Method for inactivating virus of valve leaflet material

Abstract

The invention belongs to the technical field of biomedical materials, and particularly discloses a method for inactivating viruses of a valve leaflet material. Comprising the following steps: (1) immersing fixing cloth by using a glutaraldehyde solution until the fixing cloth is fully infiltrated, unfreezing bovine pericardium sheets subjected to an anti-calcification treatment process, draining off liquid on the surfaces, and paving the bovine pericardium sheets by using the fixing cloth to obtain a layer of cloth, a layer of bovine pericardium sheets and a layer of cloth for later use; (2) cross-linking immobilization-1: putting the bovine pericardium slices treated in the step (1) into a box, pouring a glutaraldehyde solution, enabling the glutaraldehyde solution to fully infiltrate the bovine pericardium slices, enabling the height of the glutaraldehyde solution to be flush with the height of the uppermost layer of fixing cloth, and refrigerating to obtain an intermediate product; and (3) EO treatment: carrying out ethylene oxide sterilization on the intermediate product treated in the step (2) to obtain a treated product. Through cross-linking fixation and EO sterilization treatment, the advantages of thorough virus inactivation and small damage to the structure and performance of the valve leaflet material are realized.

Description

Technical Field

[0001] The present application relates to the technical field of biomedical materials, and in particular to a method for inactivating viruses in leaflet materials. Background Art

[0002] Biomaterials (such as bovine pericardium slices) must be free of viral contamination in medical applications. However, there is a window period during donor testing, which may result in viral residues. Therefore, physical or chemical methods are needed to inactivate the virus while avoiding damage to the material properties.

[0003] Currently, numerous methods exist for inactivating viruses in biomaterials. While physical methods such as high-temperature treatment (pasteurization, dry heat inactivation, and gamma-ray irradiation) can effectively inactivate viruses, they can severely damage the biomechanical properties of leaflet materials, causing them to become brittle and lose elasticity, making them unsuitable for clinical use. Among chemical methods, the commonly used organic solvent / detergent (S / D) method can effectively inactivate enveloped viruses, but is ineffective against non-enveloped viruses. Furthermore, complex post-use removal of residual reagents is required, increasing costs and contamination risks.

[0004] In addition, some traditional methods have problems such as complex operation, time-consuming, requiring special equipment or introducing foreign contaminants, which makes it difficult to meet the needs of efficient, safe and convenient virus inactivation of leaflet materials. Summary of the Invention

[0005] In order to improve the problem of incomplete virus inactivation and serious damage to the structure and performance of leaflet materials, the present application provides a method for inactivating viruses in leaflet materials.

[0006] This application provides a method for inactivating viruses in leaflet materials, which adopts the following technical solutions: A method for inactivating viruses in leaflet materials, comprising the following steps: (1) Immerse the fixing cloth in glutaraldehyde solution until the fixing cloth is fully soaked, thaw the bovine pericardium slice that has completed the anti-calcification treatment process, drain the liquid on the surface, and then use the fixing cloth to spread the bovine pericardium slice, one layer of cloth, one layer of bovine pericardium slice, and one layer of cloth, and set aside; (2) Cross-linking fixation-1: Place the bovine pericardium slice treated in step (1) into a box, pour in glutaraldehyde solution, and allow the glutaraldehyde solution to fully infiltrate the bovine pericardium slice so that the height of the glutaraldehyde solution is level with the height of the topmost fixing cloth. Refrigerate at a storage temperature of 2-8°C for 48 hours to obtain an intermediate product; (3) EO treatment: The intermediate product treated in step (2) is sterilized with ethylene oxide, and subjected to analysis or shelf storage to obtain a treated product.

[0007] By adopting the above technical solution, the bovine pericardium slice is spread using a fixing cloth. After the fixing cloth is soaked, a rigid frame is formed, which can prevent the bovine pericardium slice from wrinkling and deformation during spreading, thereby ensuring the uniformity of subsequent cross-linking; the fixing cloth serves as a carrier to keep the bovine pericardium slice flat, increase the contact area between glutaraldehyde and tissue, and the gaps between layers facilitate the subsequent penetration of glutaraldehyde solution, thereby avoiding incomplete virus inactivation caused by tissue stacking.

[0008] Glutaraldehyde solution is used for infiltration and long-term low-temperature cross-linking and fixation. By cross-linking with the amino groups of microbial proteins, its structural function is destroyed, effectively killing various microorganisms including viruses. Long-term low-temperature cross-linking (48 hours, 2-8°C) ensures that glutaraldehyde fully penetrates and inactivates viruses, and is also beneficial to maintaining the biomechanical properties of bovine pericardium materials.

[0009] Step (3) uses ethylene oxide (EO) for terminal sterilization. EO is a broad-spectrum gas sterilant that can penetrate porous materials (such as bovine pericardium sheets wrapped in fixed cloth) and destroy the DNA / RNA and proteins of microorganisms through alkylation, achieving reliable terminal sterility assurance. It supplements and strengthens the inactivation by glutaraldehyde, ensuring that the sterility required for medical devices is achieved.

[0010] After sterilization, aeration or shelf storage is performed to completely remove residual ethylene oxide and its reaction products (such as ethylene glycol and chloroethanol) to below safety limits, thereby preventing toxic reactions in patients after implantation. Aeration is typically performed in a controlled fume hood or dedicated aeration room, while shelf storage involves placing the items in a well-ventilated environment to allow the residual material to dissipate naturally.

[0011] Preferably, the preparation method of the fixing cloth comprises the following steps: stirring 50-60 parts of polyethylene terephthalate, 10-15 parts of polyethylene oxide, 5-6 parts of modified graphene, 2-4 parts of nano-titanium dioxide, 4-5 parts of modified chitosan, and 1-2 parts of a silane coupling agent at a temperature of 85-90°C for 1-2 hours to obtain a mixture, extruding and stretching to obtain polyester fibers, laying the polyester fibers, and hot pressing at 190-200°C to obtain the fixing cloth.

[0012] By adopting the above technical solution, polyethylene terephthalate is used as the matrix material to provide the mechanical strength and thermal stability of the fiber, and polyethylene oxide improves the processing fluidity of the system and reduces the melting temperature. Modified graphene enhances compatibility with polyethylene terephthalate and improves mechanical properties. Nano-titanium dioxide enhances the fiber's UV resistance and antibacterial properties. Modified chitosan improves compatibility with polyethylene terephthalate and enhances antibacterial properties, mechanical properties and biocompatibility. Silane coupling agents promote the combination of inorganic fillers (modified graphene, nano-titanium dioxide) and organic matrices (polyethylene terephthalate, polyethylene oxide).

[0013] Extrusion and cooling form nascent fibers, which are then stretched to align the molecular chains and increase fiber strength. Polyester fibers are evenly laid into a mesh structure and hot-pressed to fuse the fiber contact points, forming a three-dimensional mesh structure that enhances the mechanical properties and stability of the fixing cloth. This provides reliable mechanical support for the subsequent bovine pericardium, preventing pericardial contraction or deformation. The flexibility of the fixing cloth ensures a close fit with the pericardium, reducing stress concentration. This helps reduce the risk of pericardial calcification, enhances antibacterial efficacy, and extends service life. A preferred method for preparing modified graphene includes the following steps: dispersing graphene in sulfuric acid, stirring for 10-15 minutes, washing with water, and then dispersing it in deionized water. Adding nanosilica and gelatin, stirring at 70-75°C for 1-2 hours, and drying to obtain the modified graphene. By employing this technical solution, sulfuric acid protonates the graphene surface, introducing sulfonic acid groups and stripping away interlayer van der Waals forces, thereby enhancing hydrophilicity. Nanosilica has excellent mechanical properties and can adsorb onto the graphene surface, enhancing the stability and mechanical properties of the composite material. Gelatin forms hydrogen bonds with the oxygen-containing groups of graphene through amino and carboxyl groups, coating the graphene surface, making the nano-silica and graphene tightly combined, increasing the toughness of the modified graphene, and subsequently improving the mechanical strength of the fixed cloth.

[0014] In the subsequent application of fixed cloth in the treatment of bovine pericardium, the anti-calcification property of nano-silica reduces the risk of pericardial calcification and prolongs its service life. The anti-inflammatory properties of gelatin and the antibacterial properties of graphene work synergistically to achieve long-term antibacterial effect.

[0015] Preferably, the preparation method of the modified chitosan comprises the following steps: dispersing chitosan in an acetic acid solution, adding cassava starch, stirring at a temperature of 50-55° C. for 1-2 hours, drying, and grinding to obtain a mixture; The mixture is dispersed in deionized water, hydroxyapatite and microcrystalline cellulose are added, stirred at a temperature of 60-65° C. for 2-3 hours, dried, and ground to obtain modified chitosan.

[0016] By employing this technical solution, chitosan is protonated in an acetic acid solution, forming a transparent, viscous solution that provides active amino and hydroxyl groups for subsequent reactions. The cassava starch granules absorb water and swell, releasing amylose, which then hydrogen-bonds with the chitosan to form a semi-interpenetrating network structure, reducing the brittleness of the subsequent film. Upon drying, the composite forms a porous structure.

[0017] Ca in hydroxyapatite 2+ Forming ionic bonds with -COO- of chitosan, while its PO4 3-Hydrogen bonds are formed with the -OH groups of starch, allowing the inorganic crystalline structure of hydroxyapatite to fill the pores of the chitosan-starch matrix, improving the mechanical and compressive strength of the composite material. Microcrystalline cellulose cross-links with chitosan through hydrogen bonds, further stabilizing the bond between chitosan and hydroxyapatite and increasing the tensile strength of chitosan. This is subsequently applied to the fixing cloth, improving its mechanical properties, antibacterial properties, and thermal stability.

[0018] It was subsequently used in the process of fixing the bovine pericardium to block Ca 2+ During collagen deposition, it can remove lipid peroxidation free radicals, protect the triple helix structure of bovine pericardium collagen, inhibit thermal shrinkage, and extend the life of bovine pericardium.

[0019] Preferably, the glutaraldehyde solution is prepared by dissolving a PBS buffer solution having a pH of 7.2-7.4 in water for injection to prepare 2 L of PBS buffer solution; Take 12 mL of 50% glutaraldehyde solution, dilute to 2 L with PBS buffer solution, and mix well to obtain glutaraldehyde solution.

[0020] By employing this technical solution, cross-linking and fixation with glutaraldehyde solution reveals that the bovine pericardium, primarily composed of collagen fibers, undergoes covalent cross-linking between the two aldehyde groups of glutaraldehyde and the free amino groups in the collagen, forming a stable Schiff base structure that significantly enhances the stability of the collagen network. This cross-linking enhances the tensile strength and elastic modulus of the collagen fibers, enabling the bovine pericardium to withstand cyclic stress under physiological conditions.

[0021] Preferably, in step (2), the bovine pericardium sheet and the fixing cloth are kept fixed during the cross-linking and fixing-1 treatment.

[0022] By adopting the above technical solution, the bovine pericardium sheet is easily contracted due to surface tension in the glutaraldehyde solution. If the fixation cloth is displaced, it will cause tissue wrinkles and form a cross-linking blind area; poor fixation will lead to uneven glutaraldehyde penetration, resulting in a gradient of "local excessive cross-linking-local insufficient cross-linking", affecting the consistency of the valve mechanical properties.

[0023] Preferably, the treatment conditions for ethylene oxide sterilization are: temperature of 35-41° C. and humidity of 40-80% RH.

[0024] By adopting the above technical solution, the boiling point of EO is 10.4°C, it is in a stable gaseous state at 35-41°C, and its vapor pressure reaches 70-100kPa, which not only avoids incomplete gasification caused by low temperature (<35°C) but also prevents excessive gas expansion caused by high temperature (>41°C).

[0025] Preferably, the ethylene oxide (EO) gas concentration is 600-900 mg / L, and the treatment time is 90-360 min.

[0026] By adopting the above technical solution, when the concentration is less than 600 mg / L, the sterilization assurance level achieved within a reasonable time may only reach 10 -4 , cannot meet the requirements of medical devices.

[0027] In summary, this application has the following beneficial effects: 1. In this application, glutaraldehyde solution is used for infiltration and long-term low-temperature cross-linking and fixation. By cross-linking with the amino groups of microbial proteins, its structural function is destroyed, effectively killing various microorganisms including viruses. Low-temperature cross-linking ensures that glutaraldehyde fully penetrates and inactivates viruses, while also helping to maintain the biomechanical properties of bovine pericardium materials.

[0028] 2. The use of ethylene oxide for terminal sterilization in this application can penetrate porous materials and destroy the DNA / RNA and proteins of microorganisms through alkylation, thereby achieving reliable terminal sterility assurance.

[0029] 3. In this application, sterilization is followed by analysis or shelf storage to completely remove residual ethylene oxide and its reaction products to reduce them below the safety limit to avoid toxic reactions to patients after implantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Residual PRV titer curves of sample 1 at different sampling points.

[0031] Figure 2 Residual PRV titer curves of sample 2 at different sampling points.

[0032] Figure 3 Residual PRV titer curve of sample 3 at different sampling points.

[0033] Figure 4 Residual VSV titer curve of sample 1 at different sampling points.

[0034] Figure 5 Residual VSV titer curve of sample 2 at different sampling points.

[0035] Figure 6 Residual VSV titer curve of sample 3 at different sampling points.

[0036] Figure 7 Residual Reo3 titer curve of sample 1 at different sampling points.

[0037] Figure 8 Residual Reo3 titer curve of sample 2 at different sampling points.

[0038] Figure 9 Residual Reo3 titer curve of sample 3 at different sampling points.

[0039] Figure 10Residual PPV titer curve of sample 1 at different sampling points.

[0040] Figure 11 Residual PPV titer curve of sample 2 at different sampling points.

[0041] Figure 12 Residual PPV titer curve of sample 3 at different sampling points.

[0042] Figure 13 Residual PRV titer curve of intermediate 1 after different EO treatments.

[0043] Figure 14 Residual PRV titer curves of intermediate 2 after different EO treatments.

[0044] Figure 15 Residual PRV titer curve of intermediate 3 after different EO treatments.

[0045] Figure 16 Residual VSV titer curve of intermediate 1 after different EO treatments.

[0046] Figure 17 Residual VSV titer curve of intermediate 2 after different EO treatments.

[0047] Figure 18 Residual VSV titer curve of intermediate 3 after different EO treatments.

[0048] Figure 19 Residual Reo3 titer curve of intermediate 1 after treatment with different EOs.

[0049] Figure 20 Residual Reo3 titer curve of intermediate 2 after treatment with different EOs.

[0050] Figure 21 Residual Reo3 titer curve of intermediate 3 after different EO treatments.

[0051] Figure 22 Residual PPV titer curve of intermediate 1 after different EO treatments.

[0052] Figure 23 Residual PPV titer curve of intermediate 2 after different EO treatments.

[0053] Figure 24 Residual PPV titer curve of intermediate 3 after different EO treatments. DETAILED DESCRIPTION

[0054] The present application is further described in detail below with reference to the embodiments.

[0055] The raw materials used in the examples and comparative examples can all be obtained commercially.

[0056] Example 1 Sample number (batch number): Sample 1; Sample 2; Sample 3.

[0057] The selected indicator viruses are pseudorabies virus (PRV), vesicular stomatitis virus (VSV), reovirus (Reo3) and porcine parvovirus (PPV).

[0058] A method for inactivating viruses in leaflet materials, comprising the following steps: (1) Sample infection: Take 5 portions (1 piece / portion 5 cm × 5 cm) of each indicator virus sample from each batch and place them in a plate. Add the corresponding indicator virus solution and expose the samples by immersion for 1 hour. Then drain and remove the excess indicator virus solution. The infected samples are obtained. (2) Cross-linking fixation-1: Place the remaining contaminated samples into centrifuge tubes containing 0.3% glutaraldehyde solution according to the contaminated type and batch, place them in a refrigerator, set the temperature to 6°C, and leave them for 48 hours. Record the time to obtain the intermediate product; (3) Sample collection: Collect samples at 6h, 12h, 24h, and 48h after the “cross-linking fixation-1” treatment according to the type and batch of infection; drain the inactivation solution immediately after sampling, chop the sample and add it to 10mL of culture medium containing glycine, adjust the pH value of the solution to neutral, shake and extract for 1h, and take the extract for testing; this is used to investigate the inactivation kinetics of the virus at different inactivation times.

[0059] (4) EO treatment: Load the intermediate product into the ethylene oxide sterilizer, close the cabinet door, open the ethylene oxide sterilizer, and perform sterilization according to the procedure in the table below; after the sterilization is completed, perform analysis or shelf storage according to the procedure to obtain the processed product.

[0060] in, Preparation of 0.3% glutaraldehyde solution: Dissolve 20 g of PBS buffer (dry powder) in water for injection to prepare 2 L of PBS buffer solution; Take 12 mL of 50% glutaraldehyde solution, dilute to 2 L with PBS buffer solution, mix well to obtain 0.3% glutaraldehyde solution; filter through 0.22 μm microporous membrane and sand core filter device before use.

[0061] PBS phosphate buffer (dry powder) BL601A was purchased from biosharp, with a pH level of 7.2-7.4 and a concentration of 0.01M.

[0062] 50% glutaraldehyde solution was purchased from Tianjin Damao Chemical Reagent Factory. Intermediate product 1; Intermediate product 2; Intermediate product 3.

[0063] Between the above steps (3) and (4), the intermediate product is processed as follows: S1. Intermediate product contamination: Take 4 portions (1 tablet / portion) of each batch of intermediate product for each indicator virus, place them in a plate, add the corresponding indicator virus solution, and contaminate the samples by immersion for 1 hour. Then drain and remove the excess indicator virus solution. This is the contaminated intermediate product; S2. The remaining intermediate products after infection are packed into packaging boxes and freeze-dried; each batch of each indicator virus-infected intermediate product is divided into 3 groups for standby use; ①Put the first group of intermediates into the ethylene oxide sterilizer and treat them according to the ethylene oxide sterilization conditions: In the first step of pretreatment, the intermediate product is heated and the temperature is controlled to 38°C and the humidity is controlled to 60% RH; In the second step, EO treatment process, the ethylene oxide (EO) gas concentration is controlled at 600 mg / L and the treatment time is 90 min to perform ethylene oxide sterilization treatment.

[0064] The third step is the cleaning and ventilation process. After the sterilization process is completed, the air is cleaned 3 times, ventilated for 5 minutes, and samples are taken for inspection.

[0065] ②Put the second group of intermediates into the ethylene oxide sterilizer and treat them according to the ethylene oxide sterilization conditions: In the first step of pretreatment, the intermediate product is heated and the temperature is controlled to 38°C and the humidity is controlled to 60% RH; In the second step, EO treatment process, the ethylene oxide (EO) gas concentration is controlled at 600 mg / L and the treatment time is 180 min to perform ethylene oxide sterilization treatment.

[0066] The third step is the cleaning and ventilation process. After the sterilization process is completed, the air is cleaned 3 times, ventilated for 5 minutes, and samples are taken for inspection.

[0067] ③Put the third group of intermediates into the ethylene oxide sterilizer and treat them according to the ethylene oxide sterilization conditions: In the first step of pretreatment, the intermediate product is heated and the temperature is controlled to 38°C and the humidity is controlled to 60% RH; In the second step, EO treatment process, the ethylene oxide (EO) gas concentration is controlled at 600 mg / L and the treatment time is 360 min to perform ethylene oxide sterilization treatment.

[0068] The third step is the cleaning and ventilation process. After the sterilization process is completed, the air is cleaned 3 times, ventilated for 5 minutes, and samples are taken for inspection.

[0069] S3. After the sampling intermediates were treated with different concentrations of ethylene oxide, they were placed at room temperature for 24 hours, cut into pieces and extracted with 10 mL of culture medium by oscillation. The extracts were used as intermediate process samples for different treatment times for inspection; they were also used as process samples for sterilization treatment with different concentrations of EO to investigate the changes in the virus inactivation kinetics of the contaminated samples under treatment conditions with different concentrations of EO.

[0070] Step (1) Before the sample is infected, a fixing cloth is immersed in a 0.3% glutaraldehyde solution until the fixing cloth is fully infiltrated, the bovine pericardium slice that has completed the anti-calcification treatment process is thawed, the surface liquid is drained, and the bovine pericardium slice is spread on the fixing cloth, with a layer of cloth, a layer of bovine pericardium slice, and a layer of cloth for standby use; The bovine pericardium slices that have completed the anti-calcification treatment process include the following steps: decellularizing the sample 1, specifically, first adding it to the decellularization treatment solution A, shaking it at 4°C for 4 hours with an oscillation frequency of 120 rpm, pouring out the liquid to retain the pericardium material, and washing it with normal saline three times. The ratio of pericardium material to decellularization treatment solution A is 1:4 (weight: volume), and the decellularization treatment solution A is 0.2g EDTA, 100 micrograms of PMSF, and 0.01M Tris-HCl buffer (pH 8) are mixed; the mixture is then added to decellularization solution B, and the mixture is shaken at 4°C for 4 hours at a frequency of 120 rpm. The pericardial material is removed and washed three times with normal saline. The ratio of the pericardial material prepared in step S2 to the decellularization solution B is 1:4 (weight:volume). The decellularization solution B contains a nonionic surfactant, an anionic surfactant, and decellularization solution A. The nonionic surfactant is Triton X-100 at a concentration of 0.5%, and the anionic surfactant is deoxycholic acid at a concentration of 1%. The bovine pericardium slices that have completed the anti-calcification treatment process are obtained.

[0071] The preparation method of the fixed cloth comprises the following steps: 55 kg of polyethylene terephthalate, 13 kg of polyethylene oxide, 5 kg of modified graphene, 3 kg of nano-titanium dioxide, 4 kg of modified chitosan, and 1 kg of silane coupling agent KH-570 were stirred at 87°C for 1.5 hours to obtain a mixture, which was extruded from a spinneret at 280°C and stretched to obtain polyester fibers. The polyester fibers were laid using a web laying machine and hot pressed at 200°C to obtain a fixed cloth.

[0072] The thickness of the polyester fiber cloth is 0.4mm and the surface density is 100g / m 2 , the average diameter of polyester fiber is 18μm.

[0073] The preparation method of modified graphene includes the following steps: dispersing 6 kg of graphene in 30 L of 60% sulfuric acid solution, stirring for 12 minutes, washing with water, and then dispersing in 100 L of deionized water, adding 4 kg of nano-silica and 2 kg of gelatin, stirring at a temperature of 72° C. for 2 hours, and drying to obtain modified graphene.

[0074] 5 kg of chitosan was dispersed in 20 L of 1% acetic acid solution, 3 kg of cassava starch was added, and the mixture was stirred at 52°C for 1.5 h, dried, and ground to obtain a mixture; The mixture was dispersed in 60 L of deionized water, 3 kg of hydroxyapatite and 1 kg of microcrystalline cellulose were added, and the mixture was stirred at a temperature of 62° C. for 2.5 h, dried, and ground to obtain modified chitosan.

[0075] In addition, indicator virus selection and characteristics (1) Pseudorabies virus (PRV; ATCC VR-135): It is an enveloped DNA virus belonging to the family Herpesviridae. Its virus particles are spherical. It is a virus in the family Herpesviridae that infects a wide range of animals and is highly pathogenic, infecting a variety of animals including pigs, cattle, sheep, dogs, cats, rabbits, and mice. Pseudorabies virus is one of the more resistant herpes viruses and can survive for more than 7 days in various body fluids and on surfaces. However, it is sensitive to organic solvents and can be rapidly inactivated by 1% sodium hydroxide. It loses its activity after 3 minutes at 80°C. Its genome is double-stranded DNA (dsDNA) and is often used as an indicator virus for enveloped DNA viruses, representing this type of virus.

[0076] Virus titer: Before infecting the sample, the virus titer (TCID50) was measured to be greater than 6 logs and stored at -70°C for later use.

[0077] Cells used for detection: pig kidney passage cells (PK-15; ATCC CCL-33), conventionally cultured.

[0078] (2) Vesicular stomatitis virus (VSV; ATCC VR-1238): This is an enveloped RNA virus belonging to the family Rhabdoviridae. Its virions are bullet-shaped or cylindrical and are commonly found in cattle, pigs, horses, deer, and occasionally humans. It can usually be inactivated by exposure to 58°C for 30 minutes, ultraviolet light, or organic solvents, and 1% sodium hydroxide can rapidly inactivate it. Its genome is single-stranded RNA (ssRNA), and it is often used as an indicator virus for enveloped RNA viruses and a representative virus of this type.

[0079] Virus titer: Before infecting the sample, the virus titer (TCID50) was measured to be greater than 6 logs and stored at -70°C for later use.

[0080] Cells used for detection: African green monkey kidney cells (Vero; ATCC CCL-81), routinely cultured.

[0081] (3) Reovirus (type 3) (Respiratory enteric orphan virus, Reo3; ATCC VR-824): It is a non-enveloped RNA virus and a representative virus of the Orthoreovirus genus. It can infect many vertebrates. Reovirus has a promoting effect on certain respiratory and digestive tract diseases in humans. The virus has certain acid and lipid resistance properties and can withstand treatment at 56°C for 60 minutes. It can be quickly inactivated by 1% sodium hydroxide. The genome is double-stranded RNA (dsRNA) and is often used as an indicator virus for non-enveloped RNA viruses and as a representative virus of this type of virus.

[0082] Virus titer: Before infecting the sample, the virus titer (TCID50) was measured to be greater than 6 logs and stored at -70°C for later use.

[0083] Cells used for detection: Baby hamster kidney cells (BHK-21; ATCC CCL-10), conventional culture.

[0084] (4) Porcine parvovirus (PPV; ATCC VR-742): It is a non-enveloped DNA virus belonging to the genus Parvovirus in the family Parvoviridae. Parvovirus has the smallest known viral particles. Parvovirus is very resistant, especially to heat treatment, and can withstand heat treatment at 65°C for 30 minutes. It can be quickly inactivated by 1% sodium hydroxide. Its genome is single-stranded DNA (ssDNA), and it is often used as an indicator virus for non-enveloped DNA viruses and as a representative virus of this type of virus.

[0085] Virus titer: Before infecting the sample, the virus titer (TCID50) was measured to be greater than 6 logs and stored at -70°C for later use.

[0086] Cells used for detection: pig testicular passage cells (ST; ATCC CRL-1746), conventionally cultured.

[0087] Example 2 A method for inactivating viruses in leaflet materials, which differs from Example 1 in that modified graphene is not added in the preparation method of the fixing cloth.

[0088] Example 3 A method for inactivating viruses in leaflet materials is different from Example 1 in that modified chitosan is not added in the preparation method of the fixing cloth.

[0089] Example 4 A method for inactivating viruses in leaflet materials, which differs from Example 1 in that the preparation method of modified graphene does not add nano-silica.

[0090] Example 5 A method for inactivating viruses in leaflet materials, which differs from Example 1 in that gelatin is not added in the preparation method of modified graphene.

[0091] Example 6 A method for inactivating viruses in leaflet materials is different from Example 1 in that hydroxyapatite is not added in the preparation method of the modified chitosan.

[0092] Example 7 A method for inactivating viruses in leaflet materials is different from Example 1 in that the preparation method of modified chitosan does not add microcrystalline cellulose.

[0093] control group Sample control: (1) Sample control: Take one aliquot of each of three samples, chop them into small pieces, and extract them with 10 mL of culture medium under shaking for 1 hour. Collect the extract and sterilize it for testing. This is used to examine the effect of the sample on the presence of cells. The three sample batches are numbered as: Sample 1; Sample 2; Sample 3.

[0094] (2) Sample post-treatment control: Take one sample from each of three batches and treat it according to the process requirements (cross-linking fixation-1). After treatment, remove the sample, chop it, and extract it with 10 mL of culture medium containing glycine for 1 hour. Collect the extract and sterilize it for testing. This is used to investigate the effect of sample treatment on cell presence. (3) Virus (positive) control: This is the indicator virus used for infection, used to check whether the titer value of the indicator virus meets the requirements of the guidelines; (4) Zero-point control: After the sample is infected, it is cut into pieces and extracted with 10 mL of culture medium by oscillation. The extract is used as the zero-point control after infection and is sterilized for inspection. It is used to investigate the infection status of the sample and its effect on the virus.

[0095] Intermediate product control: (1) Intermediate control: Take one portion of each of the three batches of intermediates, chop them into small pieces, and extract them with 10 mL of culture medium for 1 hour through shaking. Collect the extracts and sterilize them for testing. This is used to examine the effects of the samples on cell survival. The intermediates are numbered as follows: Intermediate 1; Intermediate 2; Intermediate 3.

[0096] (2) Control of post-treatment intermediate product: Take one portion of each of three batches of intermediate product and treat it according to the process requirements (EO treatment). After treatment, remove the sample, chop it, and extract it with 10 mL of culture medium for 1 hour through shaking. Collect the extract and sterilize it for testing. This is used to investigate the effect of sample treatment on cell survival. (3) Virus (positive) control: This is the indicator virus used for infection, used to check whether the titer value of the indicator virus meets the requirements of the guidelines; (4) Zero-point control: After the intermediate product is contaminated, it is cut into pieces and extracted with 10 mL of culture medium for 1 hour by shaking. The extract is used as the zero-point control after contamination and sterilized for inspection; it is used to investigate the contamination of the intermediate product and its effect on the virus.

[0097] Performance testing is based on the following standards: "Medical devices of animal origin - Part 3: Confirmation of removal and inactivation of viruses and transmissible spongiform encephalopathy (TSE) agents" Pharmaceutical Industry Standard of the People's Republic of China YY / T 0771.3-2009 / ISO22442-3:2007; "Technical Review Guidelines for Registration of Medical Devices of Animal Origin" (2017 revised edition) No. 224 of 2017 issued by the State Food and Drug Administration.

[0098] The samples of the embodiment and the control group were subjected to performance tests: 1. Cell Interference Experiment and Results 1. Cell Interference Test (1) Take two portions (1 piece / portion) of each batch of samples, cut one portion into pieces and extract with 10 mL of culture medium for 1 hour, sterilize the extract and prepare for inspection as a sample control; treat the other portion according to the process requirements, cut the other portion into pieces and extract with 10 mL of culture medium containing glycine for 1 hour, sterilize the extract and prepare for inspection as a sample control after treatment.

[0099] (2) The cells for detection (PK-15, BHK-21, Vero and ST) were cultured at a rate of 1×10 5 Add 100 μL of cells / mL to columns 1-11 of a 96-well cell culture plate, and add only cell culture medium to column 12. Place the culture plate in a 37°C, 5% CO2 incubator and culture for 12-24 hours. (3) The extracts of each group of samples were diluted in a gradient series with a dilution factor of 10, i.e., 10×, 100×, 1000×; i.e., 10 -1 -10 -3 ; (4) 8 replicate wells were measured for each concentration; a cell control was set up at the same time; 10 -1 -10 -3 Serial gradient dilution sample control, 100 μL per well; normal cell control in the first column, background control in the 12th column; culture in a carbon dioxide incubator for 72 h.

[0100] (5) After culturing for at least 24 h (examine cells under a light microscope and record changes in general morphology, vacuolation, cell shedding, and membrane integrity), discard all culture medium from the 96-well plate. Add 100 μL of serum-free culture medium containing 10% CCK-8 to the 96-well plate and incubate for another 8 h.

[0101] (6) All test plates were placed in an enzyme reader for detection, with the main wavelength being λ = 450 (nm).

[0102] (7) Evaluation criteria Qualitative evaluation: Based on microscopic examination and records, if there are discrete particles in the cytoplasm, no cell lysis, and no decrease in cell proliferation (level 0); if no more than 20% of the cells are rounded, loosely attached to the wall, have no cytoplasmic particles or show morphological changes; occasional cell lysis; only slight cell growth inhibition is observed (level 1), it is judged to be no cell interference effect. If no more than 50% of the cells are rounded, have no cytoplasmic particles, and no large-scale cell lysis; no more than 50% cell growth inhibition can be observed (level 2). No more than 70% of the cell layer contains rounded cells or lysed cells; the cell layer is not completely destroyed, but more than 50% cell growth inhibition can be observed (level 3). The cell layer is almost completely detached or destroyed (level 4).

[0103] Quantitative evaluation: Level 0: RGR (cell proliferation coefficient) ≥ 100, Level 1: RGR is 75-99, Level 2: RGR is 50-74, Level 3: RGR is 25-49, Level 4: RGR is 1-24, Level 5: RGR is 0; Levels 0-1 are qualified; Level 2 should be combined with cell morphology analysis and comprehensive evaluation; Levels 3-5 are unqualified.

[0104] 2. Cell Interference Test Results After testing, the results of the cell interference test of the sample before "cross-linking fixation-1" treatment and the sample extract after "cross-linking fixation-1" treatment and neutralization are shown in Table 2-1 and Table 3-1.

[0105] Table 2-1 Statistics of cell interference levels of sample extracts before “cross-linking fixation-1” treatment Table 3-1 Statistics of cell interference levels of sample extracts after “cross-linking fixation-1” treatment The qualitative evaluation results showed that the cell interference level of the extracts of the samples before and after "cross-linking fixation-1" treatment on four cell types (PK-15, BHK-21, Vero and ST) was 0, and the samples did not interfere with the experimental results.

[0106] The quantitative evaluation results showed that the cell interference level of the extracts of the samples before and after "cross-linking fixation-1" treatment on four cell types (PK-15, BHK-21, Vero and ST) was level 1, and the samples did not interfere with the experimental results.

[0107] After testing, the results of the cell interference test of the extracts of the samples before and after EO treatment are shown in Table 2-2 and Table 3-2.

[0108] Table 2-2 Statistics of cell interference levels of sample extracts before “EO sterilization” treatment Table 3-2 Statistics of cell interference levels of sample extracts after “EO sterilization” treatment The qualitative evaluation results showed that the cell interference level of the extracts of the samples before and after "EO" treatment on four cell types (PK-15, BHK-21, Vero and ST) was 0, and the samples did not interfere with the experimental results.

[0109] The quantitative evaluation results showed that the cell interference level of the extracts of the samples before and after "EO" treatment on four cell types (PK-15, BHK-21, Vero and ST) was level 1, and the samples did not interfere with the experimental results.

[0110] 2. Virus Titer Detection Method and Result Analysis 1. Virus titer detection uses cytopathic effect assay (CPE): (1) The cells for detection were cultured at a rate of 1×10 5 The cells were added to a 96-well cell culture plate at a density of cells / mL, with 100 μL per well; the culture plate was placed in a 37°C, 5% CO2 incubator for 24 h; (2) Each control sample and the process sample were diluted in a gradient series, with the dilution factor being 10 times, i.e.: 10 -1 -10 -10 ; (3) Add 10 -1 -10 -10 Serial gradient dilutions of control samples and process-treated samples, 100 μL per well; columns 1 and 12 are normal cell controls; (4) The culture plate was cultured in a 37°C, 5% CO2 incubator, and the cell pathological changes were observed and the number of pathological cell wells was recorded until the cell pathological changes were complete.

[0111] TCID was calculated using the Karber method based on the number of wells with cytopathic effect. 50The Karber method directly gives the lower limit of detection, which is 0.5logs. The calculation formula is as follows: lgTCID 50 =xk-d[(1 / n)(r)-0.5] xk = logarithm of the maximum dilution r = total number of infected wells d = difference in logarithms of dilution n = replicate wells per dilution 2. Results Analysis (1) Calculation method of virus reduction value: Based on the titer detection values ​​before and after virus inactivation / removal, the virus reduction value of the inactivation / removal process step is calculated according to the following formula.

[0112] Virus titer reduction value = zero-point control virus titer - virus titer after process treatment.

[0113] (2) Calculation method of virus reduction coefficient: Based on the virus titer detection value and the sample volume before and after virus inactivation / removal, the virus reduction coefficient of each inactivation / removal process step is calculated according to the following formula.

[0114] Reduction coefficient R = log 10 [(V1×T1) / (V2×T2)]; Where V1 is the volume of the material before the process step begins; V2 is the volume of the material after the process steps are completed; T1 is the indicator virus titer (TCID 50 ); T2 is the indicator virus titer (TCID 50 ).

[0115] (3) In this validation experiment, the bovine pericardium slices (cross-linked fixation-1) were sheet-like tissues before and after treatment. The volume before “cross-linked fixation-1” treatment (V1) and the volume after “cross-linked fixation-1” treatment (V2) did not change substantially, and the extraction was an equal volume treatment, so the volume change V1 / V2≈1, that is, the virus reduction coefficient (4) Result determination: According to the requirements of the guidelines, the total reduction factor of virus removal / inactivation in the production process of medical devices should reach 6.00 logs or more, and in principle, at least one virus removal / inactivation step should have a reduction factor of 4.00 logs or more.

[0116] If the virus reduction factor after treatment meets the requirements of the guidelines, it can be determined that the treatment is effective in inactivating / removing the virus.

[0117] (5) Based on the residual virus titer in each sample, the virus inactivation kinetics curve of the indicator virus at different sampling points during the treatment process was drawn.

[0118] The samples were tested for pseudorabies virus (PRV), vesicular stomatitis virus (VSV), reovirus (Reo3) and porcine parvovirus (PPV): (1) Detection results of residual indicator virus PRV in samples The "cross-linking fixation-1" process was used to treat the contaminated samples. The inactivation effect on PRV is shown in Table 4-1 and Figure 1-3 .

[0119] Table 4-1 Results of pseudorabies virus detection in samples after “cross-linking fixation-1” treatment The test results in Table 4-1 show that the cell morphology and growth status in the sample control and the sample-treated control are normal, indicating that the sample has no effect on the tested cells, that is, the sample itself will not affect the virus detection results.

[0120] From Table 4-1 and Figure 1-3 It can be seen that no PRV was detected in the three batches of contaminated samples after 6 hours of treatment with "cross-linking fixation-1" and at the subsequent sampling points; after this treatment, the viral coefficient (titer) of PRV was reduced by ≥5.175logs, ≥4.925logs and ≥5.235logs respectively, and the average viral coefficient (titer) of the indicator virus PRV in the three batches of contaminated samples after treatment was reduced by ≥5.112log.

[0121] (2) Detection results of residual indicator virus VSV in samples The inactivation effect of VSV by the “cross-linking fixation-1” process on the contaminated samples is shown in Table 5-1 and Figure 4-6 .

[0122] Table 5-1 Detection results of vesicular stomatitis virus in samples after “cross-linking fixation-1” treatment The test results in Table 5-1 show that the cell morphology and growth status in the sample control and the sample-treated control are normal, indicating that the sample has no effect on the tested cells, that is, the sample itself will not affect the virus detection results.

[0123] From Table 5-1 and Figure 4-6It can be seen that no VSV was detected in the three batches of contaminated samples after 6 hours of treatment with "cross-linking fixation-1" and at the subsequent sampling points; after this treatment, the viral coefficient (titer) of VSV was reduced by ≥5.020logs, ≥5.475logs and ≥5.325logs respectively, and the average viral coefficient (titer) of the indicator virus VSV in the three batches of contaminated samples after treatment was reduced by ≥5.273log.

[0124] (3) Detection results of residual indicator virus Reo3 in samples The contaminated samples were treated with the "cross-linking fixation-1" process. The inactivation effect on Reo3 is shown in Table 6-1 and Figure 7-9 .

[0125] Table 6-1 Reovirus (type 3) detection results in samples after “cross-linking fixation-1” treatment The test results in Table 6-1 show that the cell morphology and growth status in the sample control and the sample-treated control are normal, indicating that the sample has no effect on the tested cells, that is, the sample itself will not affect the virus detection results.

[0126] From Table 6-1 and Figure 7-9 It can be seen that no Reo3 was detected in the three batches of contaminated samples after 6 hours of treatment with "cross-linking fixation-1" and at the subsequent sampling points; after this treatment, the viral coefficient (titer) of Reo3 was reduced by ≥5.175logs, ≥4.400logs and ≥5.235logs respectively, and the average viral coefficient (titer) of the indicator virus Reo3 in the three batches of contaminated samples after treatment was reduced by ≥4.937log.

[0127] (4) Detection results of residual indicator virus PPV in samples The "cross-linking fixation-1" process was used to treat the contaminated samples. The inactivation effect on PPV is shown in Table 7-1 and Figure 10-12 .

[0128] Table 7-1 Results of porcine parvovirus detection in samples after “cross-linking fixation-1” treatment The test results in Table 7-1 show that the cell morphology and growth status in the sample control and the sample-treated control are normal, indicating that the sample has no effect on the test cells, that is, the sample itself will not affect the virus detection results.

[0129] It can be seen from Table 7-1 and Figures a10-a12 that no PPV was detected at any sampling point after the three batches of contaminated samples were treated with "cross-linking fixation-1" for 6 hours or later; after this treatment, the viral coefficient (titer) of PPV was reduced by ≥4.825logs, ≥4.475logs and ≥5.235logs respectively. The average viral coefficient (titer) of the indicator virus PPV of the three batches of contaminated samples after treatment was reduced by ≥4.845log.

[0130] Analysis of results: The test results showed that PRV, VSV, Reo3 and PPV were not detected in the three batches of contaminated samples after 6 hours of treatment with "cross-linking fixation-1", and this treatment method can reduce the coefficients (titers) of PRV, VSV, Reo3 and PPV by more than 4 logs; indicating that under this treatment condition, the "cross-linking fixation-1" treatment process of artificial biological heart valves can effectively inactivate / remove the above-mentioned indicator viruses and the related viruses they represent.

[0131] The corresponding intermediate products are tested for pseudorabies virus (PRV), vesicular stomatitis virus (VSV), reovirus (Reo3) and porcine parvovirus (PPV): (1) Detection results of residual indicator virus PRV in samples The EO treatment process was used to treat the infected samples. The inactivation effect of PRV is shown in Table 4 and Figure 13-15 .

[0132] Table 4-2 Results of pseudorabies virus detection in samples after “EO” treatment The test results in Table 4-2 show that the cell morphology and growth status in the sample control and the treated sample control are normal, indicating that the sample has no effect on the test cells, that is, the sample itself will not affect the virus detection results.

[0133] From Table 4-2 and Figure 13-15 It can be seen that no PRV was detected in the three batches of contaminated samples after treatment with "EO" for 90 minutes or longer; the viral coefficient (titer) of PRV in the three batches of samples after this treatment was reduced by ≥5.235logs, ≥4.475logs and ≥4.600logs respectively, and the average viral coefficient (titer) of the indicator virus PRV in the three batches of contaminated samples after "EO" treatment was reduced by ≥4.770log.

[0134] (2) Detection results of residual indicator virus VSV in samples The “EO” treatment process was used to treat the contaminated samples. The inactivation effect on VSV is shown in Table 5-2 and Figure 16-18 .

[0135] Table 5-2 Detection results of vesicular stomatitis virus in samples after “EO” treatment The test results in Table 5-2 show that the cell morphology and growth status in the sample control and the treated sample control are normal, indicating that the sample has no effect on the tested cells, that is, the sample itself will not affect the virus detection results.

[0136] From Table 5-2 and Figure 16-18 It can be seen that no VSV was detected in the three batches of contaminated samples after being treated with "EO" for 90 minutes or longer; the viral coefficient (titer) of VSV in the three batches of samples after this treatment was reduced by ≥5.475logs, ≥5.200logs and ≥5.050logs respectively, and the average viral coefficient (titer) of the indicator virus VSV in the three batches of contaminated samples after being treated with "EO" was reduced by ≥5.242log.

[0137] (3) Detection results of residual indicator virus Reo3 in samples The "EO" treatment process was used to treat the contaminated samples. The inactivation effect on Reo3 is shown in Table 6-2 and Figure 19-21 .

[0138] Table 6-2 Detection results of reovirus (type 3) in samples after “EO” treatment The test results in Table 6-2 show that the cell morphology and growth status in the sample control and the treated sample control are normal, indicating that the sample has no effect on the test cells, that is, the sample itself will not affect the virus detection results.

[0139] From Table 6-2 and Figure 19-21 It can be seen that no Reo3 was detected in the three batches of contaminated samples after treatment with "EO" for 90 minutes or longer; after this treatment, the viral coefficient (titer) of Reo3 in the three batches of samples was reduced by ≥5.235logs, ≥4.700logs and ≥4.475logs respectively; the average viral coefficient (titer) of the indicator virus Reo3 in the three batches of contaminated samples after "EO" treatment was reduced by ≥4.803log.

[0140] (4) Detection results of residual indicator virus PPV in samples The "EO" treatment process was used to treat the contaminated samples. The inactivation effect on PPV is shown in Table 7-2 and Figure 22-24 .

[0141] Table 7-2 Results of porcine parvovirus detection in samples after “EO” treatment The test results in Table 7-2 show that the cell morphology and growth status in the sample control and the treated sample control are normal, indicating that the sample has no effect on the test cells, that is, the sample itself will not affect the virus detection results.

[0142] From Table 7-2 and Figure 22-24 It can be seen that PPV was detected in the three batches of contaminated samples after being treated with "EO" for 90 minutes or longer. After this treatment, the viral coefficient (titer) of PPV in the three batches of samples was reduced by ≥4.925logs, ≥4.925logs and ≥4.700logs respectively. The average viral coefficient (titer) of the indicator virus PPV in the three batches of contaminated samples after being treated with "EO" was reduced by ≥4.850log.

[0143] Analysis of results: The test results showed that after 90 minutes of "EO" treatment, no PRV, VSV, Reo3 and PPV were detected in the three batches of contaminated samples. This process can reduce the coefficient (titer) of the four viruses (PRV, VSV, Reo3, PPV) by more than 4 logs; this indicates that under this treatment condition, the "EO" treatment process of artificial biological heart valves can effectively inactivate / remove the above-mentioned indicator viruses and the related viruses they represent.

[0144] In summary, the test results show that after the contaminated samples were treated with "cross-linking and fixation-1", no PRV, VSV, Reo3 and PPV were detected, and the coefficients (titers) of the four viruses (PRV, VSV, Reo3, PPV) were reduced by more than 4 logs; after the contaminated samples were treated with "EO", no PRV, VSV, Reo3 and PPV were detected, and the coefficients (titers) of the four viruses (PRV, VSV, Reo3, PPV) were reduced by more than 4 logs; the two processes can make the sum of the reduction coefficients of the four viruses greater than 6 logs, which meets the requirements of the total virus reduction coefficient; it shows that the "cross-linking and fixation-1" treatment process and the "EO" treatment process of artificial biological heart valves can effectively inactivate the above-mentioned indicator viruses and the related viruses they represent (as shown in the table below).

[0145] A universal material testing machine was used to cut samples according to ASTM D638 and perform tensile tests at a tensile speed of 50 mm / min; samples were cut according to ASTM D790 and perform bending tests.

[0146] Table 8 Example data test project Tensile strength / MPa Bending strength / MPa Example 1 96 135 Example 2 82 120 Example 3 85 123 Example 4 87 126 Example 5 90 130 Example 6 89 128 Example 7 92 132 As shown in Table 8, the fixing cloth prepared in Example 1 by the present application has a tensile strength of 96 MPa and a flexural strength of 135 MPa, indicating that the fixing cloth prepared by the present application has excellent mechanical properties. The various components interact with each other to improve the mechanical strength of the fixing cloth, prevent the bovine pericardoplasty ring from collapsing or deforming, and facilitate subsequent cross-linking fixation and EO treatment.

[0147] The preparation methods for the fixing cloths in Examples 2 and 3, respectively, did not include modified graphene or modified chitosan. As shown in Table 8, the tensile strength and flexural strength test results for Examples 2 and 3 were significantly inferior to those for Example 1, indicating that the modified graphene and modified chitosan significantly improved the mechanical properties of the fibers, and thus the fixing cloth, facilitating subsequent processing of the bovine pericardium.

[0148] In the preparation methods of the modified graphene in Examples 4 and 5, no nano-silica or gelatin was added. As shown in Table 8, the tensile strength and flexural strength test results of Examples 4 and 5 were significantly worse than those of Example 1, but better than those of Example 2. This indicates that gelatin forms hydrogen bonds with the graphene's oxygen-containing groups via amino and carboxyl groups, coating the graphene surface. This allows the nano-silica to bind tightly to the graphene, increasing the toughness of the modified graphene and subsequently improving the mechanical strength of the fixed fabric.

[0149] The modified chitosans of Examples 6 and 7 were prepared without the addition of hydroxyapatite or microcrystalline cellulose. As shown in Table 8, the tensile strength and flexural strength test results of Examples 6 and 7 were significantly worse than those of Example 1, but better than those of Example 4. This indicates that the inorganic crystalline structure of hydroxyapatite fills the pores of the chitosan-starch matrix, improving the mechanical and compressive strength of the composite material. The microcrystalline cellulose cross-links with chitosan via hydrogen bonds, further stabilizing the bond between chitosan and hydroxyapatite and increasing the tensile strength of the chitosan, thereby improving the mechanical properties of the fixed fabric.

[0150] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for inactivating viruses in leaflet materials, characterized in that: The steps include: (1) Immerse the fixing cloth in glutaraldehyde solution until the fixing cloth is fully soaked, thaw the bovine pericardium slice that has completed the anti-calcification treatment process, drain the liquid on the surface, and then use the fixing cloth to spread the bovine pericardium slice, one layer of cloth, one layer of bovine pericardium slice, and one layer of cloth, and set aside; (2) Cross-linking fixation-1: Place the bovine pericardium slices treated in step (1) into a box, pour in glutaraldehyde solution, and allow the glutaraldehyde solution to fully infiltrate the bovine pericardium slices so that the height of the glutaraldehyde solution is level with the height of the topmost fixing cloth. Refrigerate at a storage temperature of 2-8°C for 48 hours to obtain an intermediate product; (3) EO treatment: The intermediate product treated in step (2) is sterilized with ethylene oxide, and subjected to analysis or shelf storage to obtain a treated product.

2. The method for inactivating viruses in leaflet materials according to claim 1, characterized in that: In step (1), the preparation method of the fixed cloth comprises the following steps: stirring 50-60 parts of polyethylene terephthalate, 10-15 parts of polyethylene oxide, 5-6 parts of modified graphene, 2-4 parts of nano-titanium dioxide, 4-5 parts of modified chitosan, and 1-2 parts of a silane coupling agent at a temperature of 85-90°C for 1-2 hours to obtain a mixture, extruding and stretching to obtain polyester fibers, laying the polyester fibers, and hot pressing at 190-200°C to obtain a fixed cloth.

3. The method for inactivating viruses in leaflet materials according to claim 2, characterized in that: The modified graphene preparation method comprises the following steps: dispersing graphene in sulfuric acid, stirring for 10-15 minutes, washing with water, and then dispersing in deionized water, adding nano-silica and gelatin, stirring at a temperature of 70-75°C for 1-2 hours, and drying to obtain the modified graphene.

4. The method for inactivating viruses in leaflet materials according to claim 2, characterized in that: The preparation method of the modified chitosan comprises the following steps: dispersing chitosan in an acetic acid solution, adding cassava starch, stirring at a temperature of 50-55° C. for 1-2 hours, drying, and grinding to obtain a mixture; The mixture is dispersed in deionized water, hydroxyapatite and microcrystalline cellulose are added, stirred at a temperature of 60-65° C. for 2-3 hours, dried, and ground to obtain modified chitosan.

5. The method for inactivating viruses in leaflet materials according to claim 1, characterized in that: Preparation of glutaraldehyde solution: Dissolve PBS buffer with a pH of 7.2-7.4 in water for injection to prepare 2 L of PBS buffer solution; Take 12 mL of 50% glutaraldehyde solution, dilute to 2 L with PBS buffer solution, and mix well to obtain glutaraldehyde solution.

6. The method for inactivating viruses in leaflet materials according to claim 1, characterized in that: In step (2), the bovine pericardium piece and the fixing cloth are kept fixed during the cross-linking fixation-1 treatment.

7. The method for inactivating viruses in leaflet materials according to claim 1, characterized in that: Treatment conditions for ethylene oxide sterilization: temperature 35-41°C, humidity 40-80%RH.

8. The method for inactivating viruses in leaflet materials according to claim 1, characterized in that: The ethylene oxide (EO) gas concentration is 600-900 mg / L, and the treatment time is 90-360 min.

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