Oral repair membrane and method of making same

Abstract

This invention discloses an oral repair membrane and its preparation method, belonging to the field of biomedical materials technology. The invention employs a freeze-thaw process to break down the cell membranes and nuclei within porcine peritoneum tissue, freeing the cell contents for subsequent chemical decellularization. Then, a composite stepwise decellularization system consisting of a first strong alkali wash, a first weak alkali neutralization wash, a second strong alkali wash, and a second weak alkali neutralization wash precisely removes cellular residues, nucleic acids, lipids, and antigenic proteins while maximally preserving the integrity of the collagen structure. Finally, a cross-linking treatment promotes the cross-linking of collagen molecules, significantly improving the mechanical toughness, tensile strength, and in vivo anti-degradation ability of the repair membrane. Combined with a sterilization process, the biosafety of the oral repair membrane is enhanced. The synergistic effect of these steps significantly improves the performance of the prepared oral repair membrane; therefore, this oral repair membrane has promising applications in oral restoration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an oral repair membrane and its preparation method. Background Technology

[0002] Oral soft tissue defects, gingival recession, poor healing of extraction sockets, and peri-implant soft tissue defects are common clinical problems in dentistry. Currently, oral restorative membrane products used clinically are mainly divided into two categories: synthetic restorative membranes and animal-derived restorative membranes; both types of restorative membranes have good application prospects in oral restoration.

[0003] However, artificially synthesized repair membranes have problems such as poor biocompatibility, poor adhesion, mismatch between degradation rate and tissue regeneration, and easy to induce inflammatory reactions; while traditional animal-derived repair membranes have problems such as incomplete decellularization, high antigen residue, insufficient mechanical strength, easy rapid degradation, rough process and poor stability.

[0004] In view of this, it is necessary to develop a new oral repair membrane and its preparation method to address the shortcomings of existing technologies. Summary of the Invention

[0005] The purpose of this invention is to provide an oral repair membrane and its preparation method. This addresses the problems of existing repair membranes, such as low mechanical strength, poor degradation resistance, and high cellular residue.

[0006] In a first aspect, the present invention provides a method for preparing an oral repair membrane, comprising the following steps: providing pretreated porcine peritoneum; subjecting the pretreated porcine peritoneum to freeze-thaw treatment to obtain freeze-thawed porcine peritoneum; subjecting the freeze-thawed porcine peritoneum to sequential washing with a first strong alkali, neutralizing with a first weak alkali, washing with a second strong alkali, and neutralizing with a second weak alkali to obtain decellularized porcine peritoneum; adding the decellularized porcine peritoneum to a crosslinking agent solution for crosslinking treatment, and after sterilization and freeze-drying, obtaining an oral repair membrane; wherein the mass-volume concentration of the first strong alkali solution used in the first strong alkali washing is higher than the mass-volume concentration of the second strong alkali solution used in the second strong alkali washing.

[0007] In this invention, the inventors discovered that by employing freeze-thaw treatment, the cell membrane and nuclear structure within porcine peritoneum tissue can be broken, freeing the cell contents and facilitating subsequent chemical decellularization. Then, a composite stepwise decellularization system consisting of a first strong alkali wash, a first weak alkali neutralization wash, a second strong alkali wash, and a second weak alkali neutralization wash precisely removes cellular residues, nucleic acids, lipids, and antigenic proteins while maximally preserving the integrity of the collagen structure. Finally, a cross-linking treatment promotes the cross-linking of collagen molecules, significantly enhancing the mechanical toughness, tensile strength, and in vivo anti-degradation ability of the repair membrane. Combined with a sterilization process, this improves the biosafety of the oral repair membrane. The synergistic effect of these steps significantly enhances the performance of the prepared oral repair membrane.

[0008] In some implementations, the preparation of the pretreated porcine peritoneum in the step of providing the pretreated porcine peritoneum includes: taking healthy fresh porcine peritoneum, trimming and removing fat, fascia, and damaged tissue, thawing at room temperature, and then soaking and washing it in NaCl solution to obtain the pretreated porcine peritoneum; wherein the mass-volume concentration of the NaCl solution is 0.5~1.5%, and the number of soaking and washing cycles includes 3~5 times, each time for 20~40 minutes.

[0009] In some implementations, the freeze-thaw process of pretreated porcine peritoneum includes: laying the pretreated porcine peritoneum flat and then freezing-thawing it; wherein the freeze-thaw process includes: freezing at a temperature of -90℃ to -70℃ for 1 to 3 hours and then thawing it with purified water; and after the freeze-thaw process is completed, the process is repeated 3 to 5 times.

[0010] In some implementation schemes, the steps of sequentially performing a first strong alkali wash, a first weak alkali neutralization wash, a second strong alkali wash, and a second weak alkali neutralization wash on the frozen-thawed pig peritoneum include: immersing the frozen-thawed pig peritoneum in a first strong alkali solution for washing; wherein the mass-volume concentration of the first strong alkali solution is 8-12%, and the first strong alkali solution includes a sodium carbonate solution; the number of washes includes 1-3 times, each time for 2-4 hours.

[0011] In some implementation schemes, the steps of sequentially performing a first strong alkali wash, a first weak alkali neutralization wash, a second strong alkali wash, and a second weak alkali neutralization wash on the frozen and thawed pig peritoneum include: immersing the pig peritoneum, which has been washed with the first strong alkali, in a first weak alkali solution for neutralization wash; wherein the mass-volume concentration of the first weak alkali solution is 2-6%, and the first weak alkali solution includes sodium bicarbonate solution; the number of washes includes 1-3 times, each time for 20-40 minutes.

[0012] In some implementation schemes, the steps of sequentially performing a first strong alkali wash, a first weak alkali neutralization wash, a second strong alkali wash, and a second weak alkali neutralization wash on the frozen and thawed porcine peritoneum include: immersing the porcine peritoneum, which has been neutralized by the first weak alkali wash, in a second strong alkali solution for further washing; wherein the mass-volume concentration of the second strong alkali solution is 0.5-1.5%, and the second strong alkali solution includes sodium hydroxide solution; the washing is performed 1-3 times, each time for 1-3 hours; after the second strong alkali wash, the process further includes washing with purified water 1-3 times, each time for 20-40 minutes.

[0013] In some implementation schemes, the steps of sequentially performing a first strong alkali wash, a first weak alkali neutralization wash, a second strong alkali wash, and a second weak alkali neutralization wash on the frozen and thawed porcine peritoneum include: immersing the porcine peritoneum washed with the second strong alkali in a second weak alkali solution for neutralization wash; wherein the mass-volume concentration of the second weak alkali solution is 2-6%, and the second weak alkali solution includes sodium bicarbonate solution; the wash is performed 3-5 times, each time for 20-40 minutes; after the second weak alkali neutralization wash, the process further includes washing with purified water 2-4 times, each time for 0.5-1.5 hours.

[0014] In some implementations, in the step of adding decellularized porcine peritoneum to a cross-linking agent solution for cross-linking treatment, followed by sterilization and freeze-drying, the cross-linking agent solution includes an EDC solution with a mass-volume concentration of 2-4%, and the cross-linking treatment time is 20-30 hours.

[0015] In some implementation schemes, the steps of adding decellularized porcine peritoneum to a cross-linking agent solution for cross-linking treatment, followed by sterilization and freeze-drying, specifically include: washing with 70-80% ethanol 1-3 times, 1-3 hours each time; and freeze-drying specifically includes: smoothing and shaping the sterilized porcine peritoneum and then freeze-drying it for 20-30 hours.

[0016] In a second aspect, the present invention provides an oral repair membrane prepared by any of the above-described preparation methods.

[0017] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention employs freeze-thaw treatment to break down the cell membranes and nuclei within porcine peritoneum tissue, freeing the cell contents and facilitating subsequent chemical decellularization. Then, a composite stepwise decellularization system consisting of a first strong alkali wash, a first weak alkali neutralization wash, a second strong alkali wash, and a second weak alkali neutralization wash precisely removes cell residues, nucleic acids, lipids, and antigenic proteins while maximally preserving the integrity of the collagen structure. Finally, a cross-linking treatment promotes the cross-linking of collagen molecules, significantly enhancing the mechanical toughness, tensile strength, and in vivo anti-degradation ability of the repair membrane. Combined with a sterilization process, this improves the biosafety of the oral repair membrane. The synergistic effect of these steps significantly enhances the performance of the prepared oral repair membrane. Therefore, this oral repair membrane has promising applications in oral restoration. Attached Figure Description

[0018] Figure 1 This is a flowchart of the preparation method of the oral repair membrane in this invention. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0021] Currently, existing repair membranes suffer from problems such as low mechanical strength, poor resistance to degradation, and high levels of cell residue.

[0022] To address the problems of low mechanical strength, poor anti-degradation performance, and high cellular residue in existing repair membranes, this invention provides an oral repair membrane and its preparation method.

[0023] In a first aspect, the present invention provides a method for preparing an oral repair membrane, comprising the following steps: providing pretreated porcine peritoneum; subjecting the pretreated porcine peritoneum to freeze-thaw treatment to obtain freeze-thawed porcine peritoneum; subjecting the freeze-thawed porcine peritoneum to sequential washing with a first strong alkali, neutralizing with a first weak alkali, washing with a second strong alkali, and neutralizing with a second weak alkali to obtain decellularized porcine peritoneum; adding the decellularized porcine peritoneum to a crosslinking agent solution for crosslinking treatment, and after sterilization and freeze-drying, obtaining an oral repair membrane; wherein the mass-volume concentration of the first strong alkali solution used in the first strong alkali washing is higher than the mass-volume concentration of the second strong alkali solution used in the second strong alkali washing.

[0024] The preparation method provided by this invention utilizes freeze-thaw treatment to break down the cell membranes and nuclei within porcine peritoneum tissue, freeing the cell contents for subsequent chemical decellularization. A multi-step decellularization system consisting of a first strong alkali wash, a first weak alkali neutralization wash, a second strong alkali wash, and a second weak alkali neutralization wash precisely removes cellular residues, nucleic acids, lipids, and antigenic proteins while maximally preserving the integrity of the collagen structure. Finally, a cross-linking treatment promotes the cross-linking of collagen molecules, significantly enhancing the mechanical toughness, tensile strength, and in vivo anti-degradation ability of the repair membrane. Combined with a sterilization process, this improves the biosafety of the oral repair membrane. The synergistic effect of these steps significantly enhances the performance of the prepared oral repair membrane. Furthermore, the preparation method provided by this invention is simple, uses inexpensive and readily available raw materials, and is suitable for large-scale industrial production.

[0025] In some implementations, the preparation of the pretreated porcine peritoneum in the step of providing the pretreated porcine peritoneum includes: taking healthy fresh porcine peritoneum, trimming and removing fat, fascia, and damaged tissue, thawing at room temperature, and then soaking and washing it in NaCl solution to obtain the pretreated porcine peritoneum; wherein the mass-volume concentration of the NaCl solution is 0.5~1.5%, and the number of soaking and washing cycles includes 3~5 times, each time for 20~40 minutes.

[0026] In this invention, by pretreating the porcine peritoneum, blood, free impurities, and surface dirt can be removed. Further gentle washing with NaCl solution can avoid damaging the surface collagen fibers and remove most of the soluble impurities, facilitating subsequent decellularization.

[0027] In some implementations, the freeze-thaw process of pretreated porcine peritoneum includes: laying the pretreated porcine peritoneum flat and then freezing-thawing it; wherein the freeze-thaw process includes: freezing at a temperature of -90℃ to -70℃ for 1 to 3 hours and then thawing it with purified water; and after the freeze-thaw process is completed, the process is repeated 3 to 5 times.

[0028] In this invention, the cell membrane and nucleus structures within the peritoneal tissue can be completely broken through multiple freeze-thaw cycles, allowing the cell contents to become free and significantly reducing the difficulty of subsequent chemical decellularization, while not damaging the three-dimensional skeleton structure of natural collagen fibers.

[0029] In some implementation schemes, the steps of sequentially performing a first strong alkali wash, a first weak alkali neutralization wash, a second strong alkali wash, and a second weak alkali neutralization wash on the frozen-thawed pig peritoneum include: immersing the frozen-thawed pig peritoneum in a first strong alkali solution for washing; wherein the mass-volume concentration of the first strong alkali solution is 8-12%, and the first strong alkali solution includes a sodium carbonate solution; the number of washes includes 1-3 times, each time for 2-4 hours.

[0030] In this invention, a high concentration of strong alkali is used to wash the porcine peritoneum to perform preliminary degreasing, lyse residual cell tissue, and degrade foreign antigen proteins.

[0031] In some implementation schemes, the steps of sequentially performing a first strong alkali wash, a first weak alkali neutralization wash, a second strong alkali wash, and a second weak alkali neutralization wash on the frozen and thawed pig peritoneum include: immersing the pig peritoneum, which has been washed with the first strong alkali, in a first weak alkali solution for neutralization wash; wherein the mass-volume concentration of the first weak alkali solution is 2-6%, and the first weak alkali solution includes sodium bicarbonate solution; the number of washes includes 1-3 times, each time for 20-40 minutes.

[0032] In this invention, after the first strong alkali cleaning, the first weak alkali neutralization cleaning can neutralize the residual strong alkali, buffer the pH value of the tissue, and prevent the strong alkali from continuously corroding the collagen fibers and reducing the performance of the membrane.

[0033] In some implementation schemes, the steps of sequentially performing a first strong alkali wash, a first weak alkali neutralization wash, a second strong alkali wash, and a second weak alkali neutralization wash on the frozen and thawed porcine peritoneum include: immersing the porcine peritoneum, which has been neutralized by the first weak alkali wash, in a second strong alkali solution for further washing; wherein the mass-volume concentration of the second strong alkali solution is 0.5-1.5%, and the second strong alkali solution includes sodium hydroxide solution; the washing is performed 1-3 times, each time for 1-3 hours; after the second strong alkali wash, the process further includes washing with purified water 1-3 times, each time for 20-40 minutes.

[0034] In this invention, by using a low-concentration second strong alkali for washing, residual nucleic acids and cell debris can be deeply removed, and foreign components that cause immune rejection can be completely eliminated.

[0035] In some implementation schemes, the steps of sequentially performing a first strong alkali wash, a first weak alkali neutralization wash, a second strong alkali wash, and a second weak alkali neutralization wash on the frozen and thawed porcine peritoneum include: immersing the porcine peritoneum washed with the second strong alkali in a second weak alkali solution for neutralization wash; wherein the mass-volume concentration of the second weak alkali solution is 2-6%, and the second weak alkali solution includes sodium bicarbonate solution; the wash is performed 3-5 times, each time for 20-40 minutes; after the second weak alkali neutralization wash, the process further includes washing with purified water 2-4 times, each time for 0.5-1.5 hours.

[0036] In this invention, by using a second weak alkali cleaning method combined with water washing, the residual decellularization reagent can be thoroughly washed away, the pH of the matrix can be adjusted to neutral, the collagen fiber structure can be stabilized, and the material can be guaranteed to be free of chemical residues and have low immunogenicity.

[0037] In some implementations, in the step of adding decellularized porcine peritoneum to a crosslinking agent solution for crosslinking treatment, followed by sterilization and freeze-drying, the crosslinking agent solution includes an EDC solution with a mass-volume concentration of 2-4% (solvent being 95% ethanol solution), and the crosslinking treatment time is 20-30 hours.

[0038] In this invention, by using EDC solution for gentle cross-linking, collagen molecules can be promoted to cross-link without introducing toxic impurities, significantly improving the mechanical toughness, tensile strength and in vivo anti-degradation ability of the repair membrane, and precisely adapting to the regeneration cycle of oral tissues.

[0039] In some implementation schemes, the steps of adding decellularized porcine peritoneum to a cross-linking agent solution for cross-linking treatment, followed by sterilization and freeze-drying, specifically include: washing with 70-80% ethanol 1-3 times, 1-3 hours each time; and freeze-drying specifically includes: smoothing and shaping the sterilized porcine peritoneum and then freeze-drying it for 20-30 hours.

[0040] In this invention, the use of ethanol for sterilization can improve the biocompatibility of the repair membrane; at the same time, combined with the freeze-drying process, the three-dimensional porous network structure of the material can be completely preserved, maintaining the flexibility and porosity of the membrane material, and the oral repair membrane prepared is soft in texture, has strong adhesion, and is suitable for various irregular wounds in the oral cavity.

[0041] In a second aspect, the present invention provides an oral repair membrane prepared by any of the above-described preparation methods.

[0042] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0043] Please see Figure 1The flowchart illustrates the preparation method of the oral repair membrane in this invention. Specifically, the preparation method of the oral repair membrane includes the following steps: providing pretreated porcine peritoneum; subjecting the pretreated porcine peritoneum to freeze-thaw treatment to obtain freeze-thawed porcine peritoneum; sequentially subjecting the freeze-thawed porcine peritoneum to a first strong alkali wash, a first weak alkali neutralization wash, a second strong alkali wash, and a second weak alkali neutralization wash to obtain decellularized porcine peritoneum; adding the decellularized porcine peritoneum to a crosslinking agent solution for crosslinking treatment, followed by sterilization and freeze-drying to obtain the oral repair membrane.

[0044] In the following embodiments and comparative examples of the present invention, the mass ratio of porcine peritoneum to the NaCl solution, sodium carbonate solution, sodium bicarbonate solution, sodium hydroxide solution, purified water, and ethanol used in the cleaning process, and the EDC solution used in the crosslinking process, is 1:15.

[0045] Example 1 A method for preparing an oral repair membrane includes the following steps: S1. Pretreatment: Take healthy fresh pig peritoneum, trim and remove fat, fascia and damaged tissue, thaw at room temperature, and soak and wash it 4 times in a 0.9% (w / v) NaCl solution for 30 minutes each time to remove blood and free impurities, and obtain pretreated pig peritoneum. S2. Freeze-thaw pretreatment: The pretreated porcine peritoneum obtained in step S1 is laid flat on a sterile tray. After it is flat and wrinkle-free, it is placed in an ultra-low temperature freezer at -80℃ for 2 hours. It is then taken out and the ice crystals are melted with purified water. The freeze-thaw process is repeated 4 times to obtain the frozen-thawed porcine peritoneum. S3. Chemical Decellularization: The frozen-thawed porcine peritoneum obtained in step S2 was washed twice in a 10% (w / v) sodium carbonate solution for 3 hours each time; then washed twice in a 4% (w / v) sodium bicarbonate solution for 30 minutes each time; then washed twice in a 1% (w / v) sodium hydroxide solution for 2 hours each time; then washed twice in purified water for 30 minutes each time; then washed four times in a 4% (w / v) sodium bicarbonate solution for 30 minutes each time; finally, washed three times in purified water for 1 hour each time; thus obtaining decellularized porcine peritoneum. S4. Chemical cross-linking, sterilization and freeze-drying: The decellularized porcine peritoneum obtained in step S3 was added to an EDC solution with a mass-volume concentration of 3% (w / v) (solvent is 95% ethanol solution) for cross-linking for 24 hours. Then it was washed with purified water 4 times, 1.5 hours each time. After that, it was taken out and washed with 75% ethanol 2 times, 2 hours each time. After that, it was taken out and washed with purified water 3 times, 1 hour each time. Finally, after flattening and shaping, it was freeze-dried for 24 hours to obtain the oral repair membrane.

[0046] Example 2 A method for preparing an oral repair membrane includes the following steps: S1. Pretreatment: Take healthy fresh pig peritoneum, trim and remove fat, fascia and damaged tissue, thaw at room temperature, and soak and wash it 5 times in a 0.9% (w / v) NaCl solution for 25 minutes each time to remove blood and free impurities, and obtain pretreated pig peritoneum. S2. Pre-freeze-thaw treatment: The pre-treated porcine peritoneum obtained in step S1 is laid flat on a sterile tray. After it is flat and wrinkle-free, it is placed in an ultra-low temperature freezer at -80℃ for 2.5 hours. After that, it is taken out and purified water is used to melt the ice crystals. The freeze-thaw process is repeated 5 times to obtain the frozen and thawed porcine peritoneum. S3. Chemical Decellularization: The frozen-thawed porcine peritoneum obtained in step S2 was washed twice in a 10% (w / v) sodium carbonate solution for 3.5 h each time; then washed three times in a 4% (w / v) sodium bicarbonate solution for 25 min each time; then washed twice in a 1% (w / v) sodium hydroxide solution for 2.5 h each time; then washed twice in purified water for 40 min each time; then washed four times in a 4% (w / v) sodium bicarbonate solution for 40 min each time; finally, washed three times in purified water for 1.5 h each time; thus obtaining decellularized porcine peritoneum. S4. Chemical cross-linking, sterilization and freeze-drying: The decellularized porcine peritoneum obtained in step S3 was added to an EDC solution with a mass-volume concentration of 3% (w / v) (solvent is 95% ethanol solution) for cross-linking for 22 hours. Then it was washed with purified water 4 times, 1.5 hours each time. After that, it was taken out and washed with 75% ethanol 2 times, 2 hours each time. After that, it was taken out and washed with purified water 3 times, 1 hour each time. Finally, after flattening and shaping, it was freeze-dried for 22 hours to obtain the oral repair membrane.

[0047] Example 3 A method for preparing an oral repair membrane includes the following steps: S1. Pretreatment: Take healthy fresh pig peritoneum, trim and remove fat, fascia and damaged tissue, thaw at room temperature, and soak and wash it 4 times in a 0.9% (w / v) NaCl solution for 35 minutes each time to remove blood and free impurities, and obtain pretreated pig peritoneum. S2. Pre-freeze-thaw treatment: The pre-treated porcine peritoneum obtained in step S1 is laid flat on a sterile tray. After it is flat and wrinkle-free, it is placed in an ultra-low temperature freezer at -80℃ for 1.5 hours. After that, it is taken out and the ice crystals are melted with purified water. The freeze-thaw process is repeated 3 times to obtain the frozen and thawed porcine peritoneum. S3. Chemical Decellularization: The frozen-thawed porcine peritoneum obtained in step S2 was washed twice in a 10% (w / v) sodium carbonate solution for 2 hours each time; then washed twice in a 4% (w / v) sodium bicarbonate solution for 30 minutes each time; then washed twice in a 1% (w / v) sodium hydroxide solution for 1 hour each time; then washed twice in purified water for 30 minutes each time; then washed four times in a 4% (w / v) sodium bicarbonate solution for 1.5 hours each time; finally, washed three times in purified water for 1 hour each time; thus obtaining decellularized porcine peritoneum. S4. Chemical cross-linking, sterilization and freeze-drying: The decellularized porcine peritoneum obtained in step S3 was added to an EDC solution with a mass-volume concentration of 3% (w / v) (solvent is 95% ethanol solution) for cross-linking for 26 hours. Then it was washed with purified water 4 times, 1.5 hours each time. After that, it was taken out and washed with 75% ethanol 2 times, 2 hours each time. After that, it was taken out and washed with purified water 3 times, 1 hour each time. Finally, after flattening and shaping, it was freeze-dried for 26 hours to obtain the oral repair membrane.

[0048] Example 4 A method for preparing an oral repair membrane includes the following steps: S1. Pretreatment: Take healthy fresh pig peritoneum, trim and remove fat, fascia and damaged tissue, thaw at room temperature, and soak and wash it three times in a 0.9% (w / v) NaCl solution for 40 minutes each time to remove blood and free impurities, and obtain pretreated pig peritoneum. S2. Freeze-thaw pretreatment: The pretreated porcine peritoneum obtained in step S1 is laid flat on a sterile tray. After it is flat and wrinkle-free, it is placed in an ultra-low temperature freezer at -80℃ for 3 hours. It is then taken out and purified water is used to melt the ice crystals. The freeze-thaw process is repeated 5 times to obtain the frozen-thawed porcine peritoneum. S3. Chemical decellularization: The frozen-thawed porcine peritoneum obtained in step S2 was washed twice in a 10% (w / v) sodium carbonate solution for 3 hours each time; then washed twice in a 4% (w / v) sodium bicarbonate solution for 30 minutes each time; then washed three times in a 1% (w / v) sodium hydroxide solution for 2 hours each time; then washed twice in purified water for 30 minutes each time; then washed four times in a 4% (w / v) sodium bicarbonate solution for 30 minutes each time; finally, washed three times in purified water for 1 hour each time; thus obtaining decellularized porcine peritoneum. S4. Chemical cross-linking, sterilization and freeze-drying: The decellularized porcine peritoneum obtained in step S3 was added to an EDC solution with a mass-volume concentration of 3% (w / v) (solvent is 95% ethanol solution) for cross-linking for 20 hours. Then it was washed with purified water 4 times, 1.5 hours each time. After that, it was taken out and washed with 75% ethanol 2 times, 2 hours each time. After that, it was taken out and washed with purified water 3 times, 1 hour each time. Finally, after flattening and shaping, it was freeze-dried for 20 hours to obtain the oral repair membrane.

[0049] Comparative Example 1 In this comparative example, the preparation method of the oral repair membrane is basically the same as that in Example 1, except that step S2 is not performed.

[0050] Comparative Example 2 In this comparative example, the preparation method of the oral repair membrane is basically the same as that in Example 1. The difference is that in step S3, the frozen and thawed porcine peritoneum is directly placed in a sodium carbonate solution with a mass-volume concentration of 10% (W / V) and washed twice, each time for 6 hours.

[0051] Comparative Example 3 In this comparative example, the preparation method of the oral repair membrane is basically the same as that in Example 1. The difference is that in step S3, the frozen and thawed porcine peritoneum is directly placed in a sodium hydroxide solution with a mass-volume concentration of 1% (W / V) and washed twice, each time for 6 hours.

[0052] Comparative Example 4 In this comparative example, the preparation method of the oral repair membrane is basically the same as that in Example 1, except that chemical cross-linking is not performed in step S4.

[0053] Performance testing The oral repair membranes prepared in Examples 1-4 and Comparative Examples 1-4 were subjected to performance tests, and the results are shown in Table 1 below.

[0054] Table 1 Performance Test Results

[0055] As shown in Table 1, the oral repair membranes prepared in Examples 1-4 exhibit good performance. In Comparative Example 1, without freeze-thaw pretreatment, incomplete cell removal resulted in a large number of residual cell nuclei and DNA residue reaching 210 ng / mg, far exceeding medical standards. This indicated significant immunogenicity, poor biocompatibility, and pore blockage, rendering the membrane unsuitable for clinical use. The results demonstrate that freeze-thaw pretreatment can improve the performance of the oral repair membrane. In Comparative Example 2, direct immersion in a high-concentration strong alkali for an extended period resulted in excessive decellularization, large-area breakage of the collagen fiber skeleton, and pore collapse. The membrane material became brittle and lacked flexibility, failing to adhere to the oral wound. Degradation was too rapid, essentially failing within one week, completely losing its oral repair function. In Comparative Example 3, direct immersion in a low-concentration strong alkali for an extended period resulted in inadequate virus inactivation, excessive DNA residue, low immunogenicity, and significantly reduced performance, rendering the membrane unsuitable for clinical use. These results indicate that the combined treatment of first cleaning with a strong alkali and then neutralizing with a weak alkali in this invention significantly improves the performance of the oral repair membrane. In Comparative Example 4, without cross-linking, the collagen structure was found to be loose and with extremely poor mechanical properties. It swelled and disintegrated rapidly upon contact with body fluids, with a degradation cycle of only 1-2 weeks, and could not support tissue regeneration. The results indicate that cross-linking treatment can significantly improve the performance of oral repair membranes.

[0056] In summary, this invention utilizes freeze-thaw treatment to break down the cell membranes and nuclei within porcine peritoneum tissue, freeing the cell contents for subsequent chemical decellularization. Then, a multi-step decellularization system employing a first strong alkali wash, a first weak alkali neutralization wash, a second strong alkali wash, and a second weak alkali neutralization wash precisely removes cellular residues, nucleic acids, lipids, and antigenic proteins while maximally preserving the integrity of the collagen structure. Finally, cross-linking treatment promotes the cross-linking of collagen molecules, significantly enhancing the mechanical toughness, tensile strength, and in vivo anti-degradation ability of the repair membrane. Combined with a sterilization process, this improves the biosafety of the oral repair membrane.

[0057] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0058] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing an oral repair membrane, characterized in that, Includes the following steps: Provide pretreated porcine peritoneum; The pretreated porcine peritoneum was subjected to freeze-thaw treatment to obtain the freeze-thawed porcine peritoneum. After sequentially performing a first strong alkali wash, a first weak alkali neutralization wash, a second strong alkali wash, and a second weak alkali neutralization wash on the frozen and thawed porcine peritoneum, decellularized porcine peritoneum was obtained. The decellularized porcine peritoneum was added to a cross-linking agent solution for cross-linking treatment, and after sterilization and freeze-drying, the oral repair membrane was obtained. Wherein, the mass-volume concentration of the first strong alkali solution used in the first strong alkali cleaning is higher than the mass-volume concentration of the second strong alkali solution used in the second strong alkali cleaning.

2. The preparation method according to claim 1, characterized in that, In the step of providing pretreated porcine peritoneum, the preparation of the pretreated porcine peritoneum includes: taking healthy fresh porcine peritoneum, trimming and removing fat, fascia and damaged tissue, thawing at room temperature, and soaking and washing it in NaCl solution to obtain the pretreated porcine peritoneum; The NaCl solution has a mass-volume concentration of 0.5-1.5%, and the soaking and rinsing are performed 3-5 times, each time for 20-40 minutes.

3. The preparation method according to claim 1, characterized in that, In the step of freezing and thawing the pretreated porcine peritoneum, the freezing and thawing process specifically includes: laying the pretreated porcine peritoneum flat and then freezing and thawing it. The freeze-thaw process specifically includes freezing for 1 to 3 hours at a temperature of -90℃ to -70℃, followed by thawing with purified water; and after the freeze-thaw process is completed, the process includes repeating the freeze-thaw process 3 to 5 times.

4. The preparation method according to claim 1, characterized in that, In the steps of sequentially performing a first strong alkali cleaning, a first weak alkali neutralization cleaning, a second strong alkali cleaning, and a second weak alkali neutralization cleaning on the frozen and thawed pig peritoneum, the first strong alkali cleaning specifically includes: immersing the frozen and thawed pig peritoneum in a first strong alkali solution for cleaning. The first strong alkaline solution has a mass-volume concentration of 8-12%, and the first strong alkaline solution includes a sodium carbonate solution; the washing is performed 1-3 times, each time for 2-4 hours.

5. The preparation method according to claim 1, characterized in that, In the steps of sequentially performing a first strong alkali cleaning, a first weak alkali neutralization cleaning, a second strong alkali cleaning, and a second weak alkali neutralization cleaning on the frozen and thawed pig peritoneum, the first weak alkali neutralization cleaning specifically includes: immersing the pig peritoneum after the first strong alkali cleaning into a first weak alkali solution for neutralization cleaning. The first weak alkaline solution has a mass-volume concentration of 2-6%, and includes sodium bicarbonate solution; the washing is performed 1-3 times, each time for 20-40 minutes.

6. The preparation method according to claim 1, characterized in that, In the steps of sequentially performing a first strong alkali cleaning, a first weak alkali neutralization cleaning, a second strong alkali cleaning, and a second weak alkali neutralization cleaning on the frozen and thawed pig peritoneum, the second strong alkali cleaning specifically includes: immersing the pig peritoneum after the first weak alkali neutralization cleaning into the second strong alkali solution for cleaning. The second strong alkali solution has a mass-volume concentration of 0.5-1.5%, and includes a sodium hydroxide solution; the washing is performed 1-3 times, each time for 1-3 hours; after the second strong alkali washing, the process also includes washing with purified water 1-3 times, each time for 20-40 minutes.

7. The preparation method according to claim 1, characterized in that, In the steps of sequentially performing a first strong alkali cleaning, a first weak alkali neutralization cleaning, a second strong alkali cleaning, and a second weak alkali neutralization cleaning on the frozen and thawed pig peritoneum, the second weak alkali neutralization cleaning specifically includes: immersing the pig peritoneum cleaned with the second strong alkali into a second weak alkali solution for neutralization cleaning. The second weak alkali solution has a mass-volume concentration of 2-6% and includes a sodium bicarbonate solution; the washing is performed 3-5 times, each time for 20-40 minutes; after neutralization and washing with the second weak alkali, the washing is further performed 2-4 times with purified water, each time for 0.5-1.5 hours.

8. The preparation method according to claim 1, characterized in that, In the step of adding the decellularized porcine peritoneum to a cross-linking agent solution for cross-linking treatment, followed by sterilization and freeze-drying, the cross-linking agent solution includes an EDC solution with a mass-volume concentration of 2-4%, and the cross-linking treatment time is 20-30 hours.

9. The preparation method according to claim 1, characterized in that, In the step of adding the decellularized porcine peritoneum to a cross-linking agent solution for cross-linking treatment, followed by sterilization and freeze-drying, the sterilization specifically includes: washing with 70-80% ethanol 1-3 times, each time for 1-3 hours; The freeze-drying process specifically includes: freezing and drying the sterilized porcine peritoneum for 20-30 hours after flattening and shaping it.

10. An oral repair membrane, characterized in that, It is prepared by the preparation method according to any one of claims 1-9.

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