Method for preparing collagen membrane for guided tissue regeneration

Abstract

Provided is a method for preparing a collagen membrane for guided tissue regeneration. By utilizing the electrochemical deposition technology and using collagen as the starting material, the method achieves the electrochemical deposition in a low-temperature environment using a direct-current source, and thus, in a constant-voltage output mode, prepares a collagen membrane of an asymmetric structure (with one dense surface and one loose and porous surface) in one step at a high voltage. Also, by means of asymmetric cross-linking, the membrane may be asymmetrically degraded in vivo, thus promoting the rapid repair of a wound while keeping a stable shielding effect on the wound. The method features ease to operate, efficiency, environment conservation, and good scalability. The obtained collagen membrane possesses excellent mechanical properties and uniform overall performance. The area of the collagen membrane of the asymmetric structure prepared by the preparation method is equal to that of the electrode plate, such that scale-up production can be achieved by using an electrode plate with a greater area, thereby improving productivity.

Description

A preparation method of collagen membrane for guiding tissue regeneration

[0001] This invention claims priority to a Chinese patent application filed with the Patent Office of China on November 20, 2023, with application number 202311545675X and invention name “A method for preparing a collagen membrane for guiding tissue regeneration”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The invention belongs to the technical field of biomedical materials and relates to a method for preparing a collagen membrane for guiding tissue regeneration. Background Art

[0003] Guided tissue regeneration membranes utilize barrier membrane technology to provide a relatively enclosed space for damaged tissue, thereby selectively guiding tissue regeneration. In the field of bone regeneration, guided tissue regeneration membranes are surgically placed within the bone defect. The soft tissue-contacting side of the membrane blocks faster-growing fibroblasts, preventing their ingress. The bone-contacting side, however, guides slower-growing osteoblasts to attach, proliferate, and differentiate, accelerating bone regeneration. Therefore, an ideal guided tissue regeneration membrane should possess an asymmetric structure, with a dense layer to block cell migration and a loose layer to guide cell attachment and proliferation.

[0004] Currently, a variety of techniques are used to construct membranes with asymmetric structures, including freeze-drying, electrospinning, 3D printing, or a combination of these techniques. Freeze-drying primarily utilizes the sublimation of ice crystals during freeze-drying to form a porous structure. While this method is simple and cost-effective, its overall pore size structure lacks uniformity and can even lack connectivity between pores. Electrospinning, through a series of equipment improvements or adjustments to process parameters, can manipulate the spatial distribution of fibers to create asymmetric structures. However, electrospinning requires rapid solvent evaporation during the spinning process, and organic solvents are typically used to dissolve the raw materials. However, organic solvents can denature natural macromolecules such as proteins, limiting the selection of suitable raw materials for electrospinning. Furthermore, the pore size range achievable by electrospinning is very small, essentially at the nanometer level. Membranes at this size are insufficient for cell / tissue infiltration and therefore lack the ability to promote tissue regeneration. 3D printing encompasses a range of technologies, including fused deposition modeling (FDM), stereolithography, and bio-3D printing. While 3D printing offers precise structural control, it places high demands on equipment and raw materials. For example, only a few thermoplastic polymers are suitable for FDM, and these polymers often have low biocompatibility. Furthermore, current asymmetric collagen membranes often degrade too quickly or too slowly at the site of the lesion, often resulting in poor repair results.

[0005] Electrochemical methods use the input of electrical signals to induce changes in the solubility of charged molecules, thereby causing self-assembly. The application of electrical signals can be precisely controlled in time and space to guide assembly. On a macro scale, the thickness of the material assembly can be quantitatively controlled by the time of electrical signal input, or the 3D structure of the assembled material can be controlled by the area of ​​electrical signal input; on a micro scale, the mesoscopic structure inside the material can be adjusted by the input mode of electrical signals (such as pulse input). This makes it possible to use electrochemical technology to construct guided tissue regeneration membranes with asymmetric structures.

[0006] The electrochemical assembly of natural macromolecules is a relatively new type of research, and the research objects include polysaccharide and protein materials such as chitosan, sodium alginate, collagen and silk fibroin. Collagen has been widely used in the field of tissue repair materials due to its high biocompatibility, multiple cell adhesion sites and high biological activity. Collagen is an amphoteric polyelectrolyte, which is positively charged at low pH and negatively charged at high pH. It can undergo phase transition and precipitation at the isoelectric point. The main mechanism of electrochemical preparation of collagen is to drive the positively charged collagen to migrate to the cathode by applying an electric field, and then use the local alkaline environment near the cathode to neutralize the charge, complete the phase transition of the collagen molecules, and obtain a deposited material on the electrode surface. Patent CN114618017A uses electrochemical deposition technology to induce the directional migration of collagen molecules and form a certain orientation arrangement on the electrode surface, thereby preparing a collagen membrane with a highly oriented and crystalline collagen fiber structure. However, the overall structure of the resulting collagen membrane is uniform and an asymmetric structure cannot be obtained. Patent CN109395175A uses electrochemical technology to prepare a chitosan-based guided tissue regeneration membrane with an asymmetric structure. It uses two-step electrodeposition to first deposit a dense chitosan hydrogel near the cathode, and then deposit loose chitosan and calcium phosphate water gel thereon to obtain a double-layer hydrogel. The process and formula are relatively complex and not suitable for scale-up production. In addition, it degrades too slowly in the body, resulting in poor wound repair effects. Patent CN107050519A uses freeze-drying to prepare a collagen membrane. After pressing, a dense layer collagen membrane is obtained. Subsequently, the collagen solution is spread on the surface of the dense layer and then freeze-dried for a second time to obtain a composite collagen membrane with an asymmetric structure. Although this method is simple in process, the overall uniformity of the resulting collagen membrane is poor, especially when preparing a large area of ​​porous layer, which is prone to uneven pore distribution problems, and the problem of loose cross-linking is prone to occur at the double-layer composite, resulting in poor batch stability of the material.

[0007] Summary of the Invention

[0008] Based on the problems and deficiencies in the prior art, the present invention aims to provide a method for preparing a collagen membrane that guides tissue regeneration. The present invention utilizes electrochemical deposition technology, uses collagen as raw material, utilizes a direct current source to perform electrochemical deposition in a low-temperature environment, and in a constant voltage output mode, produces a collagen membrane with an asymmetric structure (one side is dense, the other side is loose and porous) in one step by high voltage. At the same time, through asymmetric cross-linking, the membrane can be asymmetrically degraded after entering the body. After the porous surface contacts the wound, it first provides a cell scaffold to induce cell growth, and at the same time, matching rapid degradation occurs, continuously providing space and nutrients for cell growth and proliferation, promoting rapid wound repair; at the same time, the dense surface remains stable, playing a long-term wound shielding effect. The present invention is simple to operate, fast to prepare, green in process, and easy to scale up. The obtained collagen membrane has excellent mechanical properties and uniform overall performance. The area of ​​the asymmetric structure collagen membrane prepared by the technical solution of the present invention is equal to the area of ​​the electrode sheet. By replacing the electrode sheet with a large area, scaled-up production can be achieved and production capacity can be increased.

[0009] The technical solutions of the present invention are as follows:

[0010] In one aspect, the present invention provides a method for preparing an asymmetric structure collagen membrane, the method comprising the following steps:

[0011] S1. Electrodeposition solution configuration: Dissolve collagen in acetic acid solution, add hydrogen peroxide solution, stir to obtain electrodeposition solution, set aside;

[0012] S2. Bubble removal: The electrodeposition solution prepared in step S1 is bubble-removed and then poured into the electrodeposition tank and pre-cooled to below 3-10°C;

[0013] S3. Electrochemical deposition: Electrochemical deposition is performed at low temperature to obtain collagen film material on the electrode sheet.

[0014] S4. Physical crosslinking and demolding: The collagen membrane prepared in step S3 was dried, allowed to stand at room temperature, and then immersed in PB at room temperature. After immersion in a 37°C water bath, the drying and immersion process was repeated 2-3 times, followed by physical crosslinking and careful peeling of the collagen membrane from the electrode sheet.

[0015] S5. Asymmetric cross-linking of the stripped collagen membrane material;

[0016] S6. washing the asymmetrically cross-linked collagen membrane;

[0017] S7. Freeze-drying to obtain a collagen membrane with an asymmetric structure.

[0018] Specifically, the collagen described in step S1 is natural type I collagen.

[0019] Preferably, the collagen in step S1 is extracted from pig skin, cow skin or bovine Achilles tendon.

[0020] Specifically, the concentration of collagen in step S1 is 0.1%-1.0% w / v.

[0021] Preferably, the concentration of collagen in step S1 is 0.6% w / v.

[0022] Specifically, the concentration of the acetic acid solution in step S1 is 0.1%-3% v / v.

[0023] Preferably, the concentration of the acetic acid solution in step S1 is 1.6 v / v.

[0024] Specifically, the concentration of the hydrogen peroxide solution in step S1 is 50-200 μL / mL.

[0025] Preferably, the concentration of the hydrogen peroxide solution in step S1 is 100 μL / mL.

[0026] Specifically, the electrodeposition cell described in step S2 adopts a dual-electrode system, wherein the anode and cathode electrode plates are placed in parallel on an electrode rack, and after adjusting the distance between the two electrode plates, they are placed in the electrodeposition cell.

[0027] More specifically, the anode is a platinum mesh.

[0028] Preferably, the platinum mesh is high-purity platinum with a purity of 99.9% or higher.

[0029] More specifically, the cathode is a titanium sheet.

[0030] Preferably, the titanium sheet is high-purity titanium with a purity of 99.9% or higher.

[0031] More specifically, the electrode frame is made of glass, acrylic or quartz.

[0032] Preferably, the electrode frame is made of acrylic.

[0033] The electrode rack preferably consists of three parallel layers of acrylic frames. The frames are rectangular. The bottom surface of the base frame is sealed, with a groove in the middle for placing the titanium sheet. Bolts are installed at the four corners to hold the upper frame. The upper layer consists of two identical frames with a hollow center, which can sandwich the platinum mesh between the two frames. The four corners have circular holes with a diameter that matches the bolts of the base. An indefinite number of washers can be placed on the bolts of the bottom frame to adjust the distance between the titanium sheet and the platinum mesh. Finally, the nuts are tightened to secure the upper frame. Two handles are placed perpendicular to the base to facilitate extraction of the electrode rack.

[0034] More specifically, the anode and cathode electrode plates have the same area.

[0035] Preferably, the area of ​​the electrode plate is 1-50 cm 2 .

[0036] More specifically, the area of ​​the electrode plate is 25 cm 2 .

[0037] Preferably, the distance between the two electrode plates is 1-4 cm.

[0038] Further preferably, the distance between the two electrode plates is 2 cm.

[0039] Specifically, the bubble removal in step S2 is performed by centrifuging the electrodeposition solution 2-4 times.

[0040] More specifically, the centrifugal speed is 7000-10000 rpm.

[0041] Preferably, the centrifugal speed is 8000 rpm.

[0042] More specifically, the centrifugation time is 5-20 min.

[0043] Preferably, the centrifugation time is 5 min

[0044] More specifically, the electrodeposition cell in step S2 is made of glass, acrylic or quartz.

[0045] Preferably, the electrodeposition cell described in step S2 is made of acrylic material.

[0046] Specifically, the pre-cooling temperature in step S2 is preferably 4°C.

[0047] Specifically, the electrochemical deposition in step S3 is to connect a DC source to an electrode plate, connect the positive electrode to a platinum mesh, and connect the negative electrode to a titanium sheet, and perform electrochemical deposition in a low-temperature environment.

[0048] Specifically, the low temperature environment described in step S3 is 2-10°C.

[0049] More specifically, the low temperature environment in step S3 is 4-8°C.

[0050] Specifically, the electrochemical deposition time in step S3 is 10-30 minutes.

[0051] Preferably, the electrochemical deposition time in step S3 is 25 minutes.

[0052] Specifically, the electrochemical deposition voltage in step S3 is 5-40V.

[0053] Preferably, the electrochemical sink in step S3 is 10-25V.

[0054] Specifically, the drying in step S4 is drying in an oven.

[0055] More specifically, the oven temperature is 30-45°C.

[0056] Preferably, the oven temperature is 37°C.

[0057] Specifically, the drying time in step S4 is 20-75 minutes.

[0058] Preferably, the drying time in step S4 is 60 minutes.

[0059] Specifically, the standing time in step S4 is 5-20 minutes.

[0060] Preferably, the standing time in step S4 is 15 minutes.

[0061] Specifically, the concentration of PB in step S4 is 0.01-0.20M.

[0062] More specifically, the concentration of PB in step S4 is 0.06-0.12M.

[0063] Preferably, the concentration of PB in step S4 is 0.1M.

[0064] Specifically, the soaking time in PB in step S4 is 15-40 minutes.

[0065] Preferably, the soaking time in PB in step S4 is 25 minutes.

[0066] Specifically, the water bath temperature in step S4 is 35-40°C.

[0067] Preferably, the water bath temperature in step S4 is 37°C.

[0068] Specifically, the soaking time in the water bath environment described in step S4 is 20-40 minutes.

[0069] Preferably, the soaking time in the water bath environment described in step S4 is 30 minutes.

[0070] Specifically, the physical crosslinking described in step S4 is collagen self-assembly crosslinking.

[0071] Specifically, the asymmetric cross-linking in step S5 is UV cross-linking.

[0072] More specifically, the UV crosslinking in step S5 is performed by irradiating the substrate under UV light for 10-300 min.

[0073] Preferably, the ultraviolet crosslinking in step S5 is irradiation at an ultraviolet wavelength for 20 minutes.

[0074] Preferably, the ultraviolet cross-linking in step S5 is to place the electrode-proximal surface of the peeled collagen membrane material under ultraviolet light wavelength for irradiation.

[0075] Specifically, the cleaning in step S6 is to first flush the collagen membrane with injection water.

[0076] More specifically, the cleaning in step S6 is to place the cross-linked membrane in a relatively flat tray, and wash the collagen membrane with injection water for 20-60 minutes, and repeat 5-10 times.

[0077] Preferably, the cleaning in step S6 is to clean the collagen membrane with injection water for 20 minutes, repeated 5-10 times.

[0078] Specifically, the freeze drying in step S7 includes a freezing stage, a sublimation stage, and an analysis stage;

[0079] The freezing stage is a freezing process performed under normal pressure and low temperature conditions;

[0080] The sublimation stage is a freezing process performed under vacuum and low temperature conditions;

[0081] The analysis stage is performed under vacuum conditions at a temperature of -20 to 30°C.

[0082] Preferably, the low temperature in the freezing stage is -60°C

[0083] More specifically, the freezing treatment time in the freezing stage is 30-360 minutes.

[0084] Preferably, the freezing treatment time in the freezing stage is 120 minutes.

[0085] More specifically, the low temperature of the sublimation stage is -60 to -5°C.

[0086] Preferably, the cryogenic temperature of the sublimation stage is -10°C.

[0087] More specifically, the vacuum degree in the sublimation stage is 10-50 Pa.

[0088] Preferably, the vacuum degree in the sublimation stage is 20 Pa.

[0089] More specifically, the freezing treatment time in the sublimation stage is 6-26 hours.

[0090] Preferably, the freezing treatment time in the sublimation stage is 12 hours.

[0091] Preferably, the temperature of the desorption stage is 5-20°C

[0092] More specifically, the vacuum degree in the analysis stage is 0-20Pa.

[0093] Preferably, the vacuum degree in the analysis stage is 1 Pa.

[0094] More specifically, the parsing time of the parsing stage is 30-720 minutes.

[0095] Preferably, the parsing time of the parsing stage is 180 minutes.

[0096] On the other hand, the present application also provides a collagen membrane for guiding tissue regeneration, which is prepared by the above-mentioned preparation method.

[0097] Specifically, the collagen membrane for guiding tissue regeneration has an asymmetric structure. The collagen membrane close to the electrode has a dense structure, and the collagen membrane away from the electrode has a transport structure. The thickness of the wet collagen membrane is 400-800 μm.

[0098] In another aspect, the present application also provides the use of the above-mentioned guided tissue regeneration collagen, which is used in the preparation of tissue repair products.

[0099] Specifically, the tissue repair products include periodontal tissue regeneration membranes, bone guided regeneration membranes, artificial skin, artificial blood vessels, artificial nerve conduits, and artificial ligaments.

[0100] The beneficial effects of the present invention are:

[0101] (1) The present invention utilizes electrochemical deposition technology to prepare a collagen membrane with an asymmetric structure (one side dense and the other side loose) in one step. This method is simple to operate, fast to prepare, and a green process. The resulting collagen membrane has excellent mechanical properties and uniform overall performance.

[0102] (2) Through asymmetric cross-linking, the membrane can undergo asymmetric degradation after entering the body, promoting rapid wound repair while maintaining a stable wound shielding effect.

[0103] (3) The area of ​​the asymmetric structure collagen membrane prepared by the technical solution of the present invention is equal to the area of ​​the electrode sheet. By replacing the electrode sheet with a larger area, it is possible to achieve scaled-up production and increase production capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 shows the collagen membrane prepared by centrifugal bubble removal process.

[0105] Figure 2 shows the collagen film peeled off through the demoulding process.

[0106] Figure 3 shows the collagen membrane after cross-linking and before freeze-drying.

[0107] Figure 4 shows the collagen membrane prepared without centrifugal bubble removal process.

[0108] Figure 5 shows the collagen film peeled off without demoulding process.

[0109] FIG6 is the collagen membrane prepared in Comparative Example 3.

[0110] FIG7 is a comparison diagram of the degradation of the collagen membranes prepared in Example 1 and Comparative Example 3.

[0111] FIG8 is the asymmetric structure collagen membrane prepared in Example 1.

[0112] FIG9 is a SEM image of the asymmetric structure collagen membrane prepared in Example 1. DETAILED DESCRIPTION

[0113] The following non-limiting examples are provided to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. The following are merely illustrative of the scope of the present invention, and those skilled in the art may make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0114] When numerical ranges are given in the examples, it should be understood that, unless otherwise specified herein, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs.

[0115] The present invention is further described below by way of specific examples. Unless otherwise specified, the various chemical reagents used in the examples of the present invention were obtained through conventional commercial channels.

[0116] Example 1 Preparation of asymmetric collagen membrane

[0117] (1) Preparation of electrodeposition solution: Dissolve collagen sponge at a concentration of 0.6% in a 1.6% acetic acid solution, then add 100 μL / mL of hydrogen peroxide solution in proportion, stir evenly, and obtain the electrodeposition solution for later use;

[0118] (2) Construction of the electrodeposition cell: A dual-electrode system was used, with a titanium sheet as the cathode and a platinum mesh as the anode. Two electrode plates of the same area were placed in parallel on a special electrode rack. After adjusting the distance between the two plates, they were placed in the electrodeposition cell. The area of ​​the electrode plate was 25 cm 2 ; The distance between the two electrode plates is 2 cm.

[0119] (3) Centrifugal bubble removal: The electrodeposition solution prepared in step (1) was centrifuged at 8000 rpm for 5 min to remove bubbles. The centrifugation operation was performed twice, and then the solution was poured into the electrolytic cell prepared in step S2 and pre-cooled to below 4°C.

[0120] (4) Electrochemical deposition: Connect a DC source to the electrode plate, connect the positive electrode to the platinum mesh, and the negative electrode to the titanium sheet. Set the DC source to a constant voltage output mode, and the electrodeposition voltage is between 10-25 V. After electrochemical deposition for 25 minutes in a low temperature environment (between 4-8°C), a collagen film can be obtained on the electrode sheet. After centrifugation to remove bubbles, the electrodeposited film is smooth and flat (see Figure 1).

[0121] (5) Physical cross-linking demolding: The collagen membrane prepared in step (4) was placed in an oven at 37°C for 60 min, allowed to stand at room temperature for 15 min, and then placed in 0.1 M PB and soaked at room temperature for 25 min. It was then transferred to a water bath at 37°C and soaked for 30 min. The above drying and soaking operations were repeated 2-3 times to perform collagen self-assembly cross-linking. Finally, the collagen membrane was carefully peeled off from the electrode sheet to obtain the peeled collagen membrane (Figure 2).

[0122] (6) UV cross-linking: The electrode-proximal surface of the peeled collagen membrane prepared in step (5) was irradiated under UV wavelength for 20 minutes.

[0123] (7) Cleaning: Place the collagen membrane after irradiation with ultraviolet light in a relatively flat tray, and clean the collagen membrane with flowing injection water for 20 minutes, repeating 5-10 times.

[0124] (8) Freeze drying:

[0125] After washing, the collagen membrane undergoes three stages: pre-freezing, low-temperature freezing, and vacuum drying to obtain a collagen membrane with an asymmetric structure (Figure 8). The specific steps are as follows:

[0126] S1. Freezing stage: The collagen membrane was frozen at -60°C under normal pressure for 2 h.

[0127] S2. Sublimation stage: Freeze at -10°C and 20 Pa vacuum for 12 hours;

[0128] S3. Analysis stage: Analysis for 3 minutes at a temperature of 5-20°C and a vacuum degree of 1Pa.

[0129] Comparative Example 1 Preparation of collagen film by vacuum bubble removal process

[0130] S1. Electrodeposition solution preparation: Dissolve the collagen sponge at a concentration of 0.6% in a 1.6% acetic acid solution, then add 100 μL / mL of hydrogen peroxide solution in proportion, stir evenly, and obtain the electrodeposition solution for later use;

[0131] S2. Construction of the electrolytic cell: A dual-electrode system was used, with a titanium sheet as the cathode and a platinum mesh as the anode. Two electrode plates of the same area were placed parallel to each other on a special electrode rack. After adjusting the distance between the two plates, they were placed in the electrolytic cell. The area of ​​the electrode plate was 25 cm. 2 ; The distance between the two electrode plates is 2 cm.

[0132] S3. Vacuum centrifugal bubble removal: The electrodeposition solution prepared in step S1 is brought into the electrolytic cell prepared in step S2 and placed in a vacuum environment to remove bubbles. A total of three vacuum pumpings were performed, and the vacuum degree must be -0.1 MPa or less. After each vacuum pumping, the solution was allowed to stand for 5 minutes and then pre-cooled to below 4°C.

[0133] S4. Electrochemical deposition: Connect a DC source to the electrode plate, connect the positive electrode to the platinum mesh, and the negative electrode to the titanium sheet. Set the DC source to a constant voltage output mode with an electrochemical deposition voltage between 10-25 V. After electrochemical deposition for 25 minutes at a low temperature (4-8°C), a collagen film can be obtained on the electrode sheet. The film after vacuum bubble removal is covered with bubbles (see Figure 4), so no subsequent preparation is performed.

[0134] Comparative Example 2: Stripping of collagen film without physical cross-linking demoulding process

[0135] S1. Prepare a collagen membrane according to the method described in steps (1) to (4) in the basic example.

[0136] S2. Carefully peel off the collagen membrane prepared in S1 to obtain a peeled collagen membrane. The collagen membrane directly peeled off after electrodeposition is similar to jelly and is not easy to peel off completely (see Figure 5), so no subsequent preparation is performed.

[0137] Comparative Example 3 Preparation of collagen membrane without UV cross-linking

[0138] The only difference between Comparative Example 3 and Example 1 is that "Step (6) UV cross-linking" is deleted, and the peeled collagen membrane is directly washed and freeze-dried to prepare a collagen membrane. The asymmetric structure of the collagen membrane is not obvious (see Figure 6).

[0139] Comparative Example 4 Preparation of collagen membrane by chemical cross-linking

[0140] The only difference between Comparative Example 4 and Example 1 is that: Step (5) is different, the peeled collagen membrane is immersed in a glutaraldehyde aqueous solution (0.5% w / v) for 60 minutes to chemically cross-link it and then washed and freeze-dried to obtain an asymmetric structure collagen membrane.

[0141] Experimental Example 1 Suture Tensile Force Measurement

[0142] The suture tension of Example 1 and Comparative Examples 2 and 4 was measured using the following experimental procedures: The sample was cut into a long strip (10 mm × 25 mm) (n = 5). A No. 4-0 suture was passed through the sample 3 mm from the short edge. The suture was folded in half and tied approximately 5 cm from the perforation to prevent it from falling off. After saturating the sample with ultrapure water (approximately 30 minutes), the unthreaded end of the sample and the suture end were fixed to a mechanical testing machine at a tension rate of 100 mm / min until the sample tore. The maximum tensile load was taken as the sample tearing force. The test results are shown in Table 1.

[0143] Table 1 Suture tension test results

[0144] The results showed that the suture tensions of Example 1, Comparative Example 2, and Comparative Example 4 were 1.057 N, 0.106 N, and 0.478 N, respectively, indicating that the collagen membrane prepared by the technical solution of the present invention has better mechanical properties and enhanced suture tension.

[0145] Experimental Example 2 Degradation Time

[0146] Determine the degradation time of the collagen membranes prepared in Example 1 and Comparative Example 3, respectively. The specific process is as follows: Prepare 400 mL of 0.01 M PBS solution with a pH of 7.3 and add 0.4 g of trypsin. Take 15 mg of collagen membrane, immerse it in the PBS solution until all air is squeezed out, remove it, and absorb the surface moisture with filter paper. Add PBS solution containing trypsin to an Erlenmeyer flask and preheat it at 37°C for 1 hour. Place the collagen membrane in the Erlenmeyer flask and completely soak it. Set the shaker at 50 rpm and 37°C to 15 mg of collagen membrane and 30 mL of PBS solution. Record the start time and observe and record the final complete degradation time of the membrane.

[0147] The results (Figure 7) show that the collagen membrane prepared in Comparative Example 3 degraded too quickly, being completely degraded after 45.5 hours, while the collagen membrane prepared in Example 1 remained intact after 45.5 hours. This suggests that asymmetric crosslinking can prolong the membrane's degradation time, providing a long-term wound shielding effect.

[0148] Experimental Example 3 Appearance and Micromorphology Measurement

[0149] The prepared collagen membrane with an asymmetric structure prepared in Example 1 was observed and photographed (see FIG8 ). The microscopic morphology was further observed using a scanning electron microscope (FlexSEM 1000) and photographed (see FIG9 ).

[0150] The results show that the technical solution of the present invention can produce an asymmetric collagen membrane with a dense surface on one side and a loose surface on the other. The resulting collagen membrane has excellent mechanical properties and uniform overall performance. Furthermore, the area of ​​the collagen membrane is equal to that of the electrode sheet. By replacing the electrode sheet with a larger area, production can be scaled up and increased production capacity can be achieved.

[0151] The above detailed description is a specific description of one feasible embodiment of the present invention and is not intended to limit the scope of the present invention. It should be noted that any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the scope of protection of the patent of this invention should be based on the attached requirements.

Claims

1. A preparation method of an asymmetric structure collagen membrane, characterized in that, the preparation method comprises the following steps: S1. Preparation of the electrodeposition solution: Dissolve collagen in an acetic acid solution, add a hydrogen peroxide solution, and stir evenly to obtain the electrodeposition solution for standby; S2. Bubble removal: Remove the bubbles from the electrodeposition solution prepared in step S1, then pour it into the electrodeposition cell and pre-cool it to below 3 - 10 °C; S3. Electrochemical deposition: Conduct electrochemical deposition in an environment of 2 - 10 °C, and a collagen membrane material can be obtained on the electrode plate; S4. Physical cross-linking and demoulding: Dry the collagen membrane material prepared in step S3, let it stand at room temperature, then soak it in PB at room temperature, and then transfer it to a water bath environment at 37 °C for soaking. After repeating the drying and soaking operations 2 - 3 times, perform collagen self-assembly cross-linking, and carefully peel the collagen membrane from the electrode plate; S5. Asymmetric cross-linking of the peeled collagen membrane material; S6. Wash the asymmetric cross-linked collagen membrane; S7. Freeze-dry to obtain a collagen membrane with an asymmetric structure.

2. The preparation method according to claim 1, characterized in that, in step S1: the concentration of the collagen is 0.1% - 1.0% w / v; the concentration of the acetic acid solution is 0.1% - 3% v / v; the concentration of the hydrogen peroxide is 50 - 200 μL / mL.

3. The preparation method according to claim 1, characterized in that, the bubble removal in step S2 is to perform 2 - 4 centrifugation operations on the electrodeposition solution; the centrifugation speed is 7000 - 10000 rpm; the centrifugation time is 5 - 20 min.

4. The preparation method according to claim 1, characterized in that, the electrochemical deposition time in step S3 is 10 - 30 min; the electrochemical deposition voltage is 5 - 40 V.

5. The preparation method according to claim 1, characterized in that, the oven temperature in step S4 is 30 - 45 °C; the drying time is 20 - 75 min; the standing time is 5 - 20 min.

6. The preparation method according to claim 1, characterized in that, the PB concentration in step S4 is 0.01 - 0.2 M, and the soaking time in PB is 15 - 40 min.

7. The preparation method according to claim 1, characterized in that, the asymmetric cross-linking in step S5 is ultraviolet light cross-linking, and the ultraviolet light cross-linking is to irradiate for 10 - 300 min at the ultraviolet light wavelength.

8. The preparation method according to claim 1, characterized in that, the washing in step S6 is to wash the collagen membrane with injection water, 20 - 60 min each time, and this process is repeated 5 - 10 times.

9. A guided tissue regeneration collagen membrane, characterized in that, the guided tissue regeneration collagen membrane is prepared by the preparation method according to any one of claims 1 - 8.

10. The application of the guided tissue regeneration collagen membrane according to claim 9, characterized in that, the application is in tissue repair products.

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