An amniotic membrane biological composite material and a preparation method and application thereof

Abstract

The application provides an amniotic membrane biological composite material and a preparation method and application thereof, and the composite material comprises amniotic membrane microcarriers, mesenchymal stem cells and a polypeptide hydrogel, wherein the amniotic membrane microcarriers are 3D load type three-dimensional structures, the amniotic membrane microcarriers are loaded with the mesenchymal stem cells, and the mesenchymal stem cells are mixed and cultured with a polypeptide solution at a ratio of 1:2-1:4 to obtain a mesenchymal stem cell-amniotic membrane microcarrier-polypeptide hydrogel biological composite material. The combination of the amniotic membrane microcarriers and the hydrogel provides three-dimensional support for stem cells, perfusion into the uterine cavity can make the cells survive for a long time, meanwhile, the microcarriers and the hydrogel have a slow-release effect on the excretion of the stem cells, and are more conducive to promoting the repair effect of the mesenchymal stem cells, and can be used for endometrial repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a biocomposite material, its preparation method, and its application. Background Technology

[0002] Endometrial adhesions (IUA) refer to damage to the endometrium caused by infection, trauma, or other reasons, which may lead to complete or partial closure of the uterine cavity and / or cervix by fibrous adhesions. IUA can cause menstrual abnormalities, such as reduced menstrual flow, amenorrhea, and cyclical abdominal pain, severely jeopardizing the normal function of the female reproductive system. Secondly, cervical stenosis or occlusion can obstruct sperm passage and embryo implantation, leading to infertility; secondary infertility due to adhesions accounts for more than 8%. For pregnant women, cervical adhesions can induce placental abruption, leading to recurrent miscarriages or premature births, and even postpartum hemorrhage, significantly impacting the safety of both mother and child. Long-term adhesions causing fibrosis can further damage the uterine cavity, ultimately resulting in loss of female fertility.

[0003] Currently, clinical treatment primarily involves controlling infection with medication and surgically releasing adhesions. However, the recurrence rate after surgical release is high, necessitating the use of biomaterials, and even combinations with stem cells, as barrier materials to promote tissue repair at the wound site, inhibit inflammation, and combat fibrosis. Amniotic membrane, as a biomaterial, is used in IUA treatment due to its barrier function. The amniotic membrane matrix is ​​rich in anti-inflammatory factors (such as IL-10 and TGF-β1), which can inhibit inflammatory cell infiltration and reduce postoperative wound inflammation. Amniotic membrane can also reduce scar formation and promote functional endometrial regeneration by inhibiting fibroblast proliferation and excessive collagen deposition. Furthermore, amniotic membrane has low immunogenicity, eliminating the need for immunosuppression and reducing the risk of rejection. Experimental studies have shown that amniotic membrane transplantation can significantly reduce postoperative adhesion recurrence, promote angiogenesis and damaged tissue repair, and improve pregnancy rates. However, most commercially available amniotic membranes are freeze-dried, which have high mechanical strength but weak wound adhesion and are prone to falling off when used in the uterine cavity. Wet amniotic membranes have better adhesion but low mechanical strength and are prone to shrinking when wet, making it difficult to cover wounds in the uterine cavity. This limits the clinical use of amniotic membranes in the repair of uterine adhesions.

[0004] Stem cells possess multi-lineage differentiation, tissue repair, and anti-inflammatory and anti-fibrotic capabilities, and their application in endometrial repair and regeneration has been extensively studied. Current literature reports that intrauterine perfusion of human umbilical cord mesenchymal stem cells can increase endometrial thickness, improve endometrial blood flow, promote endometrial regeneration and repair, and increase embryo implantation and pregnancy success rates. However, this intrauterine perfusion therapy with mesenchymal stem cells makes it difficult to allow stem cells to remain in the uterine cavity for extended periods and sustain their repair effects.

[0005] Ideal biomaterials for endometrial adhesion should possess low immunogenicity, good three-dimensional structure, and biocompatibility, while also being able to load stem cells and extend their survival time in vivo. Amniotic membrane is an excellent material with low immunogenicity and high biocompatibility. It can be prepared as a microcarrier, and stem cells can be loaded onto the amniotic membrane microcarrier using 3D cell culture technology, providing structural support and a three-dimensional space for stem cell proliferation. Furthermore, self-assembled peptide hydrogels, a popular biomaterial in recent years, also exhibit good biocompatibility and low immunogenicity. Studies have shown that peptide hydrogels can provide sustained release of stem cell exocrine secretions, extending stem cell residence in vivo and enabling long-term stem cell repair functions. They are currently being applied in drug delivery, wound healing, and other fields.

[0006] The applicant's prior patent provides an amniotic membrane-MSC-peptide hydrogel composite material. Experiments have revealed the following drawbacks in practical applications: 1) The biological amniotic membrane material itself has low physical strength and easily shrivels up when exposed to liquid. Its application within the uterine cavity requires support such as a balloon, and precise application of the amniotic membrane to the wound requires high operational precision, making the surgery difficult and challenging, thus hindering practical application. 2) The primary method of using mesenchymal stem cells for endometrial repair is intrauterine perfusion. The materials prepared using this method cannot support the long-term survival of mesenchymal stem cells within the uterine cavity, resulting in limited repair efficacy. Summary of the Invention

[0007] In view of this, the main objective of the present invention is to provide an improved amniotic membrane microcarrier-mesenchymal stem cell-peptide hydrogel biocomposite material, its preparation method, and its application, in order to solve the above-mentioned technical problems.

[0008] To achieve the above objectives, as a first aspect of the present invention, a method for preparing an amniotic membrane microcarrier biocomposite material is provided, wherein mesenchymal stem cells are loaded onto an amniotic membrane microcarrier and then mixed and cultured with a polypeptide solution at a mass ratio of 1:2 to 1:4 to obtain a mesenchymal stem cell-amniotic membrane microcarrier-polypeptide hydrogel biocomposite material.

[0009] Furthermore, the above-mentioned polypeptide solution was obtained by dissolving the polypeptide powder in PBS buffer and filtering it through a 0.22 μm filter. The polypeptide powder had the sequence EILWLK-KR, was synthesized by a commercial company, and was stored at 4°C.

[0010] Furthermore, before the above-mentioned polypeptide solution was mixed with amniotic membrane as a carrier and mesenchymal stem cells for culture, the pH was adjusted to 7.4, and then filtered through a 0.22 μm filter.

[0011] Furthermore, the prepared amniotic membrane microcarrier loaded with MSCs was mixed with the polypeptide solution at a ratio of 1:2 to 1:4, and incubated in an air bath shaking bed at 30 rpm and 37°C for 30 min to obtain the sheep umbilical cord mesenchymal stem cell-amniotic membrane microcarrier-polypeptide hydrogel composite biomaterial.

[0012] Furthermore, the preparation method of the above-mentioned amniotic membrane microcarrier is as follows:

[0013] a. Wash the fresh amnion repeatedly with physiological saline until no blood remains. The amnion is obtained from a healthy donor, and informed consent is obtained from the donor. After obtaining the placenta, use medical forceps to separate the amnion from the chorionic villi tissue, remove visible blood vessels and other impurities, and rinse with physiological saline more than 5 times until there is no obvious blood on the amnion.

[0014] b. Transfer the amnion to a 50 mL sterile centrifuge tube, add an equal volume of 1.0-1.5% Trinton X-100 solution, and place the centrifuge tube in a 37℃, 120 rpm air bath shaker for 4-12 h.

[0015] c. Remove the amnion, wash it with physiological saline more than 5 times, then add an equal volume of 0.25-0.5% trypsin for digestion, and shake in a 37℃, 120 rpm air bath shaker for 4-6 hours;

[0016] d. After digestion, wash with physiological saline more than 5 times to obtain decellularized amnion;

[0017] e. Fix the amnion into a sample container and dry it using supercritical CO2 extraction to preserve the microscopic three-dimensional network structure of the amnion;

[0018] f. Place the amnion in liquid nitrogen and freeze it rapidly for 12-24 hours. Natural cracks will appear on the surface. Then remove the amnion and homogenize it into tiny particles.

[0019] g. Amniotic membrane microparticles of 200-300 μm were screened through a metal sieve, collected into 50 ml centrifuge tubes, and sterilized by irradiation to obtain amniotic membrane microcarriers.

[0020] Furthermore, in step c above, the mass fraction of trypsin is 0.25-0.5%.

[0021] Furthermore, in step e above, the CO2 flow rate is 0.1-0.8 L / min, and is maintained for 0.5-12 h.

[0022] Furthermore, the method for preparing mesenchymal stem cells in the composite material is as follows:

[0023] First, primary culture is performed:

[0024] 1) Place the umbilical cord into a 50ml centrifuge tube containing 75% alcohol and sterilize for about 1 minute. Then place it in a 15cm culture dish, pour in washing solution to rinse, and remove surface impurities with forceps. Use surgical scissors to remove 2-3cm from both ends of the ligation, and cut the remaining part into 1-2cm segments, rinsing with washing solution; remove the two arteries in the umbilical cord with ophthalmic forceps, cut the umbilical cord along the umbilical vein and remove the vein; after rinsing thoroughly with washing solution, cut the umbilical cord segment into 2mm pieces with ophthalmic scissors. 3 After disinfecting and cleaning the umbilical cord tissue block of the desired size, removing the arteries and veins, cut it into 2mm pieces. 3 The organizational blocks;

[0025] 2) Add 10ml of umbilical cord mesenchymal stem cell culture medium to a T75 culture flask, place the cut tissue pieces into the T75 flask with a spacing of 5-10mm, and incubate in a 37℃, 5% CO2 cell culture incubator. Observe the cell migration after 2-3 days and add 5-8ml of culture medium.

[0026] Then, cell passage culture was performed.

[0027] 1) When the cell confluence reaches 60%-80%, digestion and harvesting can begin;

[0028] 2) Discard the original culture medium, add 10ml of D-Hanks to wash, then discard the culture medium, add 5-8ml of 0.25% recombinant trypsin digestion solution, and digest for 1-2 minutes;

[0029] 3) Add 10ml of D-Hanks to each bottle to stop digestion. Transfer the digested cell and tissue block suspension to a 50ml centrifuge tube, filter through a 100μm cell filter, wash with 5ml of D-Hanks, and collect in a 50ml centrifuge tube.

[0030] 4) Centrifuge the cells at 500g for 5 minutes and discard the supernatant. Then resuspend the cells in 2ml of D-Hanks and seed them into T75 culture flasks at a passage ratio of 1:4. Add 10ml of umbilical cord mesenchymal stem cell culture medium and culture for 5-6 days.

[0031] 5) Repeat the digestion and passage steps above until P2 generation mesenchymal stem cells are obtained.

[0032] Furthermore, the mass fraction of trypsin used for digesting mesenchymal stem cells was 0.25%.

[0033] Furthermore, the inoculation ratio in step e is 1:4.

[0034] Furthermore, the amniotic membrane microcarriers and the loading of mesenchymal stem cells were prepared using the following steps:

[0035] 1) Weigh 0.25g of amniotic membrane microcarriers, wash twice with calcium- and magnesium-free DPBS, centrifuge at 500g for 5 min, and discard the supernatant. Rinse with umbilical cord mesenchymal stem cell culture medium and pre-equilibrate to 37℃;

[0036] 2) Discard the original culture medium of P2 generation umbilical cord mesenchymal stem cells with 80% confluence in T75 culture flasks, add 10ml of D-Hanks for washing, then discard the culture medium, add 5-8ml of 0.25% recombinant trypsin digestion solution, and digest for 1-2 minutes.

[0037] 3) Add 10ml of D-Hanks to stop digestion, transfer the digested cells to a 50ml centrifuge tube, centrifuge at 500g for 5min, and discard the supernatant;

[0038] 4) Take 10 million umbilical cord mesenchymal stem cells, add them to the pre-equilibrated amniotic membrane microcarrier, put them together into a 100ml bioreactor, add 25ml of umbilical cord mesenchymal stem cell-specific culture medium, and culture at 37℃ with a volume concentration of 5% CO2 for 24 hours with low-speed intermittent stirring at 20-50rpm.

[0039] 5) After 24 hours, take a small amount of liquid and observe the cell adhesion on the surface of the microcarrier under a microscope. Add culture medium to the reactor to a volume of 70 ml, and increase the rotation speed to 75-125 rpm to maintain microcarrier suspension.

[0040] 6) Take samples to observe cell growth and culture medium color. After culturing for 1-3 days, stop stirring, let stand for 10 minutes, discard half of the old culture medium, add 35ml of preheated umbilical cord mesenchymal stem cell culture medium, and maintain suspension culture at 75-125rpm.

[0041] 7) After culturing for 5-10 days, terminate the bioreactor, let it stand for 10 minutes, discard half of the culture medium, and transfer the remaining culture medium along with the amniotic membrane microcarrier to a 50ml centrifuge tube. Centrifuge at 500g for 5 minutes, discard the supernatant, and you will obtain the amniotic membrane microcarrier loaded with MSCs. Further, the trypsin mass fraction in step b above is 25%, and digestion is performed for 1-2 minutes.

[0042] Further, the mesenchymal stem cells from step b above are digested and centrifuged, and then 10 million mesenchymal stem cells are added to a pre-equilibrated amniotic membrane carrier.

[0043] Furthermore, the density ratio of amniotic microcarriers to mesenchymal stem cells was 1:4.

[0044] Furthermore, in step c above, mesenchymal stem cells and amniotic membrane microcarriers are stirred under a CO2 concentration of 5% by volume.

[0045] As a second aspect of the present invention, an amniotic membrane biocomposite material prepared by the above preparation method is provided, comprising amniotic membrane microcarrier, mesenchymal stem cells and polypeptide hydrogel.

[0046] Furthermore, the aforementioned amniotic membrane microcarrier is a 3D three-dimensional load structure.

[0047] Furthermore, the amniotic membrane microcarriers mentioned above are particles of 200~300μm.

[0048] As a third aspect of the present invention, the application of the above-described amniotic membrane biocomposite material in promoting the repair of endometrial damage is provided.

[0049] Compared with existing technologies, the biocomposite material, its preparation method, and its application provided by this invention have at least the following beneficial effects: This method uses decellularized amniotic membrane as raw material, which is dehydrated and freeze-pulverized to prepare a microcarrier suitable for stem cell growth. Umbilical cord mesenchymal stem cells are loaded onto the microcarrier through 3D culture, and hydrogel is added and mixed evenly to form an amniotic membrane microcarrier-mesenchymal stem cell-hydrogel composite biomaterial. The combination of amniotic membrane microcarrier and hydrogel provides three-dimensional support for stem cells. Perfusion into the uterine cavity can enable cells to survive for a long time. At the same time, the microcarrier and hydrogel have a sustained-release effect on the exocrine secretions of stem cells, which is more conducive to promoting the repair function of mesenchymal stem cells. The main product after degradation of this product is amino acids, which have good safety.

[0050] Cells loaded with microcarriers can be injected into the body's cavities via perfusion. Compared to single biological amniotic membranes or other biomaterials, the increased number of cells loaded simultaneously leads to better therapeutic effects. The procedure is simple and easy to perform, providing better coverage of the uterine lining and promoting endometrial repair. Experimental results have verified that the prepared composite biomaterial effectively promotes cell growth at sites of thin endometrial damage, prevents re-adhesion after intrauterine adhesion separation, and promotes the growth and repair of damaged endometrium. Attached Figure Description

[0051] Figure 1 This is a diagram of hematoxylin-eosin staining (HE staining) of decellularized biological amnion. 。

[0052] Figure 2 It refers to the particle size distribution of amniotic membrane microcarriers.

[0053] Figure 3 This is a flowchart of the preparation process for amniotic membrane biocomposite materials.

[0054] Figure 4 This represents the expression level of the inflammation-related cytokine IL-6 in different groups of rats.

[0055] Figure 5 This represents the expression levels of the inflammation-related cytokine TNF-α in different groups of rats.

[0056] Figure 6 This represents the expression level of the inflammation-related cytokine IL-1β in different groups of rats.

[0057] Figure 7 This represents the expression level of the fibrotic cytokine TGF-β in different groups of rats.

[0058] Figure 8 This represents the expression level of the fibrotic cytokine VEGF in different groups of rats.

[0059] Figure 9 This is a comparison of the cell load on decellularized amniotic membrane and amniotic membrane microcarriers. Detailed Implementation

[0060] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0061] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention.

[0062] Example 1: Preparation of amniotic membrane microcarriers

[0063] The amnion was obtained from a healthy donor with informed consent. After obtaining the placenta, the amnion was separated from the chorionic villi using medical forceps. Visible blood vessels and other impurities were removed, and the amnion was rinsed with physiological saline at least five times until no obvious bloodstains remained. The amnion was transferred to a 50 mL sterile centrifuge tube, and an equal volume of 1.0%–1.5% Trinton X-100 solution was added. The centrifuge tube was then placed in a 37°C, 120 rpm air bath shaker for 4–12 hours. The amnion was then transferred to a 10 mm culture dish, washed five times with physiological saline, and then digested with an equal volume of 0.25%–0.5% trypsin. The mixture was then shaken at 37°C, 120 rpm for 4–6 hours. After digestion, the amnion was washed five times with physiological saline to obtain the decellularized amnion.

[0064] The obtained decellularized amniotic membrane was immobilized in the sample container of a supercritical CO2 extraction device at a pressure of 12 MPa, a temperature of 37 °C, and a CO2 flow rate of 0.1-0.8 L / min for 0.5-12 h. The dried amniotic membrane was then carefully placed in a cryopreservation bag and rapidly frozen in liquid nitrogen for 12-24 h until natural cracks appeared on the surface. The amniotic membrane was then removed and homogenized into microparticles using a homogenizer. The amniotic membrane microparticles were then sieved through an 80-mesh metal sieve and then through a 60-mesh metal sieve to obtain particles of 200-300 μm, which were collected in 50 ml centrifuge tubes and sterilized by irradiation to obtain amniotic membrane microcarriers.

[0065] The amniotic membrane microcarriers prepared by the above methods were used to observe cell removal using hematoxylin-eosin staining. Figure 1 As can be seen, the image is predominantly eosin, indicating that the cells on the surface of the amnion have been completely removed.

[0066] Examples 2-6

[0067] The preparation steps of the amniotic membrane microcarriers in Examples 2-6 are the same as in Example 1, except that some parameters are adjusted as shown in Table 1 below. CO2 flow rate is 0.1-0.6 L / min for 1-8 h extraction, followed by liquid nitrogen freezing for at least 12 h. The resulting amniotic membrane is homogenized, sieved, and then... Figure 2 It can be seen that the yield of 200-300μm microcarriers is over 75%.

[0068] Table 1. Preparation parameters of amniotic membrane microcarriers for different embodiments

[0069]

[0070] Example 7 Preparation of umbilical cord mesenchymal stem cells

[0071] Primary culture: Place the umbilical cord in a 50ml centrifuge tube containing 75% alcohol and sterilize for approximately 1 minute. Then place it in a 15cm culture dish, pour in washing solution to rinse, and remove surface impurities with forceps. Use surgical scissors to remove 2-3cm from both ends of the ligation, and cut the remaining portion into 1-2cm segments, rinsing with washing solution. Use ophthalmic forceps to remove the two arteries and one vein of the umbilical cord. Use surgical scissors to cut the tissue into 2mm pieces in a 2ml EP tube. 3 Tissue fragments. Add 10ml of umbilical cord mesenchymal stem cell culture medium to a T75 culture flask, place the cut tissue fragments into the T75 flask with a spacing of 5-10mm, place at 37℃, and culture in a 5% CO2 cell culture incubator. Observe the cell migration after 2-3 days, and replace half of the medium to add 5-8ml of culture medium.

[0072] Cell passage: When the cell confluence reaches 60%-80%, digestion and harvesting can be performed. Remove the cell culture flask from the cell culture incubator, gently shake the flask to detach any loosely adhered tissue fragments, discard the original culture medium, add 10 mL of D-Hanks (manufacturer: Biosharp, catalog number: BL559A), wash and discard, then add 8 mL of 0.25% recombinant trypsin digestion solution and digest for 2 min. Add 10 mL of D-Hanks to stop digestion. Transfer the digested cell and tissue fragment suspension to a 50 mL centrifuge tube, filter through a 100 μm cell filter, wash with 5 mL of D-Hanks, and collect in a 50 mL centrifuge tube. Centrifuge at 500 g for 5 min and discard the supernatant. Resuspend the cells in 2 mL of D-Hanks, seed at a 1:4 passage ratio into a T75 culture flask, add 10 mL of umbilical cord mesenchymal stem cell-specific culture medium, change the medium every other day, and culture for 5 days. When the confluence of mesenchymal stem cells reaches 90%, they are digested and passaged to finally harvest P2 generation umbilical cord mesenchymal stem cells.

[0073] The obtained mesenchymal stem cells were analyzed by flow cytometry. The results showed that the positive rates of CD73, CD90, and CD105 were 99.8%, 98.1%, and 100%, respectively, the HLA-DR was 0.11%, and the sum of CD14, CD19, CD34, and CD45 was 0.08%. The results met the flow cytometry phenotypic quality standards for umbilical cord mesenchymal stem cells.

[0074] Example 8:

[0075] The primary culture steps are the same as in Example 7. During subculture, after the cell confluence reaches 80%, digestion and absorption are performed, and subsequent steps are the same as in Example 7.

[0076] Example 9: Amniotic membrane microcarrier loaded with stem cells

[0077] Weigh four 0.25g aliquots of amniotic membrane microcarriers, wash twice with calcium- and magnesium-free DPBS, centrifuge at 500g for 5 min, and discard the supernatant. Rinse with umbilical cord mesenchymal stem cell culture medium and pre-equilibrate to 37℃. Take 25, 50, 100, and 200*10 5 P2 generation umbilical cord mesenchymal stem cells with a confluence of 80% were added to pre-equilibrated amniotic membrane microcarriers and placed in 100ml bioreactors. 25ml of umbilical cord mesenchymal stem cell-specific culture medium was added, and the cells were cultured at 37℃ and 5% CO2 with intermittent stirring at 20-50rpm for 24h.

[0078] Table 2. Proportion settings for amniotic membrane microcarriers loaded with stem cells

[0079]

[0080] Take 2 ml of culture medium from each of the four experimental groups and transfer it to a 15 ml centrifuge tube. Centrifuge at 500 g for 5 min and remove the supernatant. Then, gently wash twice with 5 ml*2 PBS, centrifuge at 500 g for 5 min, and remove the supernatant. Add Hoechst 33342 / PI to a final concentration of 2 μM and incubate in the dark for 30 min. Then, wash twice with 2 ml*2 PBS, centrifuge at 500 g for 5 min, and remove the supernatant. Add 4% PFA to the centrifuge tube, fix at 4℃ for 15 min, and then centrifuge to remove the supernatant. Resuspend in 5 ml of PBS with 2% FBS, transfer to a culture dish, and mix well using the cross-hatching method. Use a fluorescence microscope to select four fields of view (top, bottom, left, and right), and take 5 images for each field of view. Analyze the microcarrier loading rate using ImageJ software. As shown in Table 3, when the cell-to-microcarrier ratio reaches 4:1, the microcarrier loading is relatively saturated. Beyond this ratio, many cells will not be able to load the microcarriers. Therefore, 4:1 is a more suitable cell-to-microcarrier ratio.

[0081] Table 3. Microcarrier loading conditions for different groups

[0082]

[0083] On the second day, the culture medium volume in the reactor was replenished to 70 ml, and the rotation speed was increased to 75-125 rpm to maintain the suspension of the microcarriers. After culturing for another 2 days, stirring was stopped, and the mixture was allowed to stand for 10 minutes. Half of the old culture medium was discarded, and 35 ml of preheated umbilical cord mesenchymal stem cell-specific culture medium was added. The mixture was then kept in suspension at 75-125 rpm. After culturing for another 3 days, the bioreactor was terminated, and after standing for 10 minutes, half of the culture medium was discarded. The remaining culture medium, along with the amniotic membrane microcarriers, was transferred to a 50 ml centrifuge tube, centrifuged at 500 g for 5 min, and the supernatant was discarded to obtain the amniotic membrane microcarriers loaded with MSCs.

[0084] Example 10: Preparation of a composite biomaterial of umbilical cord mesenchymal stem cells-amniotic membrane microcarrier-peptide hydrogel

[0085] A method for preparing an amniotic membrane biocomposite material mainly includes the following steps: first, amniotic membrane microcarriers are loaded with mesenchymal stem cells, and then mixed and cultured with a polypeptide solution at a mass ratio of 1:2-1:4 to obtain a mesenchymal stem cell-amniotic membrane microcarrier-polypeptide hydrogel biocomposite material. The preparation process is as follows: Figure 3 As shown.

[0086] Specifically, a small amount of peptide powder (EILWLK-KR) was taken and dissolved in PBS buffer (pH 6.0) at a concentration of 1.0% w / v, and then sterilized by passing through a 0.22 μm filter. The pH of the peptide solution was adjusted to 7.2, and then filtered through a 0.22 μm filter. The amniotic membrane microcarrier loaded with MSCs prepared in Example 7-Group 3 was mixed with the peptide solution at a mass ratio of 1:2, and incubated in an air bath shaking bed at 30 rpm and 37°C for 30 min to obtain the umbilical cord mesenchymal stem cell-amniotic membrane microcarrier-peptide hydrogel composite biomaterial.

[0087] Example 11:

[0088] A small amount of peptide powder (EILWLK-KR) was dissolved in PBS buffer (pH 6.0) at a concentration of 1.5% w / v and sterilized by passing through a 0.22 μm filter. The pH of the peptide solution was adjusted to 7.4, and then filtered through a 0.22 μm filter. The amniotic membrane microcarrier loaded with MSCs prepared in Example 7-Group 3 was mixed with the peptide solution at a mass ratio of 1:4. The mixture was incubated in an air bath shaking bed at 30 rpm and 37°C for 30 min to obtain the umbilical cord mesenchymal stem cell-amniotic membrane microcarrier-peptide hydrogel composite biomaterial.

[0089] Comparative Example 1: Comparison of loading capacity between decellularized amniotic membrane and amniotic membrane microcarriers

[0090] Decellularized amniotic membrane was cut into 1cm x 1cm pieces, totaling 12 pieces, and laid flat in a 6-well plate. Umbilical cord mesenchymal stem cells were then added to adjust the cell density to 1 x 102 cells using complete culture medium. 6 Add 40 μL per tablet, for a total of 40,000 cells, and then add complete culture medium to 5 ml.

[0091] Take 12 portions of 100mg amniotic membrane microcarriers (1cm) 2 Decellularized amnion membrane yields approximately 100 mg of microcarriers. Each batch contains 40,000 cells, and complete culture medium is added to a 5 ml system. The mixtures are then added separately to the bioreactor.

[0092] Digestion and cell counting were performed on days 2, 4, 6, and 8, and the average cell load was calculated. The results are as follows: Figure 9 As shown. From Figure 9 As can be seen, the number of cells that an amniotic membrane microcarrier of the same area can carry is far greater than that of decellularized amniotic membrane (after 5 days, the number of cells carried by the amniotic membrane microcarrier is up to 5 times that of decellularized amniotic membrane).

[0093] Experimental Example: Efficacy Trial of Amniotic Membrane Microcarrier-MSC-Polypeptide Hydrogel Composite Biomaterial in the Treatment of IUA

[0094] (1) Model building

[0095] Female SD rats (weighing 190±10 g) were selected and acclimatized for one week. Before surgery, the rats fasted for 12 hours. After anesthesia, laparotomy was performed. A 2 cm incision was made in the lower third of the uterus. Preoperative endometrial thickness was measured. The endometrium and part of the stroma were scraped with a scalpel until the surface felt rough. The uterus was sutured, and the abdomen was closed layer by layer. In the sham-operated group, the uterus was exposed during surgery, and the endometrial thickness was measured after incision. The wound was then sutured, and the abdomen was closed.

[0096] (2) Experimental grouping

[0097] Seventy-two rats were randomly divided into six groups: sham-operated group (control group), model group, stem cell group, amniotic membrane microcarrier group, hydrogel group, and combined treatment group (amniotic membrane microcarrier + stem cells + hydrogel). Intrauterine injection was performed on the seventh day after surgery.

[0098] Sham surgery group (control group): No drug treatment received; Model group: Uterine cavity perfused with 200 μL of normal saline; Stem cell group: Uterine cord mesenchymal stem cells (1*10) were administered via 200 μL of normal saline. 6

[0099] The treatment involved perfusion of individual rats with amniotic membrane microcarriers (0.02 g of microcarriers) in a saline solution containing 200 μL of amniotic membrane microcarriers. The combined treatment group (amniotic membrane microcarriers + stem cells + hydrogel) was perfused into the uterus of rats. Six to eight weeks after treatment, five rats from each group were used to measure endometrial thickness, glandular number, endometrial fibrosis rate, and the expression of genes related to endometrial cell proliferation regulation.

[0100] (3) Comparison of endometrial thickness, number of glands, and endometrial fibrosis rate among different groups

[0101] Mid-segment uterus was harvested and fixed, then sectioned for HE staining. SlideViewer software was used to assess endometrial thickness, glandular number, and endometrial fibrosis rate in rats. Three sections were randomly selected from each group. Endometrial thickness was measured in four random directions (up, down, left, right) on each section at 40× field of view. Three fields of view were selected from each section for glandular count, and the average value was taken. Uterine sections were then subjected to Masson staining. Three sections were randomly selected from each group, and ImageJ software was used to calculate the percentage of stromal fibrosis area to the total area, and the average value was taken.

[0102] As shown in Table 4, the endometrial thickness and glandular number of rats in the model group were smaller than those in the control group, while the uterine fibrosis rate was significantly higher in the model group. The endometrial thickness and glandular number of the amniotic membrane microcarrier group and the hydrogel group were improved compared with the model group. After receiving various combined stem cell treatments, the endometrial thickness and glandular number of rats were greater than those in the model group, while the uterine fibrosis rate was significantly lower in the model group.

[0103] Table 4. Endometrial thickness, glandular number, and endometrial fibrosis ratio in rats of each group.

[0104]

[0105] (4) Detection of expression of inflammation and fibrosis-related genes

[0106] Following the instructions of the kit, total RNA was extracted from the rat uterus using Trizol, and total cDNA was synthesized by reverse transcription. The expression levels of IL-6, TNF-α, IL-1β, TGF-β, and VEGF were detected using an RT-PCR instrument.

[0107] Pro-inflammatory cytokines (IL-6, TNF-α, IL-1β) mediate inflammatory responses by activating immune cells, and inflammation within the uterine cavity exacerbates endometrial fibrosis. For example... Figure 4-8 As shown, the expression of IL-6, TNF-α, and IL-1β in the uterus of rats in the combined treatment group was significantly lower than that in the model group, with statistical differences. This indicates that the composite biomaterial of stem cells + amniotic membrane microcarrier + hydrogel in Example 10 has a significant inhibitory effect on inflammatory factors in the uterus, and its inhibitory effect is better than that of the stem cell group. Its gene expression level is comparable to that of the control group.

[0108] TGF-β and VEGF are classic pro-fibrotic cytokines that can promote endometrial fibrosis, leading to endometrial hardening, scarring, and other pathological conditions, resulting in menstrual irregularities, infertility, and recurrent miscarriages. Studies have shown that the expression levels of TGF-β and VEGF in the uterus of rats in the combined treatment group were significantly lower than those in the model group, with statistically significant differences. This indicates that the composite biomaterial of stem cells + amniotic membrane microcarrier + hydrogel can inhibit further fibrosis of damaged endometrium and reduce uterine damage by regulating the expression of TGF-β and VEGF in the endometrium.

[0109] (5) Assessment of fertility in rats of different groups

[0110] Six rats were used in each group, and female rats in each group were mated with healthy male rats. After 25 days of gestation, three rats from each group were used to count the number of fetuses in the uterus, while the other three rats gave birth naturally, and the number of live births was counted. The results showed that the number of fetuses and the number of live births in the model group were significantly lower than those in the control group. Rats treated with the composite biomaterial of stem cells + amniotic membrane microcarrier + hydrogel in Example 10 showed a significant increase in the number of embryo implantation and live births, which was superior to that in the stem cell group. This indicates that the composite biomaterial has a good repair ability on the uterus of female rats and can improve the fertility of IUA rats.

[0111] Table 5. Number of fetuses and live-born rats in different groups of rats

[0112]

Claims

1. A method for preparing an amnion biocomposite material, characterized in that, Mesenchymal stem cells were loaded onto amniotic membrane microcarriers and then mixed with peptide solutions at a mass ratio of 1:2 to 1:4 to obtain a mesenchymal stem cell-amniotic membrane microcarrier-peptide hydrogel biocomposite material. In the polypeptide solution, the sequence structure of the polypeptide is: EILWLK-KR; The preparation method of the amniotic membrane microcarrier is as follows: a. Wash the fresh amniotic membrane repeatedly with physiological saline until no blood remains; b. Digest the amnion obtained in step a by shaking with a nonionic surfactant; c. Digest the amnion obtained in step b with trypsin by shaking. d. Wash the amnion obtained in step c with physiological saline to obtain decellularized amnion; e. The decellularized amnion obtained in step d is dried by supercritical CO2 extraction. f. After rapidly freezing the amnion obtained in step e in liquid nitrogen, remove the amnion and homogenize it into tiny particles. g. The amnion obtained in step f is screened using a metal sieve to obtain amnion microparticles; The amniotic membrane microcarriers loaded with mesenchymal stem cells were prepared using the following steps: a. Rinse the amniotic membrane microcarriers with a special culture medium for umbilical cord mesenchymal stem cells; b. Digest mesenchymal stem cells with trypsin; c. After mixing and culturing the digested mesenchymal stem cells from step b with amniotic membrane microcarriers for a period of time, centrifuge to remove the supernatant to obtain amniotic membrane microcarriers loaded with mesenchymal stem cells; and The density ratio of the amniotic membrane microcarrier to mesenchymal stem cells is 1:

4.

2. The method for preparing the amnion biocomposite material according to claim 1, characterized in that: Before the polypeptide solution is mixed with amniotic microcarriers and mesenchymal stem cells for culture, the pH is adjusted to 7.

4.

3. The method for preparing the amnion biocomposite material according to claim 1, characterized in that, In the preparation method of the amniotic membrane microcarrier, the mass fraction of the trypsin is 0.25-0.5%.

4. The method for preparing the amnion biocomposite material according to claim 1, characterized in that, In the preparation method of the amniotic membrane microcarrier, the CO2 flow rate in step e is 0.1-0.8 L / min, and the flow rate is maintained for 0.5-12 h.

5. The method for preparing the amnion biocomposite material according to claim 1, characterized in that, The method for preparing the mesenchymal stem cells is as follows: a. After disinfecting and cleaning the umbilical cord, removing the arteries and veins, cut it into 2mm pieces. 3 The organizational blocks; b. Place in a special culture medium for umbilical cord mesenchymal stem cells for primary culture; c. When the cell confluence reaches 60%-80%, digest with recombinant trypsin; d. Add D-Hanks to the digestion solution from step c to terminate digestion, filter with a cell filter, and wash with D-Hanks; e. After centrifugation, the cells were resuspended in D-Hanks medium, supplemented with umbilical cord mesenchymal stem cell-specific culture medium, and passaged to obtain P2 generation mesenchymal stem cells.

6. The method for preparing the amnion biocomposite material according to claim 5, characterized in that: The mass fraction of trypsin in step c is 0.25%.

7. The method for preparing the amnion biocomposite material according to claim 5, characterized in that: The inoculation ratio for the passage was 1:

4.

8. The method for preparing the amniotic membrane biocomposite material according to claim 1, characterized in that: The amniotic microcarrier and mesenchymal stem cell loading step b uses 0.25% trypsin and digests for 1-2 minutes.

9. The method for preparing the amnion biocomposite material according to claim 1, characterized in that: In step c, the amniotic microcarrier and mesenchymal stem cells are loaded together, and the mesenchymal stem cells and amniotic microcarrier are stirred under a volume concentration of 5% CO2.

10. An amnion biocomposite material prepared by any one of the preparation methods described in claims 1-9.

11. The amnion biocomposite material according to claim 10, characterized in that: The amniotic membrane microcarrier is a 3D three-dimensional load structure.

12. The amnion biocomposite material according to claim 10, characterized in that: The amniotic membrane microcarrier consists of particles of 200–300 μm.

13. Use of the amniotic membrane biocomposite material according to any one of claims 10-12 for the preparation of a medicament that promotes the repair of endometrial damage.

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