Porcine decellularized matrix material for the treatment of postmenopausal urogenital syndrome, its preparation method and formulation

Abstract

This invention relates to the field of biomedical materials, and discloses a porcine decellularized matrix material for treating postmenopausal urogenital syndrome, its preparation method, and formulation. The method involves inactivating porcine tissue with a virus, followed by a deantigenation process combining trypsin pretreatment and stepwise treatment with high-salt SDS and high-alkali SDS. The material is then subjected to intermediate washing with physiological saline, defatting, final washing, drying, and sterilization to obtain the material. This method can deeply remove antigenic substances such as DNA, reduce SDS and organic solvent residues, while effectively preserving the natural collagen structure and bioactive components. The resulting material has a DNA residue of less than 50 ng / mg, an SDS residue of less than 0.02%, a fat content of less than 0.5%, a bacterial endotoxin content of less than 0.5 EU / mg, a total sugar content of greater than 0.2 mg / g, and a total protein content of greater than 85%. This material can be formulated into microparticles or microspheres for preparing formulations to treat symptoms such as vaginal mucosal atrophy, dryness, and decreased elasticity associated with postmenopausal urogenital syndrome, providing both immediate support and long-term regenerative repair functions.

Description

Technical Field

[0001] This application relates to the field of biomedical materials, and more specifically, to a porcine decellularized matrix material for treating postmenopausal urogenital syndrome, as well as its preparation method and formulation. Background Technology

[0002] Menopausal urogenital syndrome (GSM) is a series of psychosomatic symptoms in women caused by declining ovarian function and estrogen levels. GSM is particularly common, manifesting as vaginal dryness, atrophy, decreased elasticity, painful intercourse, urinary frequency, and incontinence, severely impacting the quality of life and mental and physical health of middle-aged and elderly women. Currently, clinical interventions for menopausal-related vaginal atrophy and functional decline mainly include local estrogen therapy, lubricants, physical therapy (such as laser and radiofrequency ablation), and injectable fillers. However, all have limitations: hormone therapy carries potential risks and has many contraindications; physical therapy has unstable effects; and while traditional filler materials such as hyaluronic acid can improve symptoms in the short term, they degrade quickly and lack long-term regenerative capacity, making it difficult to fundamentally repair the atrophied tissue structure and function.

[0003] In recent years, acellular extracellular matrix (ECM) materials have been considered ideal injectable repair materials due to their natural origin, rich collagen and various growth factors, good biocompatibility, and tissue-inducing regeneration capabilities. Existing research has applied them to vaginal repair; for example, CN120114659A discloses a biodegradable vaginal repair injectable material using ECM microparticles and a dispersant to improve vaginal laxity. However, this technical solution does not delve into the key process control of the ECM material itself during decellularization, especially regarding the specific requirements for high biocompatibility, low immunogenicity, excellent structural preservation, and enrichment of active ingredients needed for postmenopausal atrophic vaginal tissue repair, lacking systematic optimization. Furthermore, existing decellularization processes often focus on cell removal, neglecting the deep removal of harmful substances such as residual DNA, detergents, and organic solvents. These residues may induce chronic inflammation or immune responses after long-term implantation, making them particularly unsuitable for the postmenopausal vaginal mucosa, a tissue environment with relatively reduced blood supply and weakened self-repair capabilities.

[0004] Therefore, there is an urgent need to develop a preparation process for porcine decellularized matrix materials that can efficiently and thoroughly remove immunogenic substances, retain the natural active ingredients and microstructure of ECM to the maximum extent, and have extremely low residual toxicity. Summary of the Invention

[0005] To address the aforementioned issues, this application provides a method for preparing porcine decellularized matrix material for treating postmenopausal urogenital syndrome.

[0006] The technical solution adopted in this application is as follows:

[0007] In a first aspect, this application provides a method for preparing a porcine decellularized matrix material for treating postmenopausal urogenital syndrome, comprising:

[0008] Porcine tissue was collected, cleaned, and cut into tissue blocks.

[0009] The tissue blocks were immersed in a disinfectant solution to inactivate the virus. The disinfectant solution contained at least one of anhydrous ethanol, NaCl, peracetic acid, and glutaraldehyde.

[0010] After the virus was inactivated, the tissue blocks were first soaked in trypsin solution, and then subjected to stepwise antigen removal treatment with SDS solution containing sodium chloride and SDS solution containing sodium hydroxide.

[0011] The tissue blocks after antigen removal were rinsed with physiological saline.

[0012] The tissue blocks were degreased using a mixture of chloroform and methanol.

[0013] The defatted tissue blocks were rinsed with physiological saline and purified water in the final rinse.

[0014] The cleaned tissue blocks were vacuum dried and irradiated to obtain the porcine decellularized matrix material for treating menopausal urogenital syndrome.

[0015] Furthermore, in the above-mentioned virus inactivation process, the disinfectant contains 0.5-3 wt% peracetic acid, 2-4 wt% glutaraldehyde, 10-30 wt% anhydrous ethanol and 0.5-1.5 M NaCl.

[0016] Furthermore, the above-mentioned antigen removal process includes:

[0017] Tissue blocks were soaked in a 0.2-0.5 wt% trypsin solution at 2-8℃ for 12-16 hours.

[0018] Tissue blocks were treated 2-4 times with an SDS solution of 0.4%-0.6% by mass, prepared with 0.8-1.2 M sodium chloride, at 35-38℃, for 20-40 minutes each time.

[0019] The tissue blocks were treated 2-4 times with an SDS solution of 0.4%-0.6% by mass prepared with 0.4-0.6 M sodium hydroxide at 35-38℃, with each treatment lasting 20-40 minutes.

[0020] Furthermore, in the above-mentioned intermediate cleaning, physiological saline with a concentration of 0.85-0.95wt% is used for cleaning 6-10 times, with each cleaning time being 15-25 minutes;

[0021] In the final cleaning process, the patient is first cleaned 3-5 times with physiological saline solution at a concentration of 0.85-0.95wt%, and then cleaned 3-5 times with purified water. Each cleaning cycle lasts 15-25 minutes.

[0022] Furthermore, in the above degreasing step, the volume ratio of chloroform to methanol is 1:1 to 3:1, the degreasing time is 3-5 hours, and the treatment temperature is 15-30℃.

[0023] Secondly, this application provides a porcine decellularized matrix material for treating menopausal urogenital syndrome prepared by the above-described preparation method.

[0024] Furthermore, the residual DNA content of the above materials is less than 50 ng / mg, the residual SDS content is less than 0.02%, the fat content is less than 0.5%, the bacterial endotoxin content is less than 0.5 EU / mg, the total sugar content is greater than 0.2 mg / g, and the total protein content is greater than 85%.

[0025] Thirdly, this application provides a formulation for tissue repair in menopausal urogenital syndrome, comprising the aforementioned porcine decellularized matrix material, wherein the formulation is in the form of granules, tablets or gels.

[0026] Furthermore, the particle size range of the above-mentioned granules is 50~5000μm.

[0027] Fourthly, this application also provides the use of the above-mentioned porcine decellularized matrix material in the preparation of medical devices or pharmaceutical formulations, wherein the medical devices or pharmaceutical formulations are used for:

[0028] (a) To treat or improve symptoms of vaginal mucosal atrophy, dryness, or decreased elasticity associated with postmenopausal urogenital syndrome;

[0029] (b) Medical aesthetic treatments for soft tissue filling, wrinkle improvement, or skin rejuvenation;

[0030] (c) Intra-articular injection filling, used to repair cartilage defects, improve joint lubrication or relieve symptoms of osteoarthritis.

[0031] In summary, this application has the following beneficial effects:

[0032] 1. This application pioneers a progressive antigen removal process: "trypsin pretreatment → high-salt SDS treatment → high-alkali SDS treatment." First, trypsin gently loosens cell connections. Then, the osmotic pressure difference created by high-concentration NaCl, combined with the synergistic effect of SDS, initially lyses and removes cellular components. Finally, the strongly alkaline environment of NaOH further destroys nucleic acids, inactivates viruses, and reduces endotoxins, while effectively salting out residual SDS molecules. This three-step combination is not a simple additive process but produces a significant synergistic effect, ensuring deep removal of antigenic substances (DNA, lipids) from the source. Experimental data confirms that DNA residue can be below 50 ng / mg, far exceeding industry standards.

[0033] 2. In terms of material properties, this application achieves an excellent balance between low toxicity residues and high bioactivity retention. By repeatedly using physiological saline in the intermediate and final cleaning stages, its ionic strength effectively replaces and extrudes residual substances such as SDS that may have embedded in the matrix during the aforementioned steps, thereby reducing SDS residues to below 0.02%. Simultaneously, this gentle yet efficient cleaning method, combined with the aforementioned decellularization strategy that avoids the use of highly corrosive reagents, maximizes the protection of the three-dimensional network structure of natural collagen and bioactive components such as endogenous growth factors in the extracellular matrix. This results in a material with a total protein content greater than 85%, providing a solid material basis for tissue regeneration after implantation.

[0034] 3. At the application level, this application precisely identifies and addresses specific clinical pain points in the treatment of postmenopausal urogenital syndrome. The inventiveness of this application lies not only in the process and materials themselves, but also in their explicit and rational application to the specific scenario of "treating postmenopausal urogenital syndrome." Postmenopausal vaginal tissue atrophy, reduced blood supply, and weakened repair capacity place higher demands on the safety, biocompatibility, and regenerative capacity of implant materials. The porcine decellularized matrix material prepared in this application, with its extremely low immunogenicity, near-non-toxic chemical residues, intact natural structure, and abundant active ingredients, perfectly meets the dual requirements of "long-term safety" and "active regeneration" in this scenario. The material can be further processed into granules, tablets or gels through formulation processes for use as vaginal submucosal filling. It can provide immediate support and improve symptoms such as dryness and loss of elasticity. It can also serve as a biological scaffold to continuously guide host cells to climb, proliferate and differentiate, promote the synthesis of collagen and tissue remodeling, thereby achieving a treatment upgrade from "temporary filling" to "functional repair". This provides a new and more fundamental treatment option for menopausal urogenital syndrome patients. Attached Figure Description

[0035] Figure 1 These are images showing the changes in the dermis during the preparation of the porcine decellularized matrix material in Example 1 of this application;

[0036] Figure 2 The images show the HE and Masson staining results of the porcine decellularized matrix materials in Examples 1-3 of this application. Detailed Implementation

[0037] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0038] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0039] Example 1

[0040] This embodiment provides a porcine decellularized matrix material for treating postmenopausal urogenital syndrome, and its preparation method includes:

[0041] (1) Pre-treatment: Pigskin is obtained, and the hair is scraped off, split, and cleaned. It is then cut into 0.5cm pieces. 2 The tissue blocks are cleaned, the pigskin is washed and drained.

[0042] (2) Virus inactivation: The pig skin was soaked in a disinfectant solution containing 3 wt% peracetic acid, 10 vol% anhydrous ethanol and 1M NaCl for 1 hour. After disinfection, the tissue was rinsed with purified water.

[0043] (3) Antigen removal treatment:

[0044] Soak pig skin in 0.3% trypsin at 2-8℃ for 16 hours, then wash the tissue with purified water;

[0045] After scraping off the outer layer of pigskin with a blunt instrument, the dermis is obtained. The dermis is then soaked in a 0.5% SDS solution prepared with 1M NaCl for 10 minutes, followed by fresh 1M NaCl to prepare the 0.5% SDS solution. The solution is then incubated in a 37°C water bath at 80-120 rpm with slow stirring for 30 minutes. This step is repeated four times. After processing, the water is drained.

[0046] Soak the leather in a 0.5% SDS solution prepared with 0.5M NaOH for 10 minutes, then replace with fresh 0.5M NaOH to prepare the same 0.5% SDS solution. Incubate in a 37°C water bath at 80-120 rpm with slow stirring for 30 minutes. Repeat this step four times. After treatment, drain the water.

[0047] (4) Mid-stream rinsing: Rinse with 0.9% NaCl, stirring at 80~120 rpm for 20 min at room temperature. Change the solution and rinse 8 times, then drain the water.

[0048] (5) Degreasing treatment: Add a degreasing solution of chloroform and methanol in a volume ratio of 1:1 to the dermis and stir at room temperature (80~120 rpm) for 4 hours, changing the solution every 2 hours.

[0049] (6) Final cleaning: Rinse the dermis with purified water for 1 hour. After draining the water, add 0.9% NaCl and stir at 80-120 rpm for 20 minutes at room temperature. Repeat this step 4 times. After draining the water, add purified water to the dermis and stir at 80-120 rpm for 20 minutes at room temperature. Repeat this step 4 times.

[0050] (7) Drying and sterilization: The cleaned tissue blocks were vacuum dried and irradiated for sterilization, and then pulverized into particles of 500-800 μm to obtain porcine decellularized matrix material. The changes in the dermis during its preparation process are as follows: Figure 1 As shown.

[0051] Example 2

[0052] This embodiment provides a porcine decellularized matrix material for treating postmenopausal urogenital syndrome, and its preparation method includes:

[0053] (1) Pre-treatment: Pigskin is obtained, and the hair is scraped off, split, and cleaned. It is then cut into 0.5cm pieces. 2 The tissue blocks are cleaned, the pigskin is washed and drained.

[0054] (2) Virus inactivation: Soak pig skin in a disinfectant solution containing 3% glutaraldehyde, 20% anhydrous ethanol and 1M NaCl for 1 hour. After disinfection, rinse the tissue with purified water.

[0055] (3) Antigen removal treatment:

[0056] Soak pig skin in 0.2% trypsin at 2-8℃ for 16 hours, then wash the tissue with purified water;

[0057] After scraping off the outer layer of pigskin with a blunt instrument, the dermis is obtained. The dermis is then soaked in a 0.5% SDS solution prepared with 1M NaCl for 10 minutes, followed by fresh 1M NaCl to prepare the 0.5% SDS solution. The mixture is then sonicated in a 37°C water bath for 30 minutes. This step is repeated four times. After processing, the water is drained.

[0058] Soak the leather in a 0.5% SDS solution prepared with 0.5M NaOH for 10 minutes, then replace with fresh 0.5M NaOH to prepare the same 0.5% SDS solution. Sonicate in a 37℃ water bath for 30 minutes. Repeat this step four times. After treatment, drain the water.

[0059] (4) Mid-stream cleaning: Clean with 0.9% NaCl and sonicate at room temperature for 20 minutes. Change the solution 8 times and drain the water.

[0060] (5) Degreasing treatment: Add a degreasing solution of chloroform and methanol in a volume ratio of 2:1 to the dermis and stir at room temperature (80~120rpm) for 4 hours, changing the solution every 2 hours.

[0061] (6) Final cleaning: Rinse the dermis with purified water for 1 hour. After draining the water, add 0.9% NaCl and sonicate at room temperature for 20 minutes. Repeat this step 4 times. After draining the water, add purified water to the dermis and sonicate at room temperature for 20 minutes. Repeat this step 4 times.

[0062] (7) Drying and sterilization: The cleaned tissue blocks are vacuum dried and irradiated to sterilize them, and then pulverized into particles of 500-800μm to obtain porcine decellularized matrix material.

[0063] Example 3

[0064] This embodiment provides a porcine decellularized matrix material for treating postmenopausal urogenital syndrome, and its preparation method includes:

[0065] (1) Pre-treatment: Pigskin is obtained, and the hair is scraped off, split, and cleaned. It is then cut into 0.5cm pieces. 2 The tissue blocks are cleaned, the pigskin is washed and drained.

[0066] (2) Virus inactivation: The pig skin was soaked in a disinfectant solution containing 0.5% peracetic acid, 30% anhydrous ethanol and 1M NaCl for 1 hour. After disinfection, the tissue was rinsed with purified water.

[0067] (3) Antigen removal treatment:

[0068] Soak pigskin in 0.5% trypsin at 2-8℃ for 12 hours. Rinse the tissue with purified water.

[0069] After scraping off the outer layer of pigskin with a blunt instrument, the dermis is obtained. The dermis is then soaked in a 0.5% SDS solution prepared with 1M NaCl for 10 minutes, followed by fresh 1M NaCl to prepare the 0.5% SDS solution. The solution is then incubated in a 37°C water bath with shaking at 80-120 rpm for 30 minutes. This step is repeated four times. After processing, the water is drained.

[0070] Soak the leather in a 0.5% SDS solution prepared with 0.5M NaOH for 10 minutes, then replace with fresh 0.5M NaOH to prepare the same 0.5% SDS solution. Incubate in a 37°C water bath with shaking at 80-120 rpm for 30 minutes. Repeat this step four times. After treatment, drain the water.

[0071] (4) Mid-stream rinsing: Rinse with 0.9% NaCl, shake at 80~120 rpm for 20 min at room temperature. Change the solution 8 times. Drain the water.

[0072] (5) Degreasing treatment: Add a degreasing solution of chloroform and methanol in a volume ratio of 3:1 to the dermis and shake at room temperature (80~120 rpm) for 4 hours, changing the solution every 2 hours.

[0073] (6) Final cleaning: Rinse the dermis with purified water for 1 hour. After draining the water, add 0.9% NaCl and shake at 80-120 rpm for 20 minutes at room temperature. Repeat this step 4 times. After draining the water, add purified water to the dermis and shake at 80-120 rpm for 20 minutes at room temperature. Repeat this step 4 times.

[0074] (7) Drying and sterilization: The cleaned tissue blocks are vacuum dried and irradiated to sterilize them, and then pulverized into particles of 500-800μm to obtain porcine decellularized matrix material.

[0075] Comparative Example 1

[0076] The difference between this comparative example and Example 1 lies in the order of steps. Specifically, after virus inactivation treatment, a degreasing treatment step is performed first, followed by an antigen removal treatment step. The process conditions for each step are consistent with those in Example 1.

[0077] Comparative Example 2

[0078] The difference between this comparative example and Example 1 is that the disinfectant in the virus inactivation step does not contain 1M NaCl, but only 3 wt% peracetic acid and 10 vol% anhydrous ethanol.

[0079] Comparative Example 3

[0080] The difference between this comparative example and Example 1 is that the SDS solution in the antigen removal step does not contain 1M NaCl and 0.5M NaOH.

[0081] Comparative Example 4

[0082] The difference between this comparative example and Example 1 is that pure water is used instead of 0.9% NaCl in the intermediate and final cleaning stages.

[0083] Comparative Example 5

[0084] The difference between this comparative example and Example 1 is that the degreasing solution in the degreasing step is a mixture of chloroform and methanol with a volume ratio of 0.5:1.

[0085] Performance testing

[0086] I. Staining Experiment

[0087] Raw pig skin and decellularized dermis from Examples 1-3 were sectioned and stained with hematoxylin and eosin (HE) and Masson's stain to examine nucleus removal and collagen retention. Results are as follows: Figure 2 As shown.

[0088] Figure 2 The top row shows the HE staining results, and the bottom row shows the Masson staining results. In the HE staining image, pink represents the extracellular matrix, and purple represents the cell nucleus. It can be seen that after decellularization, the cell nuclei stained purple in the original porcine skin completely disappeared, indicating that nucleic acids were removed. Only the extracellular matrix, uniformly stained pink by eosin, remains in the field of view. In the Masson staining image, red represents muscle fibers, and blue represents collagen fibers. It can be seen that after decellularization, because all cells have been removed, the areas where muscle fibers were originally located have become "cavities" or filled with loose matrix. The remaining collagen fiber network is clearly stained blue, forming the main body of the staining. Therefore, only the blue collagen fiber framework remains in the field of view, and the red muscle fibers are not visible. All these results indicate that the porcine skin cellular components were effectively removed, and the ECM scaffold was completely preserved.

[0089] II. Content Determination

[0090] 1. Measurement method:

[0091] Total protein content: determined according to the Kjeldahl method, the first method in the "National Food Safety Standard GB 5009.5-2025 Determination of Protein in Food".

[0092] Total sugar content: Determined by spectrophotometry, Method I, in accordance with GB / T 9695.31-2008 "Determination of Total Sugar Content in Meat Products".

[0093] Total fat content: determined according to the first fass extraction method in GB 5009.6-2016 National Food Safety Standard - Determination of Fat in Food.

[0094] DNA residue: Refer to the method for determination of exogenous DNA residue in Part IV, 3407 of the 2025 edition of the Pharmacopoeia of the People's Republic of China.

[0095] Bacterial endotoxin content: Detected according to the bacterial endotoxin detection method in Part IV, 1143 of the 2025 edition of the Pharmacopoeia of the People's Republic of China.

[0096] SDS content: determined by acridine orange-spectrophotometric method.

[0097] 2. Measurement Results

[0098] As shown in Table 1.

[0099] Table 1. Results of content determination of various indicators in the examples and comparative examples

[0100]

[0101] As can be seen from Table 1:

[0102] The methods for preparing porcine decellularized matrix materials in Examples 1-3 of this invention can retain nutrients to a greater extent. The high retention of total protein and total sugar content ensures the activity of the obtained decellularized matrix materials.

[0103] As can be seen from the comparison of Example 1 and Comparative Example 1, the DNA residue in Comparative Example 1 (degreasing followed by antigen removal) was significantly higher than that in Example 1, and the fat residue was also significant. This indicates that pre-degreasing may lead to difficulties in the effective penetration and complete removal of cell nuclear components by subsequent SDS. This conversely proves the rationality of the proposed sequence of "removing the core antigen components first, then removing lipids," which is the basis for achieving deep decellularization.

[0104] Combining Example 1 and Comparative Examples 2-3, it is evident that Comparative Example 2 (virus inactivation without NaCl) showed increased DNA residue, confirming that NaCl not only enhances penetration during initial treatment but also helps to initially disrupt cell structure, laying the foundation for subsequent deep treatment. Comparative Example 3 (SDS treatment without NaCl / NaOH) showed the most significant deterioration in various residue indicators: extremely high DNA residue (205.60 ng / mg), endotoxin greater than 0.5 EU / mg, and fat residue at 2.66%. This strongly demonstrates that NaCl promotes cell lysis through osmotic pressure difference, while NaOH is indispensable in providing an alkaline environment for hydrolyzing nucleic acids and fats and effectively inactivating endotoxins. These two, together with SDS, form a synergistic system, which is the core of achieving a balance between "deep clearance" and "activity retention."

[0105] As can be seen from the comparison of Example 1 and Comparative Example 4, the SDS residue in Comparative Example 4 (pure water washing) was abnormally high. This directly verifies that ions in physiological saline can competitively replace and effectively elute SDS molecules embedded in the ECM network through the "salting out" effect. This step is crucial for controlling chemical residues and ensuring the long-term biosafety of materials, and cannot be replaced by pure water washing.

[0106] As can be seen from the comparison of Example 1 and Comparative Example 5, the residual fat in Comparative Example 5 (low-ratio degreasing solution) was significantly higher than that in Example 1, indicating that the chloroform:methanol ratio is crucial to the lipid extraction efficiency. The optimized ratio of this application can more effectively remove phospholipids and neutral fats, while a ratio that is too low may lead to incomplete degreasing and affect the biocompatibility of the material.

[0107] III. In vivo experiments

[0108] In this experiment, the decellularized dermal matrix materials prepared in Example 1 and Comparative Example 3 were granulated by liquid nitrogen grinding, suspended in physiological saline, and then injected subcutaneously or intravaginally into the vagina of experimental rats. A saline injection was set up as a blank control group, and estriol as a positive control group.

[0109] The experimental procedure is as follows:

[0110] Healthy adult rats underwent ovariectomy, while a sham-operated group was established where the ovaries were not removed. After a 2-week recovery period, estrogen deficiency was confirmed in the rats. Vaginal feeding was then initiated.

[0111] Samples were harvested 4 weeks after feeding. The distal 5 mm of the vagina was placed in a custom-made soft tissue clamp, which was attached to a force sensor and connected to the machine's crosshead. The sample was preloaded to 0.15 N and then pretreated for 10 cycles at an expansion rate of 25 mm / min. After pretreatment, each sample underwent a uniaxial load failure test simulating downward expansion of the vagina and its supporting tissues at the same rate. The uniaxial load failure test simulates downward expansion of the vagina and its supporting tissues by pulling along the longitudinal axis of the distal vagina. The resulting biomechanical parameters represent the forces (loads) that the vagina and supporting tissues collectively resist downward expansion as they attempt to maintain their normal anatomical relationship. Each failure load test produced a load-expansion curve. From the curves, the biomechanical parameters were derived: linear stiffness (N / mm) and ultimate load at failure (N).

[0112] The experimental results are shown in Table 2:

[0113] Table 2. In vivo experimental results of Example 1 and Comparative Example 3

[0114]

[0115] As can be seen from Table 2:

[0116] In Example 1, the ultimate load and linear stiffness of the group recovered to levels close to the positive control (estriol), significantly better than the blank control group. This indicates that the decellularized matrix material prepared by the "pancreatic enzyme-high salt / high alkali SDS-physiological saline washing" system of this application is not a simple physical filling after implantation. Its extremely low immunogenicity (DNA residue <50 ng / mg, fat content <0.5%, bacterial endotoxin <0.5 EU / mg) and chemical toxicity (SDS residue <0.020%) avoid the interference of chronic inflammation on the repair process; while the highly preserved natural collagen structure and bioactive components (total protein >85%) provide an ideal 3D scaffold for the attachment and proliferation of host cells, effectively guiding and promoting the migration of vaginal wall fibroblasts, collagen (especially type I and III) synthesis and remodeling, thereby essentially restoring the elasticity and support of the tissue and achieving functional regeneration.

[0117] While the repair effect in Comparative Example 3 was improved, it was far inferior to that in Example 1, and only slightly better than the blank control. This directly corresponds to the key defects of the material in Comparative Example 3 itself: its extremely high DNA residue and bacterial endotoxins indicate incomplete decellularization and sterilization. After implantation, these residual foreign genetic materials and pyrogens are likely to trigger persistent subclinical immune rejection or inflammatory responses, thereby interfering with or even blocking the normal tissue regeneration process. At the same time, its more severely damaged protein structure also leads to a significant reduction in its activity as a biological scaffold. Therefore, although its physical morphology can still play a partial filling role, its core function of "actively inducing tissue regeneration" has been greatly weakened, which, from an in vivo experimental perspective, reversely verifies that the high-salt / high-alkali synergistic treatment is crucial for realizing the material's biological function.

[0118] In summary, this invention provides an innovative porcine-derived decellularized matrix material and its optimized preparation process for the treatment of postmenopausal urogenital syndrome. This process, through a synergistic system of "pancreatic enzyme pretreatment—stepwise high-salt / high-alkali SDS treatment—physiological saline-dominated washing," successfully overcomes the common challenges of incomplete antigen removal and high chemical residues in traditional decellularization methods. The resulting material exhibits low immunogenicity (DNA residue <50 ng / mg), ultra-low chemical toxicity (SDS residue <0.02%), and high preservation of its natural structure and bioactivity (total protein >85%). In vivo experiments have demonstrated that this material effectively promotes functional regeneration of vaginal tissue in postmenopausal model animals, restoring its elasticity and support, with significant effects. This invention not only provides a safe, effective, and regenerative new treatment option for postmenopausal urogenital syndrome, especially urogenital tract symptoms, but also provides innovative process ideas and material basis for other clinical fields requiring highly biocompatible repair materials, possessing significant clinical application value and market prospects.

[0119] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing a porcine-derived acellular matrix material for the treatment of menopausal urogenital syndrome, characterized in that, It includes: Take pig skin tissue, clean it, and cut it into tissue blocks; The tissue blocks were immersed in a disinfectant solution to inactivate the virus. After the virus was inactivated, the tissue blocks were first soaked in trypsin solution, and then subjected to stepwise antigen removal treatment with SDS solution containing sodium chloride and SDS solution containing sodium hydroxide. The tissue blocks after antigen removal were rinsed with physiological saline; the tissue blocks were then degreased with a mixture of chloroform and methanol. The defatted tissue blocks were rinsed with physiological saline and purified water in the final rinse. The cleaned tissue blocks were vacuum dried and irradiated to obtain the porcine decellularized matrix material for treating postmenopausal urogenital syndrome. In the virus inactivation process, the disinfectant solution contains 0.5-3 wt% peracetic acid, 2-4 wt% glutaraldehyde, 10-30 wt% anhydrous ethanol and 0.5-1.5 M NaCl.

2. The method for preparing porcine decellularized matrix material for treating postmenopausal urogenital syndrome according to claim 1, characterized in that, The antigen removal process includes: soaking the tissue block in a 0.2-0.5 wt% trypsin solution at 2-8°C for 12-16 hours; treating the tissue block 2-4 times at 35-38°C with an SDS solution of 0.4%-0.6% prepared with 0.8-1.2 M sodium chloride, each treatment lasting 20-40 minutes; and treating the tissue block 2-4 times at 35-38°C with an SDS solution of 0.4%-0.6% prepared with 0.4-0.6 M sodium hydroxide, each treatment lasting 20-40 minutes.

3. The method for preparing porcine decellularized matrix material for treating postmenopausal urogenital syndrome according to claim 1, characterized in that, The intermediate cleaning process involves rinsing with 0.85-0.95wt% physiological saline 6-10 times, with each rinsing session lasting 15-25 minutes. The final cleaning process involves rinsing with 0.85-0.95wt% physiological saline 3-5 times, followed by rinsing with purified water 3-5 times, with each rinsing session lasting 15-25 minutes.

4. The method for preparing porcine decellularized matrix material for treating postmenopausal urogenital syndrome according to claim 1, characterized in that, In the degreasing step, the volume ratio of chloroform to methanol is 1:1 to 3:1, the degreasing time is 3-5 hours, and the processing temperature is 15-30℃.

5. A porcine decellularized matrix material for treating postmenopausal urogenital syndrome, prepared by the method according to any one of claims 1-4, characterized in that, The material has a residual DNA content of less than 50 ng / mg, a residual SDS content of less than 0.02 wt%, a fat content of less than 0.5 wt%, a bacterial endotoxin content of less than 0.5 EU / mg, a total sugar content of greater than 0.2 mg / g, and a total protein content of greater than 85 wt%.

6. A preparation for tissue repair in postmenopausal urogenital syndrome, characterized in that, The formulation comprises the porcine decellularized matrix material of claim 5, wherein the dosage form of the formulation is granules, tablets or gels, and the particle size range of the granules is 50~5000μm.

7. The application of the porcine decellularized matrix material as described in claim 5 in the preparation of medical devices or pharmaceutical formulations, characterized in that, The medical device or pharmaceutical preparation is used to treat or improve symptoms associated with postmenopausal urogenital syndrome, including: vaginal mucosal atrophy, dryness, decreased elasticity and dyspareunia caused by vulvar and vaginal atrophy and atrophic vaginitis; and related urinary symptoms such as dysuria and urinary incontinence.

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