, the material was rinsed with deionized water until the pH of the washing solution was 3.0-3.5.

[0074] The acid-treated pigskin was crushed in a high-speed crusher and homogenized using a high-speed homogenizer at 8000 rpm for 3 min to obtain a uniform suspension. Pepsin (2500–3200 U / mg) was added to the suspension at a rate of 0.5 wt% of the raw material's wet weight, and the pH was adjusted to 2.5 ± 0.2. Enzymatic hydrolysis was carried out at 25 ℃ for 24 h. After hydrolysis, the mixture was heated to 60 ℃ and incubated for 20 min to inactivate the enzyme.

[0075] After cooling the system to room temperature, the pH was adjusted to 7.0±0.2 using 1 mol / L NaOH, and then coarsely filtered through a 200-mesh filter cloth to obtain a crude collagen extract.

[0076] The crude collagen extract was separated by anion exchange chromatography using a DEAE-Sepharose (diethylaminoethyl agarose gel, weak anion exchange chromatography medium) column, and the collagen elution peak was collected. The resulting eluent was desalted by ultrafiltration at 30 kDa and dialysis at 10 kDa for 48 h (with water changed every 8 h). The dialysate was then filtered sequentially through 0.45 μm and 0.22 μm microporous membranes to finally obtain a clear porcine skin type I collagen solution.

[0077] The obtained porcine skin type I collagen solution was concentrated to 10-20 mg / mL and stored at 4 ℃ for later use / lyophilized to obtain porcine skin type I collagen lyophilized powder.

[0078] (2) Preparation of collagen membrane

[0079] Take the freeze-dried pig skin type I collagen powder (or solution) prepared above, and reconstitute it with dilute hydrochloric acid solution. After the collagen solution is completely dissolved, adjust the pH to 7.0-7.5.

[0080] Phosphate-buffered saline (PBS) was added to the solution to bring the final collagen concentration to 10 mg / mL. The solution was incubated with gentle shaking in a 30 °C water bath for 16 h to form a homogeneous collagen sol. The sol was then centrifuged at 10,000 rpm for 10 min to remove insoluble matter. The collagen concentration in the solution was adjusted to 20 mg / mL, and the solution was poured into a 0.3 mm thick plate mold. The mixture was pre-frozen at −40 °C for 4 h, and then freeze-dried for 24 h to obtain dried collagen sheets.

[0081] The collagen membrane was placed in a 3:1 mass ratio (1-ethyl-3-(3-dimethylpropyl)carbodiimide hydrochloride / N-hydroxysuccinimide EDC / NHS solution and subjected to a cross-linking reaction with shaking at room temperature for 4 h. After the reaction, the membrane was washed twice with PBS and then twice with water for injection to remove residual cross-linking agent, resulting in a cross-linked and stable collagen membrane.

[0082] (3) Preparation of collagen-exosome composite membrane

[0083] The cross-linked and stabilized collagen membrane was placed in an exosome solution. The exosomes were extracted from P6 generation human umbilical cord mesenchymal stem cells (hUC-MSC) culture medium by ultracentrifugation, with a concentration of 50 μg / mL. The collagen membrane and exosome solution were incubated together at 4 ℃ with gentle shaking for 4-6 h to allow the exosomes to fully adsorb and uniformly bind to the collagen membrane structure. After incubation, the membrane and exosome solution were transferred to a lyophilization container, a lyophilization protectant (trehalose, final concentration 5% w / v) was added, and the container was pre-frozen at -40 ℃ for 4 h, followed by lyophilization for 24 h. This yielded the final collagen-exosome composite membrane. Figure 1 As shown, the collagen-exosome composite membrane prepared in Example 1 is a white or off-white thin film. The membrane is intact, with a relatively smooth surface, and no obvious cracks, curling, or delamination were observed. Figure 2 As shown, under simulated body temperature conditions, the collagen-exosome composite membrane can maintain its flexibility after humidification. It can bend under instrument clamping and maintain the integrity of the membrane without obvious breakage or fragmentation. This indicates that the composite membrane has a certain degree of flexibility and ease of operation, making it easy to grasp, transfer and attach to the uterine cavity wound during surgery.

[0084] Comparative Example 1:

[0085] To compare the performance of the collagen-exosome composite membrane of the present invention, a collagen membrane without exosome loading was prepared as a control.

[0086] 1. Preparation of collagen membranes

[0087] A porcine skin type I collagen solution was obtained according to the collagen extraction method described in Example 1. The lyophilized collagen powder was reconstituted with dilute hydrochloric acid, and the pH of the solution was adjusted to 7.0-7.5. Phosphate-buffered saline (PBS) was added to a final concentration of 10 mg / mL, and the solution was incubated with shaking in a 30 °C water bath for 16 h. Insoluble matter was then removed by centrifugation at 10,000 rpm for 10 min.

[0088] After adjusting the collagen concentration in the supernatant to 20 mg / mL, the solution was poured into a 0.3 mm thick mold, pre-frozen at −40 °C for 4 h, and then lyophilized for 24 h to obtain collagen sheets. The collagen sheets were then added to a 3:1 EDC / NHS solution and reacted with shaking at room temperature for 4 h. The mixture was then washed twice with PBS and twice with water for injection to obtain cross-linked collagen membranes.

[0089] 2. Exosome loading is not performed.

[0090] Unlike Example 1, Comparative Example 1 did not include exosome solution, did not perform shaking incubation and loading steps, and directly used cross-linked collagen membrane as control sample.

[0091] Comparative Example 2:

[0092] To verify the effect of the crosslinking reaction in step (2) of this invention on the structural stability, in vitro degradation characteristics, and exosome sustained-release behavior of the composite membrane, a collagen-exosome composite membrane without crosslinking agent was prepared as a control in this comparative example. Except for omitting the EDC / NHS crosslinking treatment and related cleaning steps, the raw material sources, solution concentrations, exosome loading conditions, and freeze-drying process were strictly consistent with those in Example 1. The specific steps are as follows:

[0093] (1) Extraction of collagen from pig skin: Same as step (1) in Example 1.

[0094] (2) Preparation of uncrosslinked collagen membrane: The lyophilized porcine skin type I collagen obtained above was reconstituted with dilute hydrochloric acid solution, and the pH of the collagen solution was adjusted to 7.0-7.5 after complete dissolution. Phosphate buffer (PBS) was added to the solution to make the final collagen concentration 10 mg / mL. The solution was gently shaken and incubated in a 30 ℃ water bath for 16 h to form a uniform collagen sol. Then, the solution was centrifuged at 10000 rpm for 10 min to remove insoluble matter. After adjusting the collagen concentration in the solution to 20 mg / mL, the solution was poured into a 0.3 mm thick plate mold and pre-frozen at −40 ℃ for 4 h. Then, it was freeze-dried in a freeze dryer for 24 h to obtain a dried collagen membrane. In this comparative example, no EDC / NHS crosslinking agent was added, and no crosslinking reaction or subsequent PBS / water for injection washing steps were performed. The dried collagen membrane was directly used as an uncrosslinked substrate for later use.

[0095] (3) Preparation of collagen-exosome composite membrane: The uncrosslinked collagen membrane was placed in an exosome solution. The exosomes were obtained from P6 generation human umbilical cord mesenchymal stem cell (hUC-MSC) culture medium and extracted by ultracentrifugation, with an exosome concentration of 50 μg / mL. The uncrosslinked collagen membrane and exosome solution were placed together at 4 ℃ and gently shaken for 4-6 h to allow the exosomes to be fully adsorbed and uniformly bound to the collagen membrane structure. After incubation, the membrane and exosome solution were transferred to a lyophilization container, a lyophilization protectant (trehalose, final concentration 5% w / v) was added, and the container was pre-frozen at -40 ℃ for 4 h, followed by lyophilization for 24 h. The uncrosslinked collagen-exosome composite membrane was thus obtained.

[0096] Example 2: In vitro degradation experiment

[0097] Enzymatic hydrolysis experiments were used to evaluate the durability of collagen sheet groups, uncrosslinked collagen-exosome complex membranes, and collagen-exosome complex membrane groups. A certain mass (W0) of sample was weighed and immersed in collagenase at a concentration of 5 U / mL. The samples were then incubated at 37°C for 1, 3, and 7 days. After treatment, the samples were washed with deionized water, centrifuged, lyophilized, and weighed (W1). The hydrolysis rate was calculated.

[0098] Enzymatic hydrolysis rate = (W0 - W1) / W0 × 100%

[0099] Results Analysis: As shown in Table 1, the cumulative enzymatic hydrolysis rates of Example 1 (collagen-exosome composite membrane crosslinked with EDC / NHS) at 1, 3, and 7 days were (20.0±1.0)%, (31.0±1.0)%, and (36.0±1.0)%, respectively, significantly lower than those of Comparative Example 2 (uncrosslinked collagen-exosome composite membrane) (28.0±1.0)%, (46.0±1.0)%, and (61.0±1.5)% (p<0.01). The enzymatic hydrolysis kinetic curves of Example 1 and Comparative Example 1 (crosslinked pure collagen membrane) highly overlapped (p>0.05), indicating that the exosome loading did not interfere with the enzymatic stability of the collagen matrix. The above data demonstrate that the EDC / NHS crosslinking step introduced in this invention effectively masks the specific cleavage sites of collagenase by forming a stable intermolecular amide bond network, reducing the degradation rate of the composite membrane by approximately 40% in the simulated intrauterine enzyme environment after 7 days.

[0100] Table 1 Comparison of Enzymatic Hydrolysis Rate

[0101]

[0102] Example 3: Animal experiments to verify endometrial thickness

[0103] Nine healthy female Bama pigs were selected and randomly divided into three groups of three:

[0104] (1) Model control group;

[0105] (2) Collagen membrane assembly;

[0106] (3) Collagen-exosome complex membrane group.

[0107] First, an injury model was established in the uterus using standard methods. After the injury was established, appropriate materials were implanted into the injury site. Animals were dissected at weeks 4, 8, and 12. Sufficient tissue from the uterine cavity defect repair area and surrounding tissue was removed. The endometrial thickness was evaluated by α-SMA immunohistochemical staining, and the tissue samples were stained with hematoxylin and eosin (HE) to observe the tissue repair.

[0108] The endometrial thickness of each group of animals was measured at three time points: week 4, week 8, and week 12. The results are as follows: Figure 3 As shown, the endometrial thickness in the model group was low at all time points, indicating insufficient repair after injury.

[0109] The collagen membrane group showed a significantly increased endometrial thickness compared to the model group at week 4, suggesting that the collagen membrane has a certain tissue repair promoting effect on the damaged site. As time progressed to week 8 and week 12, the endometrial thickness in this group remained relatively stable, both higher than that in the model control group.

[0110] The collagen-exosome composite membrane group showed a more significant intima thickening effect at all time points. By week 4, the intima thickness in this group was already higher than that in the collagen membrane group; this trend continued into weeks 8 and 12, indicating that the combined use of exosomes and collagen membranes can further enhance tissue repair capabilities.

[0111] Overall, collagen membranes promoted the recovery of endometrial thickness, and the addition of exosomes further enhanced the repair effect, making tissue thickening more pronounced and stable. These results validate the potential advantages of collagen-exosome composite membranes in endometrial repair.

[0112] Example 4: Animal experiments to verify the number of large intimal vessels

[0113] The aforementioned steps are the same as in Example 3, using SlideViewer software to open the HE-stained slide scan. Figure 4 As shown, the number of clearly identifiable blood vessels was counted on 5X field images, and the average number of large intimal vessels in each group was calculated.

[0114] like Figure 5 As shown, in week 4 of the experiment, the number of large blood vessels in the collagen exosome composite membrane group was higher than that in the collagen membrane group and higher than that in the model group; in week 8 of the experiment, the number of large blood vessels in the collagen membrane group was slightly lower than that in the model group, while the number of large blood vessels in the collagen exosome composite membrane group was the highest; the results in week 12 of the experiment were the same as those in week 4, and statistical analysis showed significant differences between the groups.

[0115] Analysis of the number of large blood vessels in the endometrium leads to the following conclusion: collagen exosome complex membranes are more conducive to promoting the formation of large blood vessels in the endometrium.

[0116] Example 5: Surface modification and performance regulation of collagen-exosome composite membranes

[0117] Materials and reagents: Collagen-exosome composite membrane (prepared in Example 1), dopamine (DA), bioactive powder (optional), β-tricalcium phosphate (β-TCP), bioactive glass, PBS buffer

[0118] Steps: First impregnation solution preparation: Add 2 g of dopamine to 500 mL of purified water, adjust the pH to 8.5 using Na₂CO₃, and stir for 10 min. Add 50 g of β-TCP powder (particle size D). 50 = 20 μm, D 90 = 30 μm), mechanically stirred for 12 h to disperse uniformly. The first layer of membrane was impregnated by placing the freeze-dried collagen-exosome membrane in the first impregnation solution and ultrasonically dispersed for 10 min. Excess liquid was removed, and the membrane was dried at 60 ℃ for 3 h to obtain the preliminary surface-modified membrane.

[0119] The second impregnation solution was prepared by adding 2 g of dopamine to 500 mL of purified water, adjusting the pH to 8.5, and then adding 50 g of bioactive glass powder (particle size D). 50 = 5 μm, D 90 = 8 μm), and stirred for 12 h to disperse evenly. For the second membrane impregnation, the first modified membrane was immersed in the second impregnation solution and ultrasonically dispersed for 10 min. Excess liquid was removed, and the membrane was dried at 60 ℃ for 3 h.

[0120] After a second drying process, the membrane was freeze-dried for 24 hours to obtain the final porous composite membrane.

[0121] The prepared porous composite membrane exhibits the following properties: Pore size distribution: 50-300 μm, accounting for >85%, which is beneficial for the sustained release of exosomes and adhesion to endometrial cells. Surface hydrophilicity: Contact angle <30°, promoting the penetration of intrauterine fluid and blood. Mechanical properties: Tensile strength 0.8-1.2 MPa, suitable for intrauterine adhesion.

[0122] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A collagen-exosome composite membrane material for uterine cavity repair, characterized in that, The collagen-exosome composite membrane material is prepared by combining type I collagen with mesenchymal stem cell-derived exosomes. The type I collagen matrix is ​​obtained by film formation and cross-linking of animal-derived type I collagen, which is obtained by extraction and purification of animal tissue. The mesenchymal stem cell-derived exosomes express one or more exosome marker proteins among CD9, CD63, and TSG101.

2. The collagen-exosome composite membrane material according to claim 1, characterized in that, The crosslinking agent used in the crosslinking treatment is 1-ethyl-3-(3-dimethylpropyl)carbodiimide hydrochloride / N-hydroxysuccinimide; Preferably, the mass ratio of 1-ethyl-3-(3-dimethylpropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 2:1 to 5:1; Preferably, the particle size of the mesenchymal stem cell-derived exosomes is 30~150 nm.

3. The method for preparing the collagen-exosome composite membrane material according to any one of claims 1 to 2, characterized in that, The preparation method includes the following steps: Step S1: Animal tissues are subjected to acid swelling, tissue disruption, pepsin hydrolysis, enzyme inactivation, pH adjustment to acidic, adsorption, ultrafiltration, dialysis, concentration, secondary ultrafiltration dialysis, and freeze drying to obtain type I collagen; Step S2: Acid-soluble treatment of type I collagen, pH adjustment, dilution with phosphate buffer, self-assembly, centrifugation, pouring into a mold, freeze-drying, to obtain type I collagen membrane material; Step S3: The mixture containing type I collagen membrane material and cross-linking agent is subjected to cross-linking reaction and washed to obtain collagen solution; Step S4: Mix the collagen solution and the mesenchymal stem cell-derived exosome solution, freeze-dry, and obtain the collagen-exosome composite membrane material.

4. The preparation method according to claim 3, characterized in that, The preparation method further includes impregnating the collagen-exosome composite membrane material with an impregnation solution to obtain a porous collagen-exosome composite membrane material. Preferably, the impregnation solution comprises water, dopamine, and bioactive powder; Preferably, the bioactive powder is selected from at least one of bioactive glass, chitosan, and sodium hyaluronate.

5. The preparation method according to claim 4, characterized in that, The immersion treatment time is 5-30 minutes; Preferably, the pore size of the porous collagen-exosome composite membrane material is 50~300μm.

6. The preparation method according to claim 3, characterized in that, In step S1, the animal tissue is derived from at least one of pig skin, pig Achilles tendon, cow skin, and cow Achilles tendon.

7. The preparation method according to claim 3, characterized in that, In step S2, the pH value is adjusted to 6-8; Preferably, the phosphate buffer dilution brings the final concentration of type I collagen to 1-10 mg / mL; Preferably, the self-assembly temperature is 18~30℃; Preferably, the thickness of the type I collagen membrane material is 0.1~0.5 mm.

8. The preparation method according to claim 3, characterized in that, In step S3, the crosslinking agent is 1-ethyl-3-(3-dimethylpropyl)carbodiimide hydrochloride / N-hydroxysuccinimide; Preferably, the mass ratio of 1-ethyl-3-(3-dimethylpropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 2:1 to 5:1; Preferably, the crosslinking reaction is carried out at a temperature of 20-30°C for 5-24 hours. Preferably, the cleaning includes washing with phosphate buffer solution 1 to 3 times, followed by washing with water 1 to 3 times.

9. The preparation method according to claim 3, characterized in that, In step S4, the concentration of the mesenchymal stem cell-derived exosome solution is 10~100 μg / mL; Preferably, the mesenchymal stem cell-derived exosomes in the mesenchymal stem cell-derived exosome solution express one or more exosome marker proteins among CD9, CD63, and TSG101; Preferably, the freeze-drying includes pre-freezing, primary drying, and secondary drying, wherein the pre-freezing temperature is -40℃ to -80℃, the primary drying temperature is -30℃ to -10℃, and the secondary drying temperature is 15℃ to 25℃; the total freeze-drying time is 24 to 36 hours. Preferably, in step S4, a freeze-drying protectant is added to the mixture of collagen solution and mesenchymal stem cell-derived exosome solution before freeze-drying; Preferably, the freeze-drying protectant is selected from at least one of trehalose, sucrose, mannitol, and sodium trehalose; Preferably, the final concentration of the freeze-drying protectant in the mixed system is 2%~10% (w / v).

10. The collagen-exosome composite membrane material prepared by the preparation method of the collagen-exosome composite membrane material according to any one of claims 1 to 2 or any one of claims 3 to 9 is used for uterine cavity repair.

Images