Pericardial dry film and method of making same

Abstract

The application discloses a preparation method of a dry pericardium film. The preparation method comprises the steps of pretreatment, crosslinking, post-treatment and drying of pericardium material, wherein the post-treatment comprises the step of softener soaking. The application also comprises the post-treatment of the pericardium material, the post-treatment step comprising the softener soaking. The softener soaking can reduce film collapse in the drying process, reduce the adhesion between fibers, increase the inter-fiber space and enhance the fiber activity, thereby improving the toughness of the dry film made of the pericardium material.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical materials and medical devices, in particular to a dry pericardial membrane and a preparation method thereof. BACKGROUND

[0002] Cardiovascular disease is the leading cause of death, among which, heart valve disease is a significant cause of morbidity and mortality, which is caused by a variety of diseases, such as rheumatic fever and congenital defects. Heart valve disease can occur in any one valve or a combination of four valves.

[0003] The treatment of heart valve disease includes open chest valve replacement surgery and percutaneous heart valve replacement surgery. Heart valve replacement is a surgery that replaces a mechanical valve made of synthetic material or a biological valve made of biological tissue, simply called valve replacement. The existing artificial biological valve is generally cross-linked with glutaraldehyde, and then sewn on a metal stent, and then stored in a glutaraldehyde solution. Before the interventional valve replacement surgery, it needs to be cleaned, compressed and assembled several times. This preparation process is complicated and can easily lead to an increase in additional risks of surgery. The biological valve stored in the glutaraldehyde solution for a long time is prone to residual glutaraldehyde, which increases the calcification and toxicity of the biological valve. Moreover, the preservation of glutaraldehyde requires cleaning before use on site, and after cleaning, compression and loading into the delivery system on site, which increases the time of the surgery. Therefore, developing a dry membrane of the biological valve that is free from glutaraldehyde solution and pre-compressed and loaded into the valve delivery system can better solve the above problems. However, the dry biological valve pre-loaded into the valve delivery system is prone to compression damage due to the long time of mechanical compression, so the toughness of the biological valve in the dry loaded state is required to be higher, and the toughness of the existing artificial biological valve is still insufficient. SUMMARY

[0004] The present application aims to provide a dry pericardial membrane and a preparation method thereof to solve the technical problem of insufficient toughness of the artificial biological valve in the prior art.

[0005] In order to achieve the above-mentioned purpose, according to one aspect of the present application, a preparation method of a dry pericardial membrane is provided. The preparation method comprises the steps of pretreatment, cross-linking, post-treatment and drying of the pericardial material, wherein the post-treatment comprises the step of soaking with a softener.

[0006] Further, the softener comprises glycerol; preferably, the softener further comprises one or more selected from the group consisting of PEG, histidine, glycine, lysine, sodium hyaluronate, mannitol, sorbitol, stearic acid, glyceryl stearate, polyglycerol, wherein the molecular weight M w ≤ 1500 D.

[0007] Further, the glycerol is 8% to 30% by mass of the softening agent, and one or more selected from the group consisting of PEG, histidine, glycine, lysine, sodium hyaluronate, mannitol, sorbitol, stearic acid, glycerol stearate, and polyglycerol is 0.5% to 10% by mass of the softening agent; preferably, the solvent of the softening agent is one or a mixture of two or more of water, ethanol, and isopropyl alcohol; preferably, the soaking time of the softening agent is 2 to 72 hours.

[0008] Further, the post-treatment step further comprises a protein soaking step, which is performed before or simultaneously with the softening agent soaking.

[0009] Further, the protein used in the protein soaking step is one or more selected from the group consisting of gelatin, collagen, elastin, and glycoprotein; preferably, the mass percentage concentration of the protein in the protein solution is 0.1% to 5%; preferably, the protein soaking time is 2 to 72 hours.

[0010] Further, the pre-treatment step comprises one or more selected from the group consisting of acid soaking, alkali soaking, or acid and alkali soaking without sequence requirement, decellularization, and protein soaking; preferably, the acid or alkali used in the acid soaking or alkali soaking is one or more selected from hydrochloric acid, acetic acid, oxalic acid, citric acid, sodium hydroxide, and sodium carbonate, and more preferably, the concentration of the acid or alkali is 2 to 10 mM, and the acid or alkali soaking time is 5 to 120 minutes; preferably, the decellularization solution used in the decellularization is a mixed solution of sodium dodecyl sulfate and triton X-100, the concentration of sodium dodecyl sulfate in the decellularization solution is 0.25% to 2%, the concentration of triton X-100 is 0.25% to 2%, and the decellularization time is 24 to 72 hours; preferably, the protein used in the protein soaking step is one or more selected from the group consisting of gelatin, collagen, elastin, and glycoprotein; preferably, the mass percentage concentration of the protein in the protein solution is 0.1% to 5%; preferably, the protein soaking time is 2 to 72 hours.

[0011] Further, the cross-linking is glutaraldehyde cross-linking, the concentration of the used glutaraldehyde is 0.2% to 1%, the cross-linking time is 1 to 5 days, and the cross-linking temperature is room temperature.

[0012] Further, the temperature in the drying step is room temperature, the drying time is 2 to 72 hours, and then air drying or vacuum drying is performed for 1 to 5 days; preferably, the temperature of the air drying or vacuum drying is 30 to 50°C.

[0013] Further, the pericardial material is porcine pericardium or bovine pericardium.

[0014] According to another aspect of the present application, a dry pericardial membrane is provided. The dry pericardial membrane is prepared by the method described above.

[0015] According to the technical solution of the present application, the post-treatment of the pericardial material is also included. The post-treatment step includes a softener soaking. The softener soaking can reduce the membrane collapse during the drying process, reduce the adhesion between fibers, increase the inter-fiber space, and enhance the fiber mobility, thereby improving the toughness of the dry membrane made of the pericardial material. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 A comparison of the dry pericardial membrane prepared in Example 1 before (left) and after (right) kneading;

[0017] Figure 2 A comparison of the dry pericardial membrane prepared in Comparative Example 1 before (left) and after (right) kneading. DETAILED DESCRIPTION

[0018] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments.

[0019] According to a typical embodiment of the present application, a method for preparing a dry pericardial membrane is provided. The method includes a pre-treatment, a cross-linking, a post-treatment, and a drying of the pericardial material. The post-treatment includes a softener soaking.

[0020] According to the technical solution of the present application, the post-treatment of the pericardial material is also included. The post-treatment step includes a softener soaking. The softener soaking can reduce the membrane collapse during the drying process, reduce the adhesion between fibers, increase the inter-fiber space, and enhance the fiber mobility, thereby improving the toughness of the dry membrane made of the pericardial material.

[0021] According to a typical embodiment of the present application, the softener includes glycerol. Preferably, the softener also includes one or more selected from the group consisting of PEG (molecular weight M w ≤1500D), histidine, glycine, lysine, sodium hyaluronate, mannitol, sorbitol, stearic acid, glyceryl stearate, and polyglycerol. The glycerol can play a moisturizing role, and the other components can prevent the membrane from collapsing and reduce the adhesion between fibers.

[0022] Preferably, the glycerol is present in the softening agent in a mass percentage of 8% to 30%, and one or more of the following is present in the softening agent in a mass percentage of 0.5% to 10%: PEG, histidine, glycine, lysine, sodium hyaluronate, mannitol, sorbitol, stearic acid, glycerol stearate, and polyglycerol. In this concentration range, the water content of the pericardial membrane is maintained, and the water absorption of the membrane is not too strong, so that the membrane is not too wet. In addition, the toughness of the membrane is improved, effectively preventing the membrane from becoming hard and brittle.

[0023] Preferably, the solvent in the softening agent is one or a mixture of two or more of water, ethanol, and isopropyl alcohol. In this way, the softening agent has a strong affinity for the membrane and easily penetrates into the pericardial membrane, distributing more evenly in the membrane. More preferably, the softening agent is soaked for 2 to 72 hours.

[0024] According to a typical embodiment of the present application, the post-treatment step further comprises a protein soaking step, which can be performed before or simultaneously with the softening agent soaking. The protein used in the protein soaking step is one or more selected from the group consisting of gelatin, collagen, elastin, and glycoprotein. Preferably, the mass percentage of the protein in the protein solution is 0.1% to 5%. Preferably, the protein soaking time is 2 to 72 hours. Protein soaking can improve the elasticity and toughness of the pericardial membrane.

[0025] That is, in the present application, the post-treatment step can have only one step of softening agent soaking, or two steps of softening agent soaking and protein soaking.

[0026] According to a typical embodiment of the present application, the pre-treatment step comprises one or more selected from the group consisting of acid soaking, alkali soaking, or acid-alkali soaking without sequence requirement (acid-alkali soaking without sequence requirement means that acid soaking can be performed first, followed by alkali soaking, or alkali soaking can be performed first, followed by acid soaking), decellularization, and protein soaking. Preferably, the acid or alkali used in the acid soaking or alkali soaking is one or more selected from the group consisting of hydrochloric acid, acetic acid, oxalic acid, citric acid, sodium hydroxide, and sodium carbonate (as understood by those skilled in the art, the acid and the alkali cannot be mixed at the same time). More preferably, the concentration of the acid or alkali is 2 to 10 mM, and the acid or alkali soaking time is 5 to 120 minutes. Preferably, the decellularization solution used in the decellularization is a mixture of sodium dodecyl sulfate and triton X-100, the concentration of sodium dodecyl sulfate in the decellularization solution is 0.25% to 2%, the concentration of triton X-100 is 0.25% to 2%, and the decellularization time is 24 to 72 hours. This step can remove the cells and oil on the pericardial membrane.

[0027] In order to further optimize the preparation method, the protein used in the protein soaking step in the pretreatment step is one or more selected from the group consisting of gelatin, collagen, elastin and glycoprotein; preferably, the mass percentage concentration of the protein in the protein solution is 0.1% to 5%; preferably, the protein soaking time is 2 to 72 hours. The protein soaking can improve the elasticity and toughness of the pericardial membrane.

[0028] According to a typical embodiment of the present application, the cross-linking is glutaraldehyde cross-linking, the concentration of the glutaraldehyde used is 0.2% to 1%, the cross-linking time is 1 to 5 days, and the cross-linking temperature is room temperature.

[0029] According to a typical embodiment of the present application, the temperature in the drying step is room temperature, the drying time is 2 to 72 hours, and then air drying or vacuum drying is performed for 1 to 5 days; preferably, the temperature for the air drying or vacuum drying is 30 to 50°C.

[0030] According to a typical embodiment of the present application, the pericardial material is pig pericardium or bovine pericardium.

[0031] According to a typical embodiment of the present application, a pericardial dry membrane is provided. The pericardial dry membrane is prepared by using any of the above-mentioned pericardial dry membrane preparation methods. The pericardial dry membrane not only has improved toughness, but also has improved elasticity and flatness. The beneficial effects of the present application will be further illustrated in the following examples.

[0032] Example 1

[0033] 2 mM sodium hydroxide was used to soak the fresh pericardial membrane for 5 minutes, and then the pericardial membrane was washed with purified water. Then, the pericardial membrane was soaked in 0.1% sodium dodecyl sulfate and 0.1% triton X-100 for 2 hours, and then soaked in 0.1% collagen for 0.5 hours. The pericardial membrane was cross-linked in a 0.05% glutaraldehyde solution for 6 hours at 0°C, and then washed with purified water. The pericardial membrane was soaked in 0.5% collagen for 0.5 hours and then soaked in 5% glycerol for 0.5 hours. Then, the pericardial membrane was dried at room temperature for 4 hours, and then air dried at 30°C for 1 week. The obtained pericardial dry membrane is shown in the left part of Figure 1 Figure 1. After being rubbed, the pericardial dry membrane is shown in the right part of Figure 1 Figure 2. As can be seen from Figure 1 , the pericardial dry membrane prepared by the method of the present application is relatively flat, and can maintain a flat state even after being rubbed.

[0034] Example 2

[0035] 10 mM acetic acid soak fresh pericardium for 120 min, washed with purified water, then soaked with 5% sodium dodecyl sulfate and 5% triton X-100 for 72 h, cross-linked in 0.05% glutaraldehyde solution for 72 h, cross-linking temperature is 37°C, washed with purified water. 10% collagen soak for 1 week, 50% glycerol and 0.1% glyceryl stearate mixture soak for 3 weeks. Then dry at room temperature for 4 h, 40°C air dry for 4 h, the appearance of the pericardium dry film and the appearance after rubbing are similar to Example 1.

[0036] Example 3

[0037] 5 mM sodium hydroxide soak fresh pericardium for 30 min, washed with purified water, then 5 mM acetic acid soak fresh pericardium for 30 min, washed with purified water, 1% sodium dodecyl sulfate and 1% triton X-100 soak for 48 h, 0.5% collagen soak for 1 week. Cross-linked in 0.625% glutaraldehyde solution for 72 h, cross-linking temperature is 25°C, washed with purified water. 10% glycerol and 5% PEG soak for 48 h. Then dry at room temperature for 24 h, 50°C air dry for 72 h, the appearance of the pericardium dry film and the appearance after rubbing are similar to Example 1.

[0038] Example 4

[0039] 0.5% sodium dodecyl sulfate soak fresh pericardium for 48 h, cross-linked in 0.25% glutaraldehyde solution for 72 h, cross-linking temperature is 0°C, washed with purified water. 2% collagen soak for 12 h, 10% glycerol and 30% glycine mixture soak for 72 h. Then dry at room temperature for 48 h, 40°C air dry for 72 h, the appearance of the pericardium dry film and the appearance after rubbing are similar to Example 1.

[0040] Example 5

[0041] 1% sodium dodecyl sulfate and 1% triton X-100 soak for 48 h, 1% collagen soak for 24 h. Cross-linked in 0.25% glutaraldehyde solution for 72 h, cross-linking temperature is 25°C, washed with purified water. 8% glycerol, 10% PEG, 2% sorbitol soak for 48 h. Then dry at room temperature for 12 h, 40°C air dry for 72 h, the appearance of the pericardium dry film and the appearance after rubbing are similar to Example 1.

[0042] Example 6

[0043] 2 mM acetic acid soak fresh pericardium for 30 min, rinse with purified water, then soak fresh pericardium with 10 mM sodium hydroxide for 30 min, rinse with purified water. Soak with 5% sodium dodecyl sulfate and 5% triton X-100 for 2 h, soak with 5% collagen for 24 h. Crosslink in 1% glutaraldehyde solution for 72 h, crosslinking temperature is 37 °C, rinse with purified water. Soak with 8% glycerol, 10% PEG, 5% polyglycerol for 72 h. Then dry at room temperature for 24 h, dry at 40 °C for 1 week, the appearance of the dry pericardium film and the appearance after rubbing are similar to Example 1.

[0044] Example 7

[0045] 6 mM sodium hydroxide soak fresh pericardium for 5 min, rinse with purified water, then soak with 1% sodium dodecyl sulfate and 1% triton X-100 for 12 h, soak with 0.1% collagen for 0.5 h. Crosslink in 0.05% glutaraldehyde solution for 6 h, crosslinking temperature is 0 °C, rinse with purified water. Soak with 1% collagen and 15% glycerol for 5 h. Then dry at room temperature for 4 h, dry at 30 °C for 1 week, the appearance of the dry pericardium film and the appearance after rubbing are similar to Example 1.

[0046] Comparative Example 1

[0047] 2 mM sodium hydroxide soak fresh pericardium for 5 min, rinse with purified water, then soak with 0.1% sodium dodecyl sulfate and 0.1% triton X-100 for 2 h. Crosslink in 0.05% glutaraldehyde solution for 12 h, crosslinking temperature is 0 °C, rinse with purified water. Then dry at room temperature for 4 h, dry at 30 °C for 1 week, the dry pericardium film obtained is as shown in the left image of Figure 2 . After rubbing, the dry pericardium film is as shown in the right image of Figure 2 . It can be seen that the flatness of the dry pericardium film is poor, and the flatness decreases after rubbing. Figure 2

[0048] The tensile testing machine was used to detect the breaking strength of the film strips obtained in each example and comparative example, and the results are recorded in Table 1 as follows:

[0049] Table 1: Breaking strength of dry films obtained in each example and dry films obtained by crosslinking treatment only

[0050]

[0051]

[0052] As can be seen from Table 1, the dry film samples obtained by the steps in each example have a large degree of improvement in breaking force without affecting the elongation, and the thickness of the dry film is smaller, indicating that the sample has better toughness and is more convenient for the pericardium to complete delivery in a compressed state.​

[0053] From the above description, it can be seen that the above-mentioned embodiments of the present application achieve the following technical effects:

[0054] The preparation method is used for improving the toughness of the dry film prepared from the pericardium material after removing cells and oil on the pericardium material by acid and alkali soaking, a mixed solution of sodium dodecyl sulfate and triton X-100. As can be seen from the above two comparison charts, the pericardium material processing technology disclosed in the patent improves the dry film state, especially the toughness.

[0055] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a pericardial dry membrane, characterized in that, The process includes pretreatment, cross-linking, post-treatment, and drying steps for pericardial materials, wherein the post-treatment includes a fabric softener soaking step; The pretreatment includes sequential acid soaking, alkaline soaking, decellularization and protein soaking, or the pretreatment includes sequential alkaline soaking, acid soaking, decellularization and protein soaking. The acid or alkali used in the acid or alkali soaking is one or more selected from hydrochloric acid, acetic acid, oxalic acid, citric acid, sodium hydroxide and sodium carbonate, the concentration of the acid or alkali is 2~10mM, and the soaking time is 5~120min. The protein used in the protein soaking step of the pretreatment step is one or more selected from the group consisting of gelatin, collagen, elastin and glycoprotein; the mass percentage concentration of the protein in the protein solution is 0.1%~5%; and the protein soaking time is 2~72h. The fabric softener includes glycerin, and further includes one or more selected from the group consisting of PEG, histidine, glycine, lysine, sodium hyaluronate, mannitol, sorbitol, stearic acid, glyceryl stearate, and polyglycerol, wherein the molecular weight M of the PEG is... w ≤1500 Da; The glycerin accounts for 8% to 30% of the mass percentage of the fabric softener, and one or more of the ingredients selected from the group consisting of PEG, histidine, glycine, lysine, sodium hyaluronate, mannitol, sorbitol, stearic acid, glyceryl stearate, and polyglycerol account for 0.5% to 10% of the mass percentage of the fabric softener. The softener is soaked for 2 to 72 hours.

2. The preparation method according to claim 1, characterized in that, The solvent in the fabric softener is a mixture of one or more of water, ethanol, and isopropanol.

3. The preparation method according to claim 1 or 2, characterized in that, The post-processing step further includes a protein soaking step, which is performed before or simultaneously with the fabric softener soaking.

4. The preparation method according to claim 3, characterized in that, The protein used in the protein soaking step of the post-processing step is one or more selected from the group consisting of gelatin, collagen, elastin and glycoprotein; the mass percentage concentration of the protein in the protein solution is 0.1% to 5%; and the protein soaking time is 2 to 72 hours.

5. The preparation method according to claim 1, characterized in that, The decellularization solution used is a mixture of sodium dodecyl sulfate and Triton X-100, with the concentration of sodium dodecyl sulfate ranging from 0.25% to 2% and the concentration of Triton X-100 ranging from 0.25% to 2%. The decellularization time is 24 to 72 hours.

6. The preparation method according to claim 1, characterized in that, The crosslinking is glutaraldehyde crosslinking, with a glutaraldehyde concentration of 0.2% to 1%, a crosslinking time of 1 to 5 days, and a crosslinking temperature of room temperature.

7. The preparation method according to claim 1, characterized in that, The drying step is performed at room temperature for 2 to 72 hours, followed by forced-air drying or vacuum drying for 1 to 5 days; the forced-air drying or vacuum drying temperature is 30 to 50°C.

8. The preparation method according to claim 1, characterized in that, The pericardial material is either pig pericardium or cow pericardium.

9. A pericardial dry membrane, characterized in that, It is prepared by the method of any one of claims 1 to 8.

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