A composite amnion for improving post-treatment contracture of tissue repair

Abstract

The application discloses a composite amnion for improving contracture after tissue repair and a preparation method thereof. The composite amnion comprises a recombinant humanized collagen nanofiber membrane in a middle layer and human acellular amnions attached to two sides of the nanofiber membrane. The composite amnion is prepared by electrospinning technology to prepare the recombinant humanized collagen nanofiber membrane and then pressing the nanofiber membrane and the human acellular amnions. The composite amnion has excellent mechanical properties and biological activity. Animal experiment results show that the composite amnion can effectively inhibit radiotherapy-induced capsular contracture, and the comprehensive effect is obviously better than that of single-component materials in terms of anti-inflammatory, anti-fibrosis, delayed degradation speed and improved capsular compliance. The composite amnion can be used for improving breast prosthesis capsular contracture, especially radiotherapy-induced capsular contracture.

Description

Technical Field

[0001] The invention relates to the field of biomedical materials technology, specifically to a composite membrane for tissue repair and its application in the preparation of medical devices, particularly in the prevention or improvement of capsular contracture after breast implantation. Background Technology

[0002] In the fields of regenerative medicine and tissue engineering research, good biomaterials for tissue repair and regeneration need to possess good biocompatibility, biodegradability, excellent physicochemical properties, and an ideal three-dimensional network structure, while also exhibiting properties that promote angiogenesis and soft tissue repair. However, many implantable biomaterials, such as breast implants, often trigger unpredictable immune responses and excessive foreign body reactions, leading to tissue inflammation, contractures, and other complications.

[0003] Breast cancer is one of the most common malignant tumors in women. With the improvement of treatment levels, patients, while pursuing higher survival rates, are also placing higher demands on postoperative quality of life and physical reconstruction. Implant-assisted breast reconstruction has become a commonly used procedure due to its minimal surgical trauma and rapid recovery. However, as a foreign body, the implant can trigger a foreign body reaction, eventually leading to the formation of a fibrous capsule around the implant. When this fibrous capsule becomes excessively fibrotic and contracts, it results in capsular contracture, clinically manifested as breast hardening, deformation, and pain. In severe cases, repeat surgery is required, causing significant physical and psychological suffering and financial burden on patients. Studies show that approximately 40% of patients undergoing implant-assisted breast reconstruction require a second surgery due to severe capsular contracture.

[0004] To address this challenge, breast patches have been introduced clinically. Commonly used breast patches are divided into two categories: biological patches and non-biological patches. Biological patches, such as acellular dermal matrix (ADM), not only provide adequate tissue coverage, but their biological activity also helps reduce capsular contracture. However, currently mainstream human or animal-derived ADMs have inherent drawbacks such as scarcity, high cost, potential immunogenicity, and the risk of disease transmission. Non-biological patches, such as synthetic patches, are cheaper, but generally lack biological activity, cannot actively induce tissue repair, and their degradation products may induce chronic inflammation, making their preventive effect on capsular contracture controversial.

[0005] Human acellular amniotic membrane (HdAM), as a natural biomaterial, exhibits extremely low immunogenicity, abundant bioactive factors, and good anti-inflammatory and anti-fibrotic properties, showing potential as an ideal patch. However, its poor mechanical strength and rapid in vivo degradation limit its application in sites requiring long-term support. The emergence of recombinant humanized collagen has brought hope for solving the immunogenicity problem of animal-derived collagen, but it also suffers from insufficient mechanical properties or mismatched degradation rates when formed into a film on its own.

[0006] Therefore, there is an urgent need in this field to develop a novel biocomposite membrane patch that combines excellent mechanical properties, controllable degradation rate, good biocompatibility, and active anti-inflammatory and anti-fibrotic biological functions, thereby effectively preventing and improving capsular contracture. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a composite membrane with enhanced mechanical properties, controllable degradation, good biocompatibility, and effective inhibition of membrane contracture, as well as its preparation method and application.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a composite membrane (CC) for improving postoperative contracture after tissue repair surgery. The composite membrane comprises a recombinant humanized collagen nanofiber membrane in the middle layer, and decellularized amniotic membranes attached to both sides of the recombinant humanized collagen nanofiber membrane, forming a three-layer structure enclosed by decellularized amniotic membranes on both the inner and outer sides. The average fiber diameter of the recombinant humanized collagen nanofiber membrane is 100-150 nm, preferably 113.11 ± 2.08 nm.

[0010] Preferably, the average fiber diameter of the composite membrane is 10-20 nm, and more preferably 15.44 ± 0.24 nm.

[0011] Preferably, the composite membrane exhibits excellent mechanical properties and a controllable degradation rate, with an elastic modulus of not less than 8 MPa, and a degradation rate of not more than 60% after 7 days of in vitro degradation in collagenase solution, preferably between 50% and 60%.

[0012] Secondly, the present invention provides a method for preparing the composite membrane: human decellularized amnion and recombinant humanized collagen nanofiber membrane are sequentially stacked to form a three-layer structure, subjected to pressing treatment, and freeze-dried to obtain the composite membrane.

[0013] The preparation method of the recombinant humanized collagen nanofiber membrane includes the following key steps: 1. Dissolve recombinant humanized collagen at a concentration of 30-40% (w / v) in a 50% (v / v) ethanol aqueous solution, and electrospin at a injection speed of 0.1-0.2 mm / min to obtain the original nanofiber membrane. 2. Perform plasma treatment on the fiber membrane to activate its surface. The plasma treatment parameters are: oxygen inlet pressure 0.1 MPa, inlet flow rate 30 ml / min, chamber pressure 25 Pa, radio frequency power 20 W, and treatment time 5 min. 3. Crosslink the original fiber membrane using a crosslinking solution (solvent: 90.0% anhydrous ethanol) with a concentration of 1.5 WT and an EDC to NHS molar ratio of 5:2. In a third aspect, the present invention provides the application of the composite membrane in the preparation of medical devices. The medical device is preferably a breast patch or breast implant, used to prevent or improve postoperative contracture after tissue repair, especially radiotherapy-induced capsular contracture.

[0014] The human decellularized amnion (HdAM) involved in this invention is the innermost layer of the human placenta. It is a transparent tissue without nerves, blood vessels, or lymphatic vessels, and is tough. The decellularized amnion is obtained after complete decellularization.

[0015] The recombinant humanized collagen involved in this invention is obtained by using the collagen of human skin as the original gene sequence, optimizing and screening the advantageous parts (such as parts with strong water solubility, high biological activity and / or good permeability), and performing codon optimization and splicing recombination.

[0016] The recombinant humanized collagen nanofiber membrane (RCC) involved in this invention refers to a porous thin film material with an average fiber diameter in the nanometer range, prepared by electrospinning and other techniques using recombinant humanized collagen as the main raw material. Its microstructure consists of a large number of nanofibers randomly interlaced to form a three-dimensional network structure.

[0017] The beneficial effects of this invention are as follows:

[0018] 1. Process Innovation, Structural Innovation, and Performance Synergy: By optimizing electrospinning parameters (such as specific injection speed), crosslinking agent ratio, and introducing plasma treatment, RCC with an average fiber diameter of 100-150 nm is formed. The composite amniotic membrane (CC) obtained after RCC and HdAM are pressed together has a significantly thinner fiber diameter, with an average diameter of approximately 15.44±0.24 nm. The fibers are tightly packed, forming a large number of pores, constituting a unique "HdAM-RCC-HdAM" three-layer composite structure. This combines the excellent bioactivity of HdAM with the nanofiber scaffold advantages of RCC, significantly enhancing mechanical properties (elastic modulus increased by more than ten times) while maximizing the preservation of its anti-inflammatory and anti-fibrotic biological functions, and effectively slowing down the degradation rate. 2. Significant Effects: Animal experiments have shown that this composite membrane can significantly inhibit radiotherapy-induced capsular contracture, manifested as the thinnest capsule thickness, reduced collagen deposition, downregulation of fibrosis markers (TGF-β, α-SMA), reduced inflammatory response (decreased MPO, decreased IL-10), improved capsule compliance (decreased type I / III collagen ratio), and promotion of angiogenesis (α-SMA). + Increased vascular density) and overall superior effects compared to single-component materials. The composite membrane of this invention can be specifically used for the prevention and treatment of capsular contracture of breast implants, especially for radiotherapy-induced capsular contracture and applications requiring long-term tissue repair. 3. Safety and ethical advantages: Utilizing recombinant humanized collagen and human amniotic membrane avoids the risk of animal-borne disease transmission and serious ethical controversies. The source is relatively abundant, quality is controllable, the process is environmentally friendly, and reproducibility is good, possessing industrialization potential. Attached Figure Description

[0019] Figure 1 These are morphological observation images of human decellularized amnion (HdAM), recombinant humanized collagen nanofiber membrane (RCC), and composite membrane (CC).

[0020] Figure 2 These are scanning electron microscope (SEM) images of HdAM, RCC, and CC, showing the microscopic surface morphology and fiber diameter distribution of the material.

[0021] Figure 3 The graph shows the mechanical property test results for HdAM, RCC, and CC. Figure 4 This is an in vitro degradation curve of HdAM, RCC, and CC in collagenase solution.

[0022] Figure 5 This diagram illustrates the adhesion and proliferation of different cells on HdAM, RCC, and CC as determined by the Calcein-AM / PI double staining method.

[0023] Figure 6 This is a general observation diagram of radiotherapy-induced capsular contracture.

[0024] Figure 7 This is an image showing the effect of CC in reducing radiotherapy-induced capsule formation and collagen deposition.

[0025] Figure 8 This image shows the effect of CC reducing fibrosis at the capsule and promoting angiogenesis.

[0026] Figure 9 This is an image showing the effect of CC in reducing radiotherapy-induced inflammation of the capsule tissue.

[0027] Figure 10 This is an experimental result of CC improving the compliance of the capsule tissue (the ratio of type I to type III collagen content). Detailed Implementation

[0028] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that modifications can be made to the described embodiments without departing from the principle of the present invention, and these modifications should also be considered to fall within the scope of protection of the present invention.

[0029] Example 1: Preparation of human decellularized amnion (HdAM)

[0030] Rinse the amniotic membrane thoroughly to remove blood stains and place it in a stainless steel dish. Prepare 500 mL of 0.1% (w / v) neutral protease PBS solution and thoroughly soak the amniotic membrane surface. Incubate at 37°C for 30 min. After incubation, wash three times with PBS. Gently scrape off cell debris from both sides using a cell scraper. Rinse the obtained decellularized amniotic membrane (HdAM) with PBS for 3 min and set aside. Confirm the absence of residual cells by HE staining and scanning electron microscopy.

[0031] Example 2: Preparation of recombinant humanized collagen nanofiber membrane (RCC)

[0032] Recombinant humanized collagen was dissolved in a 50% (v / v) ethanol aqueous solution at a concentration of 35% (w / v) and stirred overnight at room temperature to prepare a clear and transparent electrospinning solution. The spinning solution was fixed in the electrospinning apparatus using a 10 mL syringe equipped with a 21G stainless steel needle. The spinning parameters were set as follows: reciprocating stroke 80 mm, translational speed 250 mm / min, spinning solution injection speed 0.15 mm / min, roller receiver speed 100 rpm / min, positive voltage +9 kV, negative voltage -7 kV. Electrospinning was performed to obtain the original recombinant humanized collagen nanofiber membrane (RCC). Scanning electron microscopy showed that the RCC fibers were continuous and uniform, with an ideal porosity and an average diameter of approximately 113.11 ± 2.08 nm.

[0033] Example 3: Preparation of recombinant humanized collagen composite amniotic membrane (CC)

[0034] The RCC surface obtained in Example 2 underwent plasma treatment: The RCC was placed flat on the quartz plate holder of the plasma cleaner, and the surface of the nanofiber membrane was marked. The membrane was then placed into the plasma cleaner chamber. The oxygen valve was opened, and the plasma cleaner was turned on. The vacuum pump was turned on, and the pressure value in the vacuum gauge was observed. When the pressure in the chamber dropped to 300 mTorr, the angle valve was opened to allow oxygen to enter. The pressure was stopped when it rose to 350-450 mTorr. The RFlevel was rotated to MED, and treatment lasted for 5 minutes. After timing, the RFlevel was rotated to OFF. The vacuum pump was turned off, and the angle valve was rotated until the chamber door opened. The nanofiber membrane was removed and placed in a disposable petri dish for storage away from light. The other side of the nanofiber membrane was treated in the same manner. The plasma treatment parameters were: oxygen inlet pressure 0.1 MPa, inlet flow rate 30 ml / min, chamber pressure 25 Pa, RF power 20 W, and treatment time 5 min.

[0035] Chemical crosslinking of RCC with EDC / NHS: EDC and NHS were dissolved in 90.0% anhydrous ethanol at a molar ratio of 5:2 to prepare a 1.5 WT EDC / NHS crosslinking solution. 10 mL of this crosslinking solution was used to thoroughly wet RCC (5 cm × 8 cm), and the reaction was carried out at 20°C for 24 h – 48 h. After crosslinking, the RCC was removed, washed three times with PBS, and then vacuum dried.

[0036] HdAM was laid flat on silicone paper (matrix side up), and cross-linked RCC was laid flat on top of it. Then, HdAM (matrix side down) was completely placed over the RCC to form a three-layer structure. This composite structure was placed on a pneumatic press and pressed under 27 kPa. After pressing, it was placed at 4°C for 20-24 hours. After the composite membrane reaction was complete, the silicone paper was removed, the membrane was washed three times with PBS, and then freeze-dried to obtain a dried composite membrane (CC). Scanning electron microscopy showed that the CC surface fibers were tightly packed, with an average diameter of approximately 15.44 ± 0.24 nm.

[0037] The morphologies of the prepared human decellularized amniotic membrane (HdAM), recombinant humanized collagen nanofiber membrane (RCC), and composite membrane (CC) are as follows: Figure 1 As shown. SEM images of the surface morphology of the three materials and frequency histograms of fiber diameter distribution for RCC and CC are shown below. Figure 2 As shown. Figure 2In the image, ac is the overall scanning electron microscope image of HdAM, RCC, and CC, with a scale bar of 30 μm; df is a local scanning image of HdAM, RCC, and CC, with a scale bar of 5 μm; g and h are the fiber diameter distribution frequency histograms of RCC and CC.

[0038] Example 4: Performance Testing of Composite Membranes

[0039] Mechanical properties were tested for HdAM, RCC, and CC. The results are as follows: Figure 3 As shown, CC has the highest elastic modulus, reaching 10.16±1.27 MPa, which is significantly higher than HdAM (0.88±0.8 MPa) and RCC (2.23±0.12 MPa). CC's ultimate tensile strength is 3.52±0.59 MPa, which is also significantly better than the other two groups.

[0040] In vitro degradation experiment results are as follows Figure 4 As shown, after incubation in collagenase solution for 7 days, HdAM was almost completely degraded (degradation rate 89.1%), while the degradation rate of CC was only 55.1% and that of RCC was 60.6%, proving that CC has a more stable resistance to degradation.

[0041] The results of the cell compatibility assay (Calcein-AM / PI double staining method) are as follows: Figure 5 As shown, mouse mononuclear macrophage leukemia cells Raw264.7, human skin fibroblasts (HSF), and human umbilical vein endothelial cells (HUVEC) can all adhere, grow, and proliferate normally on CC, demonstrating that CC has good biocompatibility.

[0042] Example 5: Validation of the effect of composite membrane on alleviating radiotherapy-induced capsular contracture in an animal model

[0043] Animal model making:

[0044] 1. Prosthesis implantation

[0045] The experiment was divided into 4 groups: simple prosthesis implantation group (Blank group), HdAM, RCC and CC combined prosthesis implantation group, with 6 rats in each group;

[0046] Anesthesia: After inducing anesthesia in rats with 3%–5% isoflurane, the rats were placed on a small animal operating table and anesthesia was maintained with 2–3% isoflurane. The rats were kept warm with a thermal blanket. The hair on the upper right back was shaved off, covering an area of ​​approximately 5 × 5 cm.

[0047] Disinfection and draping: After disinfecting with 5% povidone-iodine three times, drape with sterile surgical towels;

[0048] Implantation: A surgical incision of about 3 cm was made on the back near the second pair of mammary glands. The implant and the material-encapsulated implant were inserted into the mammary fat pad of the rats. In the RCC and CC groups, the implant was fixed to the surrounding tissue.

[0049] Suturing: Subcutaneous tissue and skin are sutured layer by layer, and the incision is covered with sterile gauze. Aseptic techniques are strictly followed during the surgical procedure.

[0050] Postoperative care: Administer ceftiofur sodium (0.2 mg / kg) for anti-infection and meloxicam (0.3 mg / kg) for analgesia for 3 consecutive days. During this period, closely monitor the wound healing and observe the implantation site for signs of infection such as increased skin temperature, redness, and swelling.

[0051] 2. Radiotherapy-induced capsular contracture

[0052] Seven days after implantation, the incision was found to be completely healed, with no signs of redness, swelling, fever, or increased skin temperature around the implant, indicating infection. X-ray irradiation was then performed on the implantation site to induce capsular contracture. The specific procedures are as follows:

[0053] (1) Anesthesia: Tribromoethanol was administered via intraperitoneal injection for anesthesia;

[0054] (2) X-ray irradiation: The rat was placed flat on the stage of the small animal irradiation instrument. The height of the emitter and the stage was adjusted. The irradiation range was 4*4 cm. The parts that did not need to be irradiated were blocked with lead plates. The X-ray energy was adjusted to 20 Gy for a single irradiation.

[0055] (3) After irradiation, keep the experimental animals warm and encourage them to wake up;

[0056] (4) Within one week after irradiation, observe the mental state of the experimental animals every 2 to 3 days, and check for any redness, swelling, ulceration or other radiation dermatitis damage at the irradiated site.

[0057] (5) Three months later, the capsule tissue around the prosthesis was sampled.

[0058] (6) Prepare paraffin sections from the collected specimens for subsequent research.

[0059] Experimental results

[0060] 1. General observation of the capsule tissue; experimental results are as follows: Figure 6 As shown: Both the Blank group (without radiotherapy) and the Blank-IR group (with radiotherapy) exhibited significant double-capsule formation, with fewer vessels on the capsule surface in the Blank-IR group compared to the Blank group; no significant double-capsule phenomenon was observed in the other radiotherapy groups. Angiogenesis in the capsule of the CC-IR group was significantly increased compared to the other groups.

[0061] 2. Capsule tissue thickness and collagen deposition, experimental results are as follows: Figure 7 (a) is a representative image of capsule thickness; b) is a representative image of collagen deposition in the capsule; c) is a statistical analysis of capsule thickness; d) is a statistical analysis of collagen deposition. HE staining showed that the capsule thickness in the Blank-IR group was significantly increased compared to the Blank group, indicating successful radiotherapy-induced capsule contracture modeling. The capsule thickness in the Blank-IR group was 226.25±23.18 μm, while that in the Blank group was 149.29±6.57 μm, a statistically significant difference. The capsule thicknesses of the different material groups receiving radiation were as follows: HdAM group: 105.34±10.40 μm; RCC group: 135.63±6.16 μm; CC group was the thinnest, at only 55.16±6.43 μm. The difference between Blank-IR and the other radiotherapy-receiving groups was statistically significant.

[0062] Masson staining results showed that the collagen fibers in the Blank-IR group were dense, appearing as coarse cords arranged in parallel; the amount of collagen fibers deposited in the other three radiotherapy groups was significantly less than that in the Blank-IR group; the collagen density in the HdAM group was greater than that in the RCC and CC groups, and the fiber arrangement was more compact; the collagen fibers in the RCC and CC groups were fine and loosely arranged, and scattered red-stained cellulose and other tissues could be seen in the collagen, and tissue-integrated materials could be seen in the capsule tissue.

[0063] 3. CC reduces fibrosis at the capsule site and promotes angiogenesis, such as... Figure 8 (a is a representative graph of TGF-β and α-SMA expression in the capsule tissue; b is a statistical analysis graph of TGF-β; c is a statistical analysis graph of α-SMA; d is a statistical analysis graph of α-SMA + vascular density) As shown: TGF-β is a direct inducing factor of fibrosis. The expression of TGF-β in the capsule of each group showed that the expression of TGF-β in the Blank-IR capsule was significantly increased compared with the Blank group, and the difference was statistically significant; the expression of TGF-β in the capsule tissue of the other three groups was lower than that in the Blank-IR group, and the difference was statistically significant.

[0064] Sustained activation of myofibroblasts is the initiating factor for capsular contracture. Analysis of α-SMA expression in the capsular tissue of each group revealed that α-SMA expression was significantly increased in the Blank-IR group receiving radiotherapy compared to the Blank group (statistically significant); α-SMA expression was decreased in the combined implantation material group compared to the Blank-IR group (also statistically significant). α-SMA+ vessel density was significantly increased in the RCC and CC groups compared to other groups.

[0065] 4. CC reduces radiation-induced inflammation of the capsule tissue, such as... Figure 9(Figure a shows the expression of inflammatory cytokine MPO and anti-inflammatory cytokine IL-10 in the capsule tissue; figure b shows the statistical analysis of MPO expression; figure c shows the statistical analysis of IL-10 expression.) As shown, capsule contracture is a result of continuous inflammatory stimulation. As illustrated, compared with the Blank group, radiotherapy significantly induced tissue inflammation, and MPO expression increased significantly. MPO in the Blank-IR group increased threefold compared to the Blank group, a statistically significant difference. MPO expression in the HdAM-IR and RCC-IR groups was 60% of that in the Blank-IR group. The CC group showed the most significant decrease in MPO, approximately one-third of that in the Blank-IR capsule, a statistically significant difference, suggesting that CC can significantly alleviate radiotherapy-induced capsule tissue inflammation.

[0066] IL-10, as an anti-inflammatory cytokine, plays an important role in the regulation of inflammation. This study examined the expression of IL-10 in the capsule tissue. As shown in the figure, compared to the Blank group, among the four radiotherapy groups, IL-10 expression was similar and slightly decreased in the HdAM-IR and CC-IR groups, while expression was significantly reduced in the Blank-IR and RCC-IR groups, approximately 50% of that in the HdAM-IR and CC-IR groups.

[0067] 5. CC improves the compliance of radiotherapy-induced capsule tissues, such as... Figure 10 (a) Graph showing the expression of type I and III collagen content in the capsule tissue of the Blank group; b) Graph showing the expression of type I and III collagen content in the capsule tissue of each radiotherapy group; c) Statistical analysis of the difference in the ratio of type I and III collagen content in the capsule tissue. As shown, the ratio of type I to type III collagen content in the capsule tissue is a good indicator for predicting capsule compliance. Immunofluorescence co-staining of type I / III collagen revealed varying degrees of increase in type I collagen in the capsule tissue of each of the four radiation-treated groups. This was particularly evident in the Blank-IR group, where, compared to the Blank group, both the inner and outer capsules contained a large amount of type I collagen, especially in the inner capsule where the type I collagen fibers were large and tightly packed. The results of the type I / III collagen content ratio in each group showed that the type I / III collagen content in the Blank-IR group increased 4.3 times compared to the Blank group, a statistically significant difference. The content ratio in the HdAM-IR group was similar to that in the Blank group at 4.4%. The content ratios in the RCC-IR and CC-IR groups were significantly lower than those in the Blank-IR group, at 2.9% and 2.5%, respectively. The differences in the type I / III collagen content ratio between the three material groups and the Blank-IR group were statistically significant.

Claims

1. A composite amniotic membrane for improving postoperative contracture after tissue repair surgery, characterized in that, The composite amniotic membrane comprises a recombinant humanized collagen nanofiber membrane in the middle layer, and decellularized human amniotic membranes attached to both sides of the recombinant humanized collagen nanofiber membrane. The decellularized human amniotic membrane and the recombinant humanized collagen nanofiber membrane are sequentially stacked to form a three-layer structure, then pressed and freeze-dried to obtain the composite amniotic membrane. The recombinant humanized collagen nanofiber membrane is obtained by electrospinning, with an average fiber diameter of 100-150 nm. The average fiber diameter of the pressed composite amniotic membrane is 10-20 nm. The contracture refers to radiotherapy-induced contracture after breast tissue repair surgery.

2. The composite amnion as described in claim 1, characterized in that... The preparation method of the recombinant humanized collagen nanofiber membrane includes the following steps: (1) Electrospinning: Recombinant humanized collagen was dissolved in 50% (v / v) ethanol aqueous solution at a concentration of 30-40% (w / v) to prepare an electrospinning solution. Electrospinning was carried out at a solution injection speed of 0.1-0.2 mm / min to obtain the original recombinant humanized collagen nanofiber membrane. (2) Plasma treatment: The recombinant humanized collagen nanofiber membrane is subjected to plasma treatment to obtain the target recombinant humanized collagen nanofiber membrane. (3) Crosslinking reaction: The plasma-treated recombinant humanized collagen nanofiber membrane is immersed in a crosslinking solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to carry out the crosslinking reaction, thereby obtaining a crosslinked recombinant humanized collagen nanofiber membrane; the concentration of the crosslinking solution is 0.05-0.5 M, and the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to N-hydroxysuccinimide (NHS) is 5:

2.

3. The composite amnion as described in claim 2, characterized in that... The electrospinning parameters for the preparation method of the recombinant humanized collagen nanofiber membrane are as follows: reciprocating stroke is 80 mm, translation speed is 250 mm / min; spinning solution injection speed is 0.15 mm / min; rotational speed of the roller receiver is 100 rpm, positive voltage is +9 kV, and negative voltage is -7 kV.

4. The composite amnion as described in claim 2, characterized in that... The specific steps of the crosslinking reaction in the preparation method of the recombinant humanized collagen nanofiber membrane are as follows: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are dissolved in 90% anhydrous ethanol at a molar concentration ratio of 5:2 to prepare a 0.1 M crosslinking solution. The original recombinant humanized collagen nanofiber membrane was fully immersed in the crosslinking solution for 24 hours. After washing and vacuum drying, the crosslinked recombinant humanized collagen nanofiber membrane was obtained.

5. The composite amnion as described in claim 2, characterized in that... The plasma treatment parameters for the preparation method of the recombinant humanized collagen nanofiber membrane are as follows: oxygen inlet pressure 0.1 MPa, inlet flow rate 30 ml / min, chamber pressure 25 Pa, radio frequency power 20 W, and treatment time 5 min.

6. The method for preparing the composite amnion as described in claim 1, characterized in that, Includes the following steps: (1) Provide decellularized human amnion; (2) Provide recombinant humanized collagen nanofiber membranes; (3) Human decellularized amnion and recombinant humanized collagen nanofiber membrane are stacked in sequence to form a three-layer structure, and then pressed and freeze-dried to obtain the composite amnion.

7. The use of the composite amniotic membrane as described in claim 1 in the preparation of breast patches or breast implants or breast implant capsules.

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