Biological dry film, use thereof, and preparation method therefor

Abstract

A biological dry film, use thereof, and a preparation method therefor. The residual amount of aldehyde in the biological dry film is ˂ 3 mg / g. The biological dry film has good biological safety, biocompatibility, anti-calcification capacity, and effectiveness; meanwhile, the biological dry film can rapidly rehydrate. Moreover, when the biological dry film is used in combination with a degradable material, the degradable material can be prevented from being corroded or degraded due to absorbing moisture in the dry film during the instrument storage process. When the biological dry film is applied to a heart valve, even if stored for a long time, the biological dry film does not absorb moisture, so that the degradable material in the heart valve can be prevented from being corroded or degraded, thereby ensuring the stability, safety, and effectiveness of the performance of the heart valve during the storage process.

Description

Biological dry film and its application and preparation method Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to a biological dry film and an application and preparation method thereof. Background Art

[0002] Valvular heart disease is a common heart disease, with valve damage caused by rheumatic fever being the most common. With the aging population, senile valvular disease, as well as valvular lesions caused by coronary heart disease and myocardial infarction, are becoming increasingly common. Valvular heart disease refers to single-valve or multi-valve lesions caused by rheumatic fever, myxomatous degeneration, degenerative changes, congenital malformations, ischemic necrosis, infection, or trauma, affecting normal blood flow, leading to abnormal heart function and ultimately heart failure.

[0003] Treatments for heart valve disease include open-chest valve replacement surgery and percutaneous heart valve replacement surgery. Open-chest surgery is highly invasive, carries high risks, slows recovery, and requires extracorporeal circulation, making it unacceptable to many patients. Percutaneous heart valve replacement surgery, with its minimal invasiveness and low risks, is becoming the dominant approach for future valve surgery.

[0004] Biological heart valves refer to a type of biomedical material used to replace diseased heart valves in the human body. Percutaneous replacement biological heart valves consist of a valve frame and a valve. The valve is cross-linked with glutaraldehyde, cut, and then sewn onto the valve frame. It is then sterilized and preserved with glutaraldehyde solution. During implantation, the delivery system needs to be assembled and cleaned on site, which prolongs the operation time and increases the risk to the patient. Glutaraldehyde residue will remain in the valve, leading to short-term toxicity and long-term calcification risk. In addition, long-term immersion of the heart valve in glutaraldehyde solution can easily lead to degradation or corrosion of the valve frame, which is unstable in the liquid.

[0005] Prior art has developed bioprosthetic valves into dry films that can be preserved without glutaraldehyde solutions. For example, CN103933612A discloses a surgically implanted biological tissue. This treatment method involves contacting the biological tissue with a non-aqueous treatment solution containing a polyol and a C1-C3 alcohol, and removing a portion of the treatment solution from the treated tissue. This treatment method can maintain the tissue in a substantially dry state. However, simple alcohol dehydration treatment methods cannot effectively control the residual water content and residual organic reagent content in the tissue. CN109172867B discloses a rapidly rehydrating dry bioprosthetic heart valve, but the introduction of hydrophilic molecules on its surface increases the safety risk of the bioprosthetic heart valve after implantation. CN112220971B also discloses a rapidly rehydrating dry bioprosthetic heart valve, but the introduction of multiple organic substances such as hydrophilic monomers, hydrogel monomers, and initiators during its preparation process also increases the safety risk of the bioprosthetic heart valve after implantation. Summary of the Invention

[0006] In order to overcome the defects and shortcomings of the above-mentioned prior art, the present invention provides a biological dry film and its application and preparation method, which has good biosafety, biocompatibility, anti-calcification ability and effectiveness, and can be quickly rehydrated and has high softness after rehydration.

[0007] The technical solution of the present invention provides a biological dry film, wherein the residual amount of aldehydes in the biological dry film is less than 3 mg / g.

[0008] In some embodiments, the residual amount of aldehydes in the bio-dry film is less than 0.1 mg / g; in other embodiments, the residual amount of aldehydes in the bio-dry film is less than 10 μg / g.

[0009] In some embodiments, the moisture content of the bio-dry film is ≤15 wt.%; in other embodiments, the moisture content of the bio-dry film is <10 wt.%; in still other embodiments, the moisture content of the bio-dry film is ≤5 wt.%.

[0010] In some embodiments, the total content of organic solvents in the bio-dry film is ≤0.5 wt.%.

[0011] In some embodiments, the weight loss rate T of the bio-dry film before and after preparation is ≥40%; in other embodiments, the weight loss rate T of the bio-dry film before and after preparation is ≥50%; in still other embodiments, the weight loss rate T of the bio-dry film before and after preparation is ≥58%.

[0012] In some embodiments, the volume V of the biofilm after preparation is 后 ≤Volume before preparation V 前 90%;

[0013] In some embodiments, the length / width / thickness of the prepared biological dry film is ≤ 90% of the length / width / thickness before preparation.

[0014] In some embodiments, the color of the biological dry film is beige, white, wool or ivory.

[0015] In some embodiments, the bio-dry film can be rehydrated in physiological saline for ≤5 minutes.

[0016] In some embodiments, the softness of the bio-dry film after rehydration in physiological saline is ≥ 90%, and / or the length / width / thickness of the bio-dry film after rehydration in physiological saline is ≥ 95% of the length / width / thickness before rehydration.

[0017] In some embodiments, the precursor of the biological dry film includes at least one of the pericardium, heart valve, blood vessel, ligament, muscle, intestine, skin, peritoneum, pleura, Achilles tendon or venous valved conduit of an animal; further, the precursor of the biological dry film includes at least one of the pericardium, heart valve, blood vessel, ligament, muscle, intestine or skin, peritoneum, pleura, Achilles tendon or venous valved conduit of a pig, cow, horse, donkey, mule or sheep.

[0018] The present invention also provides an application of the biological dry film on at least one of a biological patch, an artificial blood vessel, an artificial pipeline and a heart valve.

[0019] In some embodiments, the heart valve includes a valve frame.

[0020] The present invention further provides a method for preparing the biodry film, comprising subjecting the biodry film precursor to supercritical drying treatment.

[0021] The present invention, through supercritical drying of a biodry film precursor, produces a biodry film with excellent biosafety, biocompatibility, anti-calcification ability, and effectiveness, while also being able to rapidly rehydrate. When used in conjunction with a degradable material, the biodry film prevents the degradable material from absorbing moisture from the dry film during device storage, which can lead to corrosion and degradation of the degradable material. When applied to heart valves, the biodry film can prevent corrosion or degradation of the degradable material within the heart valve, thereby ensuring the stability, safety, and effectiveness of the heart valve during storage. The biodry film produced by this method has an aldehyde residue content of less than 3 mg / g, resulting in excellent safety, biocompatibility, and anti-calcification ability after implantation in the human body. Furthermore, the biodry film has a low water content and, when used in conjunction with a degradable material, does not absorb water even after prolonged storage, thus preventing the degradable material from absorbing moisture from the dry film during device storage, which can lead to corrosion and degradation of the degradable material. This method eliminates the need for additional organic reagents, reducing safety risks associated with biofilm implantation. Upon rehydration, the biofilm returns to its original size and quickly softens after implantation, resulting in excellent conformability and enhanced effectiveness following subsequent implantation. The resulting biofilm is significantly smaller than before dehydration, facilitating pre-installation in smaller delivery systems, allowing for smaller blood vessels and improved implant compatibility. Furthermore, upon implantation into a diseased area, the biofilm returns to its original size in physiological solutions, without compromising its usability or adding any additional risks.

[0022] In some embodiments, the temperature of the supercritical drying is 31.2°C to 69°C, and the pressure is 7.38Mpa to 30Mpa; further, the temperature of the supercritical drying is 31.2°C to 40°C, and the pressure is 7.38Mpa to 10Mpa; further, the temperature of the supercritical drying is 35°C to 40°C, and the pressure is 8Mpa to 10Mpa.

[0023] In some embodiments, the supercritical drying time is 0.2 h to 24 h, and the number of cycles is 1 to 99 times.

[0024] In some embodiments, the supercritical drying time is 0.5 h to 5 h, and the number of cycles is 2 to 20 times.

[0025] In some embodiments, the supercritical drying medium includes at least one of carbon dioxide, methane, ethane, and propane.

[0026] In some embodiments, the biodry film precursor further comprises a cross-linking treatment step before supercritical drying.

[0027] In some embodiments, the crosslinking agent used in the crosslinking treatment includes an organic aldehyde.

[0028] In some embodiments, the bio-dry film precursor further comprises at least one dehydration step before the supercritical drying treatment.

[0029] In some embodiments, the dehydrating agent used in the dehydration treatment is an organic alcohol.

[0030] The present invention also provides a method for preparing the biological dry film, comprising the following steps:

[0031] cross-linking the bio-dry film precursor;

[0032] Cutting the biofilm precursor;

[0033] subjecting the biofilm precursor to at least one alcohol dehydration treatment;

[0034] The bio-dry film precursor is subjected to supercritical drying treatment;

[0035] The biofilm precursor is sterilized. DETAILED DESCRIPTION

[0036] The following descriptions are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. For example, the examples illustrate biological dry films, but this does not necessarily imply that the present invention is solely applicable to biological dry films. It should be noted that variations and improvements based on this inventive concept, as readily apparent to those skilled in the art, fall within the scope of the present invention. Reagents and instruments used without manufacturer identification are commercially available conventional products.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the art of the present invention. The terms used in the specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0038] The present invention provides a biodry film, particularly a biodry film for heart valves, wherein the residual amount of aldehydes in the biodry film is less than 3 mg / g; further, the residual amount of aldehydes in the biodry film is less than 0.1 mg / g; further, the residual amount of aldehydes in the biodry film is less than 10 μg / g; further, the residual amount of aldehydes in the biodry film is less than 5 μg / g; further, the residual amount of aldehydes in the biodry film is less than 1 μg / g; further, the residual amount of aldehydes in the biodry film is less than 0.05 μg / g; further, the residual amount of aldehydes in the biodry film is below the detection limit. Glutaraldehyde is a highly active hazardous substance. When glutaraldehyde reacts with hemoglobin, it undergoes a condensation reaction with amino acid residues in hemoglobin (especially lysine and histidine) to form glutaraldehyde-hemoglobin adducts. This adduct causes hemoglobin to polymerize, thereby forming hemoglobin aggregates. These aggregates alter the structure and function of hemoglobin, thereby affecting the binding and release of oxygen. The polymerization of hemoglobin will cause its deposition and accumulation in red blood cells, thereby affecting the morphology and function of red blood cells. Red blood cells with polymerized hemoglobin become more fragile and unstable, easily ruptured and dissolved, leading to the occurrence of hemolysis. In addition, polymerized hemoglobin will also cause red blood cell deformation, increase its adhesion in blood vessels, thereby affecting the flow and oxygen supply capacity of blood. The phenomenon of glutaraldehyde polymerization of hemoglobin is particularly prominent in some diseases, such as diabetes, kidney disease and chronic inflammation. In these diseases, the generation of glutaraldehyde increases, resulting in a significant increase in the polymerization of hemoglobin. This further aggravates the development and progression of the disease and may lead to a series of serious complications. In the present invention, by controlling the residual amount of aldehydes in the biological dry film, the biological dry film has better safety, biocompatibility and anti-calcification ability after being implanted in the human body.

[0039] In some embodiments, the moisture content of the bio-dry film is ≤15 wt.%; further, the moisture content of the bio-dry film is <10 wt.%; further, the moisture content of the bio-dry film is ≤5 wt.%; further, the moisture content of the bio-dry film is ≤2.5 wt.%; further, the moisture content of the bio-dry film is ≤1 wt.%; further, the moisture content of the bio-dry film is ≤0.5 wt.%; further, the moisture content of the bio-dry film is less than 0.2 wt.%, further, the moisture content of the bio-dry film is less than 0.5 wt.%. The water content of the biodry membrane is less than 0.15 wt %. Furthermore, the water content of the biodry membrane is less than 0.1 wt %. Furthermore, the water content of the biodry membrane is less than 0.08 wt %. Furthermore, the water content of the biodry membrane is less than 0.05 wt %. Furthermore, the water content of the biodry membrane is less than 0.04 wt %. Furthermore, the water content of the biodry membrane is less than 0.03 wt %. Furthermore, the water content of the biodry membrane is less than 0.02 wt %. Furthermore, the water content of the biodry membrane is less than 0.01 wt %. Extensive experiments have shown that excessively high water content causes the biodry membrane to become hard, making it impossible to rehydrate and restore its original size. This means that the biodry membrane cannot quickly become soft after implantation in the human body, resulting in poor conformability of the biodry membrane, which further affects its effectiveness after subsequent implantation in the human body. By controlling the moisture content within a certain range, the present invention can achieve rapid rehydration of the biological dry film, thereby improving the effectiveness of the biological dry film. When used in combination with a degradable material, it prevents the degradable material from absorbing moisture in the dry film during the storage of the device, causing corrosion or degradation of the degradable material. When applied to heart valves, it ensures the stability, safety and effectiveness of the heart valve performance during the preservation process.

[0040] It should be noted that the water in the biological dry film of the present invention refers to free water, which means water that can be removed using alcohol.

[0041] In some embodiments of the present invention, the total content of organic solvents in the bio-dry film is ≤0.5 wt.%; further, the total content of organic solvents in the bio-dry film is not greater than 0.25 wt.%; further, the total content of organic solvents in the bio-dry film is not greater than 0.2 wt.%; further, the total content of organic solvents in the bio-dry film is not greater than 0.15 wt.%; further, the total content of organic solvents in the bio-dry film is not greater than 0.1 wt.%; further, the total content of organic solvents in the bio-dry film is not greater than 0.08 wt.% Furthermore, the total content of organic solvents in the biodry film is no more than 0.06 wt%; further, the total content of organic solvents in the biodry film is no more than 0.05 wt%; further, the total content of organic solvents in the biodry film is no more than 0.04 wt%; further, the total content of organic solvents in the biodry film is no more than 0.03 wt%; further, the total content of organic solvents in the biodry film is no more than 0.02 wt%; further, the residual amount of organic matter in the biodry film is no more than 0.01 wt%. Excessive residual organic matter in the biodry film, on the one hand, makes the biodry film susceptible to water absorption after long-term storage. When used in combination with degradable materials, it can accelerate the corrosion or degradation of the degradable materials when applied to heart valves, leading to valve frame fracture during valve expansion, affecting the stability, safety, and effectiveness of heart valve performance. On the other hand, excessive residual organic matter poses short-term toxicity and long-term calcification risks, affecting the safety, biocompatibility, and calcification capacity of the biodry film after implantation into the human body. By controlling the amount of residual organic matter within an appropriate range, a biological dry film with good biosafety, biocompatibility, anti-calcification ability and effectiveness can be obtained, while also being able to quickly rehydrate. When used in combination with degradable materials, when applied to heart valves, it can prevent the degradable materials from absorbing moisture from the dry valve during the storage of the device, causing the degradable materials to corrode and degrade. For example, when the biological dry film is applied to a degradable valve frame to make a degradable heart valve, it can effectively prevent the corrosion or degradation of the valve frame, thereby ensuring the stability, safety and effectiveness of the performance of the heart valve during the storage process.

[0042] It should be noted that the organic solvents remaining in the biological dry film of the present invention include but are not limited to alcohols, such as isopropyl alcohol, ethanol, glycerol, and the like.

[0043] In some embodiments of the present invention, the bio-dry film has a weight loss rate T of ≥40% before and after preparation; a weight loss rate T of ≥50% before and after preparation; and a weight loss rate T of ≥58% before and after preparation. In other embodiments, the bio-dry film has a weight loss rate T of 40% ≤ ≤ 90% before and after preparation. The weight loss rate of the biodry film before and after preparation is controlled within an appropriate range, and the solvent content, moisture content, residual organic solvent content, and residual aldehyde content of the biodry film are controlled within an extremely low range. The volume of the biodry film of the present invention is reduced compared to that before dehydration, which facilitates pre-installation in a delivery system with a smaller diameter, is suitable for use in smaller blood vessels, and has better implant compatibility. The biodry film also has good biosafety, biocompatibility, anti-calcification ability, and effectiveness, while being able to rehydrate quickly. When used in combination with a degradable material, the biodry film prevents the degradable material from absorbing moisture from the dry valve during device storage, thereby preventing corrosion and degradation of the degradable material. When used in heart valves, the biodry film can prevent corrosion or degradation of the degradable material of the heart valve, thereby ensuring the stability, safety, and effectiveness of the performance of the heart valve during storage.

[0044] In some embodiments of the present invention, the volume V of the prepared biological dry film is 后 Less than or equal to the volume before preparation V 前 In other embodiments, the volume of the biofilm after preparation is 50% V 前 ≤V 后 ≤72.9%V before.

[0045] In some embodiments of the present invention, the length / width / thickness of the biodry film after preparation is less than or equal to 90% of the length / width / thickness before preparation; in other embodiments, the length / width / thickness of the biodry film after preparation is less than or equal to 89% of the length / width / thickness before preparation; in other embodiments, the length / width / thickness of the biodry film after preparation is less than or equal to 88% of the length / width / thickness before preparation; in other embodiments, the length / width / thickness of the biodry film after preparation is less than or equal to 87% of the length / width / thickness before preparation; in other embodiments, the length / width / thickness of the biodry film after preparation is less than or equal to 85% of the length / width / thickness before preparation; in other embodiments, the length / width / thickness of the biodry film after preparation is less than or equal to 84% of the length / width / thickness before preparation.

[0046] In some embodiments of the present invention, the length, width, and thickness of the bio-dry film after preparation are less than or equal to 90% of their pre-preparation length, width, and thickness, and greater than or equal to 80% of their pre-preparation length, width, and thickness. The finished bio-dry film of the present invention is significantly smaller in volume than before dehydration, facilitating pre-installation in smaller diameter delivery systems, compatibility with smaller blood vessels, and improved implant compatibility. Furthermore, upon implantation in a diseased area, the valve returns to its original size in a physiological solution, without compromising its usability or adding any additional risks.

[0047] It should be noted that the aforementioned parameters such as weight loss rate, volume, length / width / thickness of the bio-dry film before preparation refer to the values ​​corresponding to the corresponding indicators before the bio-dry film undergoes the dehydration step. The prepared bio-dry film refers to the bio-dry film after the sterilization step. It is understood that "before dehydration" refers to the bio-dry film before the dehydration step. For example, if the bio-dry film has undergone cross-linking, alcohol dehydration, or supercritical dehydration, the pre-prepared bio-dry film refers to the bio-dry film after cross-linking and alcohol dehydration. If the cross-linking and alcohol dehydration also involve a modification step, the bio-dry film refers to the bio-dry film after modification and before alcohol dehydration.

[0048] It can be understood that in some embodiments, the length of the biological dry film after preparation is less than or equal to 90% of the length before preparation; in some embodiments, the width of the biological dry film after preparation is less than or equal to 90% of the width before preparation; in some embodiments, the thickness of the biological dry film after preparation is less than or equal to 90% of the thickness before preparation; in some embodiments, the length and width of the biological dry film after preparation are respectively less than or equal to 90% of the length and width before preparation; in some embodiments, the length and thickness of the biological dry film after preparation are respectively less than or equal to 90% of the length and thickness before preparation; in some embodiments, the width and thickness of the biological dry film after preparation are respectively less than or equal to 90% of the width and thickness before preparation; in some embodiments, the length, width, and thickness of the biological dry film after preparation are respectively less than or equal to 90% of the length, width, and thickness before preparation.

[0049] In some embodiments, the rehydration time of the bio-dry film in normal saline is ≤5 minutes; in other embodiments, the rehydration time of the bio-dry film in normal saline is ≤4 minutes; in other embodiments, the rehydration time of the bio-dry film in normal saline is ≤3 minutes; in other embodiments, the rehydration time of the bio-dry film in normal saline is ≤2 minutes; in some embodiments, the rehydration time of the bio-dry film in normal saline is ≤0.5 minutes ≤5 minutes.

[0050] In some embodiments, the softness of the bio-dry film after rehydration in physiological saline is ≥90%; in other embodiments, the softness of the bio-dry film after rehydration in physiological saline is ≥92%; in other embodiments, the softness of the bio-dry film after rehydration in physiological saline is ≥93%; in some embodiments, the softness of the bio-dry film after rehydration in physiological saline is ≥94%; in other embodiments, the softness of the bio-dry film after rehydration in physiological saline is ≥95%; in other embodiments, the softness of the bio-dry film after rehydration in physiological saline is ≥97%; in some embodiments, the softness of the bio-dry film after rehydration in physiological saline is ≥98%; in other embodiments, the softness of the bio-dry film after rehydration in physiological saline is ≥99%; in other embodiments, the softness of the bio-dry film after rehydration in physiological saline is 100%.

[0051] In some embodiments, the length / width / thickness of the bio-dry film after rehydration in physiological saline is ≥ 95% of the length / width / thickness before rehydration. In other embodiments, the length / width / thickness of the bio-dry film after rehydration in physiological saline is ≥ 96% of the length / width / thickness before rehydration; in other embodiments, the length / width / thickness of the bio-dry film after rehydration in physiological saline is ≥ 97% of the length / width / thickness before rehydration; in other embodiments, the length / width / thickness of the bio-dry film after rehydration in physiological saline is ≥ 98% of the length / width / thickness before rehydration. As described above, the length / width / thickness of the bio-dry film of the present invention after rehydration in physiological saline refers to at least one of the length, width, and thickness of the bio-dry film after rehydration in physiological saline.

[0052] The biological dry film of the present invention can restore its original size after being rehydrated and can quickly become soft after being implanted in the body. The biological dry film has good conformability, thereby improving its effectiveness after subsequent implantation in the human body.

[0053] In some embodiments of the present invention, the color of the biological dry film is beige, white, wool or ivory.

[0054] In some embodiments of the present invention, the precursor of the biofilm comprises at least one of the following: pericardium, heart valves, blood vessels, ligaments, muscles, intestines, skin, peritoneum, pleura, Achilles tendon, or venous valved conduits from an animal. Furthermore, the precursor of the biofilm comprises at least one of the following: pericardium, heart valves, blood vessels, ligaments, muscles, intestines, skin, peritoneum, pleura, Achilles tendon, or venous valved conduits from pigs, cows, horses, donkeys, mules, or sheep. Furthermore, the precursor of the biofilm can be at least one of porcine pericardium, bovine pericardium, sheep pericardium, equine pericardium, donkey pericardium, or mule pericardium. The biofilm precursor herein specifically refers to the raw material of the biofilm.

[0055] The biological dry film provided by the present invention can be applied to biological patches, artificial blood vessels, artificial pipes, and heart valves.

[0056] In some embodiments of the present invention, the heart valve further includes a valve frame. Furthermore, the heart valve further includes a degradable valve frame; wherein the material of the degradable valve frame can be a degradable metal material or an absorbable polymer material. For example, in some embodiments, the degradable valve frame is a pure iron valve frame, an iron-based alloy valve frame, a pure magnesium valve frame, a magnesium-based alloy valve frame, a pure zinc valve frame, a zinc-based alloy valve frame or a degradable polylactic acid valve frame. The biological dry film of the present invention is suitable for both permanent valve frames and degradable valve frames; further, the biological dry film provided by the present invention is particularly suitable for degradable valve frames to make degradable heart valves, which can ensure that the heart valve is not easy to absorb water during long-term storage, thereby effectively preventing the corrosion or degradation of the degradable valve frame, thereby improving the effectiveness of the heart valve.

[0057] The present invention also provides a method for preparing the above-mentioned biological dry film, which includes supercritical drying treatment of the biological dry film precursor. The present invention can obtain a biological dry film that has good biosafety, biocompatibility, anti-calcification ability and effectiveness, and can be quickly rehydrated by supercritical drying the biological dry film precursor. When the biological dry film is used in combination with a degradable material, it prevents the degradable material from absorbing moisture in the dry valve during the storage of the device, causing the degradable material to corrode and degrade. For example, when the biological dry film is applied to a degradable valve frame to make a degradable heart valve, it can fully prevent the corrosion or degradation of the degradable valve frame, thereby ensuring the stability, safety and effectiveness of the performance of the heart valve during the storage process. The "biological dry film precursor" here refers to the raw material or semi-finished product before the biological dry film is supercritically dried.

[0058] Furthermore, the organic solvent content of the biodry film prepared by this method is no more than 0.5 wt.%, and when used in combination with a degradable material, the biodry film prevents the biodry material from absorbing moisture from the dry valve during device storage, causing the biodry material to corrode and degrade. When applied to heart valves, this prevents corrosion or degradation of the biodry material, thereby ensuring the stability, safety, and effectiveness of the heart valve during storage. This method does not require the introduction of additional organic reagents and can control the organic content of the finished biodry film within an extremely low range, reducing the safety risks of the biodry film after implantation. After rehydration, the biodry film can return to its original size, meaning it quickly becomes soft after implantation, and the biodry film has good conformability, improving its effectiveness after subsequent implantation into the human body. The volume of the finished biodry film prepared by this method of the present invention is reduced compared to the volume before dehydration, which facilitates pre-installation in smaller diameter delivery systems, compatibility with smaller blood vessels, and better implant compatibility. At the same time, when implanted in the lesion area, the valve will return to its original size in physiological solution, without affecting its use or adding additional risks.

[0059] In some embodiments of the present invention, the supercritical drying temperature is 31.2° C. to 69° C., and further, the supercritical drying temperature is 31.2° C. to 40° C., 33° C. to 40° C., 33.5° C. to 40° C., 34° C. to 40° C., 34.5° C. to 40° C., 35° C. to 40° C., 36° C. to 40° C., 37° C. to 40° C., 38° C. to 40° C., 39° C. to 40° C., 35° C. to 50° C., or 35° C. to 60° C. In specific examples, the critical point drying temperature may be 31.2° C., 32° C., 32.5° C., 33° C., 33.5° C., 34° C., 34.5° C., 35° C., 35.5° C., 36° C., 36.5° C., 37° C., 37.2° C., 38° C., 39° C., 40° C., 45° C., 50° C., or 60° C.

[0060] In some embodiments of the present invention, the critical point drying pressure is 7.38 MPa to 30 MPa. Further, the critical point drying pressure can be 7.38 MPa to 10 MPa, 7.5 MPa to 10 MPa, 8 MPa to 10 MPa, 8.6 MPa to 9.7 MPa, 9 MPa to 10 MPa, 7.5 MPa to 15 MPa, 7.5 MPa to 20 MPa, or 7.5 MPa to 30 MPa. In specific examples, the critical point drying pressure can be 7.38 MPa, 7.5 MPa, 7.6 MPa, 8 MPa, 8.6 MPa, 9 MPa, 9.3 MPa, 9.7 MPa, 10 MPa, 15 MPa, 20 MPa, 25 MPa, or 30 MPa. By keeping the temperature and pressure of supercritical drying within this range, organic residues and moisture in the biofilm can be effectively removed, and the organic solvent content, moisture content, and residual aldehyde content in the biofilm can be effectively controlled to extremely low levels. The biodry film prepared by the present invention will not absorb water even after long-term storage, which can prevent the degradation or corrosion of the degradable valve frame during storage. This method does not require the introduction of other organic reagents, and can control the organic content of the biodry film, reducing the safety risk of the biodry film after implantation in the body. After the biodry film is rehydrated, it can restore its original size, that is, it can quickly become soft after implantation in the body. The biodry film has good conformability, which improves its effectiveness after subsequent implantation in the human body. The volume of the prepared biodry film is reduced, which is conducive to pre-installation in a smaller diameter delivery system, suitable for smaller blood vessels, and better implantation matching. At the same time, when implanted in the diseased area, the valve will restore its original size in the physiological solution, which will not affect its use or increase additional risks.

[0061] In some embodiments of the present invention, the supercritical drying time is 0.5 h to 24 h. Further, the supercritical drying time is 1 h to 2 h, 1 h to 5 h, 1 h to 10 h, 2 h to 10 h, or 3 h to 15 h. In specific examples, the supercritical drying time can be 0.5 h, 1 h, 1.5 h, 2 h, 5 h, 10 h, 15 h, 20 h, or 24 h.

[0062] In some embodiments of the present invention, the number of supercritical drying cycles is 1 to 99. Furthermore, the number of supercritical drying cycles is 5 to 20. Extensive experimental verification has demonstrated that the moisture content of the biofilm can be effectively controlled to no more than 15 wt.%, the residual organic solvent content to no more than 0.5 wt.%, and the residual aldehyde content in the biofilm to be less than 3 mg / g. The preparation method provided by the present invention is simple to operate, requires minimal production steps, and is easily practicable and industrially applicable.

[0063] In some embodiments of the present invention, the supercritical drying medium includes at least one of carbon dioxide, methane, ethane and propane. Specifically, the present invention uses easily liquefied gases such as carbon dioxide as supercritical drying media. Generally, water is not a suitable fluid for treating bioactive tissue materials because water has a very high critical point (374°C, 3200psi). Heating biological materials at such high temperatures will destroy their biological activity. Therefore, the present invention provides supercritical drying of biological valve materials using easily liquefied gases such as carbon dioxide (hereinafter referred to as supercritical fluids). The supercritical fluid used in the present invention has a relatively low critical point, is relatively easy to reach and is compatible with biological materials, and is an excellent non-polar solvent that can dissolve lipids, oils and fats in the material. Above the critical point, supercritical fluids such as carbon dioxide can basically penetrate into the entire material to remove components such as alcohol and lipids.

[0064] In some embodiments of the present invention, the bio-dry film precursor is further subjected to a cross-linking step prior to supercritical drying. Cross-linking the bio-dry film precursor can improve biological tissue stability, reduce immunogenicity, and enhance mechanical properties and durability. In a specific example, animal pericardium is cross-linked.

[0065] In some embodiments of the present invention, the crosslinking agent used in the crosslinking treatment includes an organic aldehyde, which can be an organic aldehyde capable of crosslinking amine groups of proteins in biological tissues. In specific examples, the crosslinking agent includes at least one of glutaraldehyde, formaldehyde, glyceraldehyde, paraformaldehyde, and dialdehyde starch.

[0066] In some embodiments of the present invention, the volume concentration of the crosslinking agent is 0.5-5%. Specifically, the volume concentration of the crosslinking agent can be 0.5%, 1%, 1.2%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 5%. When the volume concentration of the crosslinking agent is controlled within an appropriate range, stable crosslinking of most collagen tissues can be achieved, thereby improving the structural stability of the biofilm precursor and reducing or even eliminating immunogenicity.

[0067] The pH of the cross-linking agent is 7 to 8. Specifically, the pH may be 7, 7.2, 7.6, 7.8 or 8.

[0068] The cross-linking temperature is 20-45°C; specifically, the cross-linking temperature can be 20°C, 22°C, 25°C, 30°C, 35°C, 38°C, 40°C, or 45°C.

[0069] The crosslinking time is 1 d to 7 d; specifically, the crosslinking time can be 1 d, 2 d, 3 d, 4 d, 5 d, 6 d or 7 d.

[0070] In some embodiments, the biofilm precursor is cross-linked in a 0.5-5% volume concentration glutaraldehyde aqueous solution at a pH of 7-8 and 20-45° C. for 1-7 days.

[0071] In some embodiments of the present invention, the biodry film precursor is decellularized before cross-linking. Conventional methods can be used for decellularization, or commercially available decellularized biodry film precursor materials can be used, which is not a limitation of the present invention.

[0072] In some embodiments of the present invention, the biofilm precursor is cross-linked and then cut into leaflets. Conventional methods are used, and the present invention is not limited thereto. For example, the biofilm precursor can be cut using a die-cutter or laser machine. Specifically, a VLS4.75-SYS laser etching system from UNIVERSAL can be used to cut porcine pericardium into leaflets at 3.5% energy, 1.5% speed, and a 0.05 mm spot size.

[0073] In some embodiments of the present invention, the biofilm precursor further undergoes at least one dehydration treatment prior to supercritical drying. This dehydration treatment preliminarily removes most of the water, glutaraldehyde, and alcohol (e.g., isopropyl alcohol) and other reagents, thereby increasing the biofilm's flexibility. Combined with supercritical drying, this treatment further removes the remaining water, glutaraldehyde, and alcohol (e.g., isopropyl alcohol) and other reagents, resulting in a total organic solvent content of no more than 0.5wt%, a water content of less than 15wt%, and a residual amount of aldehydes in the biofilm of less than 3mg / g. This method yields a biofilm with good biosafety, biocompatibility, anti-calcification ability, and effectiveness, while also enabling rapid rehydration. Furthermore, when used in conjunction with a degradable material, such as in a heart valve, the biofilm can prevent corrosion or degradation of the degradable material, thereby ensuring the stability, safety, and effectiveness of the heart valve's performance. Moreover, the biodry film prepared by this method is smaller in size, which is conducive to pre-installation in a smaller diameter delivery system, suitable for smaller blood vessels, and better implant compatibility. At the same time, when implanted into the diseased area, the biodry film will return to its original size in the physiological solution, which will not affect its use or increase additional risks.

[0074] In some embodiments of the present invention, the bio-dry film precursor is subjected to at least one dehydration treatment after the cross-linking treatment and before the supercritical drying treatment.

[0075] In some embodiments of the present invention, dehydration is performed using an alcohol solution dehydration method.

[0076] Specifically, the alcohol solution may include at least one of methanol, ethanol, polyethylene glycol, n-propanol, isopropanol, glycerol, n-butanol, n-pentanol, n-undecanol, n-dodecanol, 2-propanol, 2-butanol, 2-hexanol, cyclohexanol, and tert-butanol. In specific examples, the alcohol solution may be a single alcohol or a compound alcohol solution, such as a glycerol solution or a compound alcohol solution of ethanol, isopropanol, polyethylene glycol, and glycerol, or a compound alcohol solution of ethanol and glycerol.

[0077] In some embodiments of the present invention, the dehydration step utilizes a gradient concentration alcohol solution for dehydration and drying. Gradient concentration dehydration refers to sequential dehydration in alcohol solutions of varying concentrations. Compared to dehydration in a single concentration solution, sequential dehydration in alcohol solutions of varying concentrations can effectively improve dehydration efficiency and enhance the flexibility of the bio-dry film precursor.

[0078] Specifically, the dehydration treatment is to soak the biological dry film precursor in alcohol solutions with increasing concentrations of 25% to 100%.

[0079] In a specific example, the biofilm precursor is sequentially immersed in (a) a mixed alcohol solution of 25% ethanol, 25% isopropanol, 25% polyethylene glycol, and 25% glycerol; or (b) a mixed alcohol solution of 45% ethanol, 45% isopropanol, 5% polyethylene glycol, and 5% glycerol for 20-30 hours at 25°C to 55°C. Alternatively, the biofilm precursor is sequentially immersed in a mixture of 50% ethanol and 50% glycerol for 24 hours, and then in a mixture of 90% ethanol and 10% glycerol for 24 hours.

[0080] It should be noted that the dehydration step can be repeated once or multiple times using a treatment solution of an alcohol solution with a concentration gradient. For example, the dehydration step can be repeated once or multiple times using a treatment solution of an alcohol solution with the same concentration gradient. The dehydration step can also be repeated once or multiple times using treatment solutions of alcohol solutions with different concentration gradients.

[0081] In some embodiments of the present invention, after supercritical drying, the bio-dry film precursor is sterilized and packaged. Conventional methods are used for sterilization and packaging, which are not limited by the present invention. For example, sealed packaging may involve packaging the bio-dry film precursor in a liquid-free container, such as a packaging bag, in an environment with a relative humidity of less than 30% or in an inert atmosphere, such as nitrogen or argon.

[0082] Specifically, sterilization refers to sterilization using ethylene oxide, electron beam radiation, or gamma ray irradiation.

[0083] In some embodiments of the present invention, the method for preparing the biological dry film may include the following steps:

[0084] S100: cross-linking the biofilm precursor;

[0085] S200: cutting the biofilm precursor;

[0086] S300: subjecting the bio-dry film precursor to at least one alcohol dehydration treatment;

[0087] S400: subjecting the biofilm precursor to supercritical drying;

[0088] S500: sterilizing the biofilm precursor.

[0089] In the above steps for preparing the biological dry film, the order of each step is not completely in accordance with the above steps. For example, in some embodiments, S200 may be performed first and then S100. In some other embodiments of the present invention, S300 may be performed first and then S200, and so on.

[0090] The present invention subjects a cross-linked biodry film precursor to alcohol dehydration and supercritical drying. The resulting biodry film has an organic solvent content of no more than 0.5 wt.%, a water content of less than 15 wt.%, and a residual aldehyde content of less than 3 mg / g. This yields a biodry film with excellent biosafety, biocompatibility, anti-calcification capability, and effectiveness, while also being rapidly rehydrated. When used in conjunction with a degradable material, such as a heart valve, it can prevent corrosion or degradation of the degradable material. The biodry film obtained by this method does not absorb water even after prolonged storage. When used in conjunction with a degradable material, it prevents the degradable material from absorbing moisture from the dry film during device storage, potentially leading to corrosion and degradation. For example, when used in a heart valve, this prevents corrosion or degradation of the degradable material, thereby ensuring the stability, safety, and effectiveness of the heart valve during performance preservation. This method eliminates the need for additional organic reagents and can control the organic content of the biofilm, reducing safety risks after implantation. Furthermore, the biofilm can return to its original size upon rehydration, quickly softening after implantation. This improved conformability enhances the effectiveness of subsequent implantation. The biofilm prepared by the method of the present invention is smaller in size, facilitating preinstallation in smaller diameter delivery systems, compatibility with smaller blood vessels, and improved implant compatibility. Furthermore, upon implantation into the affected area, the valve returns to its original size in a physiological solution, without compromising its use or adding any additional risks.

[0091] In order to facilitate understanding of the present invention, the following describes the key points of the present invention in conjunction with some specific embodiments. It is understood that the relevant embodiments are only some examples of the present invention and do not constitute a limitation on the scope of application.

[0092] Test method:

[0093] Thickness test: measured with a flat head micrometer thickness gauge.

[0094] Weight loss rate test: Use a 1 / 1000 electronic balance to weigh the mass M1 of the biofilm before dehydration and the mass M2 of the biofilm after sterilization. Weight loss rate = (M1-M2) / M1*100%.

[0095] Length and width test: The leaflet size is measured with a vernier caliper.

[0096] Moisture content test: For samples with higher water content, a volumetric Karl Fischer titrator can be used for testing, while for samples with lower water content, a coulometric Karl Fischer titrator can be used for testing.

[0097] Glutaraldehyde residue test: Take the sample, wash it three times with normal saline (500 mL normal saline each time), wipe it dry, press 6 cm 2 / mL was added with ethanol, and the mixture was analyzed by GCMS after ultrasonication at room temperature.

[0098] Organic solvent content test: Refer to the residual solvent determination method in 0861 of the 2020 edition of the "Chinese Pharmacopoeia" (Volume IV), weigh a certain amount of sample, put it into a headspace bottle, seal it, and use a headspace-gas chromatograph (FID detector) for testing.

[0099] Softness Test: Take a rectangular biofilm strip 10 mm long, 1 mm wide, and approximately 0.2 mm thick. Measure the length and width with a vernier caliper, and the thickness with a micrometer. Place the strip horizontally on a 3 mm diameter cylindrical test rod, perpendicular to and located at the midpoint of the strip's long side. Allow the strip to sag naturally under gravity, and measure the horizontal distance between the inner ends of the strip using a vernier caliper.

[0100] Rehydrated dry film softness test: Take a rectangular dry film specimen with a length of 10 mm, a width of 1 mm, and a thickness of about 0.2 mm, soak the specimen in physiological saline for 5 minutes, and perform a softness test on the specimen. Compare it with a wet film with the same thickness after rehydration (biofilm without any dehydration treatment) to calculate the softness of the dry film after treatment.

[0101] Softness = (softness of dry film after rehydration / softness of wet film of the same thickness) * 100%

[0102] Example 1

[0103] The method for preparing the biological dry film of this embodiment comprises the following steps:

[0104] (1) Fresh porcine pericardium was cross-linked in a glutaraldehyde solution at 25°C, pH 7.4, and 0.5% volume concentration for 72 h. After cross-linking, the pericardium was cut into leaflets with a thickness of 0.2 mm using a laser cutter.

[0105] (2) The leaflets were sequentially immersed in a mixture of 50% ethanol and 50% glycerol for 24 h, 90% ethanol and 10% glycerol for 24 h, and then air-dried at room temperature for 24 h.

[0106] (3) The dehydrated leaflets were subjected to supercritical drying for 1 hour; the supercritical drying parameters were: dry carbon dioxide as the drying medium, temperature of 32°C, pressure of 10 MPa, and 1 cycle to obtain a biological dry film.

[0107] (4) The biofilm was sealed with nitrogen and sterilized with ethylene oxide, and the thickness of the biofilm was measured to be 0.18 mm.

[0108] The biological dry film prepared in this example is white, with a weight loss rate of 50%, V 后 is the volume before preparation V 前72.9%; the length, width and thickness of the biological dry film prepared in step 4) are 90%, 90% and 90% of the length, width and thickness of the dry film prepared in step (1), respectively.

[0109] The biological dry film prepared in this example had a moisture content of 4.6 wt.%, an organic solvent content of 0.40 wt.%, and a glutaraldehyde content of 2 mg / g. The rehydration time was 4 minutes, and the softness after rehydration was 93%. The length, width, and thickness of the dry film after rehydration were 96%, 96%, and 96% of the length, width, and thickness of the dry film before rehydration, respectively.

[0110] The biological dry film prepared in this embodiment is applied to a heart valve, wherein the heart valve includes an iron-based valve frame. The heart valve does not absorb water when stored for 2 years, and the iron-based valve frame does not corrode. After the heart valve is implanted in a goat, it can be quickly rehydrated and restored to its original size without the occurrence of thrombosis, hyperplasia, and calcification. It can meet the anti-fatigue requirements for more than 10 years, and the valve functions normally, with good biocompatibility, biosafety, and mechanical properties.

[0111] Example 2

[0112] The method for preparing the biological dry film of this embodiment comprises the following steps:

[0113] (1) Fresh porcine pericardium was cross-linked in a glutaraldehyde solution at 25°C, pH 7.4, and 0.5% volume concentration for 72 h, and then cut into leaflets with a thickness of 0.20 mm using a laser cutter.

[0114] (2) The leaflets were immersed in a mixture of 50% ethanol and 50% glycerol for 24 h, 90% ethanol and 10% glycerol for 24 h, and then dried at room temperature for 24 h.

[0115] (3) The dehydrated leaflets were subjected to supercritical drying for 2 h; the supercritical drying parameters were: completely dry carbon dioxide as the drying medium, a temperature of 33 °C, a pressure of 9 MPa, and three cycles to obtain a bio-dry film, the thickness of which was measured to be 0.17 mm.

[0116] (4) The biofilm is sealed with nitrogen and sterilized with ethylene oxide.

[0117] The biological dry film prepared in this example is white, with a weight loss rate of 55%, V 后 V 前 61.4%; the length, width and thickness of the biological dry film prepared in step (4) are 85%, 85% and 85% of the length, width and thickness of the dry film prepared in step (1), respectively.

[0118] The biofilm prepared in this example had a moisture content of 3 wt.%, an organic solvent content of 0.35 wt.%, and a glutaraldehyde content of 1.5 mg / g. The rehydration time was 4 minutes, and the softness after rehydration was 94%. The length, width, and thickness of the dry film after rehydration were 96%, 97%, and 97% of the length, width, and thickness of the dry film before rehydration, respectively.

[0119] The biological dry film prepared in this embodiment is applied to a heart valve, which includes an iron-based valve frame. The heart valve does not absorb water and the iron-based valve frame does not corrode after being stored for 2 years. After being implanted in a goat, the heart valve can be quickly rehydrated and restored to its original size without the occurrence of thrombosis, hyperplasia, and calcification. It can meet the anti-fatigue requirements for more than 10 years, and the valve functions normally, with good biocompatibility, biosafety, and mechanical properties.

[0120] Example 3

[0121] The method for preparing the biological dry film of this embodiment comprises the following steps:

[0122] (1) Fresh bovine pericardium was cross-linked in a glutaraldehyde solution at 25°C, pH 7.4, and 0.5% volume concentration for 72 h, and then cut into leaflets with a thickness of 0.2 mm using a laser cutter.

[0123] (2) The leaflets were sequentially immersed in a complex alcohol solution of 25% ethanol, 25% isopropanol, 25% polyethylene glycol and 25% glycerol for 24 h; a complex alcohol solution of 30% ethanol, 30% isopropanol, 20% polyethylene glycol and 20% glycerol for 24 h; and a mixture of 45% ethanol, 45% isopropanol and 10% glycerol for 24 h, and then air-dried at room temperature for 24 h.

[0124] (3) The dehydrated leaflets were subjected to supercritical drying for 5 h; the supercritical drying parameters were: completely dry carbon dioxide as the drying medium, a temperature of 34°C, a pressure of 8 MPa, and 5 cycles to obtain a dry biofilm, and the thickness of the biofilm was measured to be 0.16 mm.

[0125] (4) The biofilm is sealed with nitrogen and sterilized with ethylene oxide.

[0126] The biological dry film prepared in this example is white, with a weight loss rate of 58%, V 后 V 前 51.2%; the length, width and thickness of the biological dry film prepared in step (4) are 80%, 80% and 80% of the length, width and thickness of the dry film prepared in step (1), respectively.

[0127] The biofilm prepared in this example had a moisture content of 1.9 wt.%, an organic solvent content of 0.42 wt.%, and a glutaraldehyde content of 8.1 μg / g. The rehydration time was 2 minutes, and the softness after rehydration was 98%. The length, width, and thickness of the dry film after rehydration were 98%, 98%, and 99% of the length, width, and thickness of the dry film before rehydration, respectively.

[0128] The biodry film of this embodiment is applied to a heart valve, which includes an iron-based alloy valve frame. The heart valve does not absorb water and corrodes after being stored for two years. After being implanted in a goat, the heart valve can quickly rehydrate and restore its original size without thrombosis, hyperplasia, or calcification. It can meet fatigue resistance requirements for more than 10 years, and the valve functions normally, with good biocompatibility, biosafety, and mechanical properties.

[0129] Example 4

[0130] The method for preparing the biological dry film of this embodiment comprises the following steps:

[0131] (1) Fresh porcine pericardium was cross-linked in a glutaraldehyde solution at 25°C, pH 7.4, and 0.5% volume concentration for 72 h, and then cut into leaflets with a thickness of 0.2 mm using a laser cutter.

[0132] (2) The leaflets were immersed in a mixture of 50% ethanol and 50% glycerol for 24 h, 90% ethanol and 10% glycerol for 24 h, and then dried at room temperature for 24 h.

[0133] (3) Dehydrate the leaflets with 100% ethanol for 20 minutes.

[0134] (4) The leaflets after secondary dehydration were subjected to supercritical drying for 2 h. The supercritical drying parameters were: completely dry carbon dioxide as the drying medium, temperature of 38 °C, pressure of 10 MPa, and number of cycles of 10 times to obtain a bio-dry film. The thickness of the bio-dry film was measured to be 0.17 mm.

[0135] (5) The biofilm is sealed with nitrogen and sterilized with ethylene oxide.

[0136] The biological dry film prepared in this example is white, with a weight loss rate of 59%, V 后 V 前 The length, width and thickness of the biological dry film prepared in step (5) are 85%, 85% and 85% of the length, width and thickness of the dry film prepared in step (1), respectively.

[0137] The biofilm prepared in this example had a moisture content of 1.8 wt.%, an organic solvent content of 0.44 wt.%, and a glutaraldehyde content of 6.3 μg / g. The rehydration time was 2 minutes, and the softness after rehydration was 98%. The length, width, and thickness of the dry film after rehydration were 98%, 98%, and 98% of the length, width, and thickness of the dry film before rehydration, respectively.

[0138] The biological dry film prepared in this embodiment is applied to a heart valve, which includes an iron-based valve frame. The heart valve does not absorb water after being stored for 2 years, and the zinc-based alloy valve frame is not corroded. After the heart valve is implanted in a goat, it can be quickly rehydrated and restored to its original size without thrombosis, hyperplasia, or calcification. It can meet the anti-fatigue requirements for more than 10 years, and the valve functions normally, with good biocompatibility, biosafety, and mechanical properties.

[0139] Example 5

[0140] The method for preparing the biological dry film of this embodiment comprises the following steps:

[0141] S1: Fresh porcine pericardium was cross-linked in a glutaraldehyde solution at 25°C, pH 7.4, and 0.5% volume concentration for 72 h, and then cut into leaflets with a thickness of 0.2 mm using a laser cutter.

[0142] S2: The leaflets were sequentially immersed in a mixture of 50% ethanol and 50% glycerol for 24 h, and 90% ethanol and 10% glycerol for 24 h, and then air-dried at room temperature for 24 h.

[0143] S3: The leaflets were dehydrated with 100% ethanol for 30 minutes.

[0144] S4: The leaflets after secondary dehydration were subjected to supercritical drying for 1 hour. The supercritical drying parameters were: completely dry carbon dioxide as the drying medium, temperature of 39°C, pressure of 10 MPa, and 20 cycles to obtain a bio-dry film. The thickness of the bio-dry film was measured to be 0.16 mm.

[0145] S5: The biofilm is sealed by nitrogen filling and sterilized by ethylene oxide.

[0146] The biofilm prepared in this example is white, and the weight loss rate of the biofilm is 65%. 后 V 前 51.2%; the length, width and thickness of the biological dry film prepared in step (4) are 80%, 80% and 80% of the length, width and thickness of the dry film prepared in step (1), respectively.

[0147] The biofilm prepared in this example had a moisture content of 1.0 wt.%, an organic solvent content of 0.45 wt.%, and a glutaraldehyde content of 8.5 μg / g. The rehydration time was 1 minute, and the softness after rehydration was 100%. The length, width, and thickness of the dry film after rehydration were 100%, 100%, and 100% of the length, width, and thickness of the dry film before rehydration, respectively.

[0148] The biological dry film prepared in this embodiment is applied to a heart valve, which includes an iron-based valve frame. The heart valve does not absorb water after being stored for 2 years, and the iron-based valve frame is not corroded. After being implanted in a goat, the heart valve can be quickly rehydrated and restored to its original size without the occurrence of thrombosis, hyperplasia, and calcification. It can meet the anti-fatigue requirements for more than 10 years, and the valve functions normally, with good biocompatibility, biosafety, and mechanical properties.

[0149] Example 6

[0150] The method for preparing the biological dry film of this embodiment comprises the following steps:

[0151] S1: Fresh porcine pericardium was cross-linked in a 0.5% volume concentration glutaraldehyde solution at 25°C, pH 7.4 for 72 h, and then cut into leaflets with a thickness of 0.21 mm using a laser cutter.

[0152] S2: The leaflets were sequentially immersed in a mixture of 50% ethanol and 50% glycerol for 24 h, and 90% ethanol and 10% glycerol for 24 h, and then air-dried at room temperature for 24 h.

[0153] S3: The leaflets were dehydrated with 100% isopropyl alcohol for 20 minutes.

[0154] S4: The leaflets after secondary dehydration were subjected to supercritical drying for 1 hour. The supercritical drying parameters were: completely dry carbon dioxide as the drying medium, temperature of 40°C, pressure of 8 MPa, and number of cycles of 5 to obtain a bio-dry film. The thickness of the bio-dry film was measured to be 0.17 mm.

[0155] S5: The biofilm is sealed by nitrogen filling and sterilized by ethylene oxide.

[0156] The biofilm prepared in this example is white, and the weight loss rate of the biofilm is 56%. 后 V 前 51.8%; the length, width and thickness of the biological dry film prepared in step (4) are 80%, 80% and 81% of the length, width and thickness of the dry film prepared in step (1), respectively.

[0157] The biofilm prepared in this example had a moisture content of 2.0 wt.%, an organic solvent content of 0.46 wt.%, and a glutaraldehyde content of 8.6 μg / g. The rehydration time was 2 minutes, and the softness after rehydration was 96%. The length, width, and thickness of the dry film after rehydration were 97%, 98%, and 99% of the length, width, and thickness of the dry film before rehydration, respectively.

[0158] The biological dry film prepared in this embodiment is applied to a heart valve, which includes an iron-based valve frame. The heart valve does not absorb water and the iron-based valve frame does not corrode after being stored for 2 years. After being implanted in a goat, the heart valve can be quickly rehydrated and restored to its original size without the occurrence of thrombosis, hyperplasia, and calcification. It can meet the anti-fatigue requirements for more than 10 years, and the valve functions normally, with good biocompatibility, biosafety, and mechanical properties.

[0159] Example 7

[0160] The method for preparing the biological patch of this embodiment comprises the following steps:

[0161] 1) Fresh porcine pericardium was cross-linked in a 0.5% volume concentration glutaraldehyde solution at 25° C. and pH 7.4 for 72 h. After cross-linking, the pericardium was cut into leaflets with a thickness of 0.2 mm using a laser cutter.

[0162] 2) The leaflets were sequentially immersed in a mixture of 50% ethanol and 50% glycerol for 24 h, and then in 90% ethanol and 10% glycerol for 24 h, and then air-dried at room temperature for 24 h.

[0163] 3) subjecting the dehydrated leaflets to supercritical drying for 2 hours; wherein the supercritical drying parameters are: dry carbon dioxide as the drying medium, a temperature of 33° C., a pressure of 10 MPa, and 5 cycles to obtain a dry biological patch.

[0164] 4) The biological patch was sealed with nitrogen and sterilized with ethylene oxide, and the thickness of the biological patch was measured to be 0.17 mm.

[0165] The biological patch prepared in this example is white, with a weight loss rate of 50%, V 后 V 前 The length, width and thickness of the biological patch prepared in step (4) are 86%, 85% and 86% of the length, width and thickness of the biological patch prepared in step (1), respectively.

[0166] The biofilm prepared in this example had a moisture content of 4.4 wt.%, an organic solvent content of 0.48 wt.%, and a glutaraldehyde content of 8.9 μg / g. The rehydration time was 4 minutes, and the softness after rehydration was 93%. The length, width, and thickness of the dry film after rehydration were 96%, 96%, and 96% of the length, width, and thickness of the dry film before rehydration, respectively.

[0167] After implantation into goats, the biological patch can quickly rehydrate and restore its original size, with few postoperative complications, no calcification, good mechanical properties, and good biocompatibility, biosafety and mechanical properties.

[0168] Example 8

[0169] The method for preparing the biological dry film of this embodiment comprises the following steps:

[0170] S1: Fresh porcine pericardium was cross-linked in a 0.5% volume concentration glutaraldehyde solution at 25°C, pH 7.4 for 72 h, and then cut into leaflets with a thickness of 0.21 mm using a laser cutter.

[0171] S2: Soak the leaflets in a mixture of 50% ethanol and 50% glycerol for 24 hours.

[0172] S3: The dehydrated leaflets were subjected to supercritical drying for 0.2 h, wherein the supercritical drying parameters were: completely dry carbon dioxide as the drying medium, temperature of 32° C., pressure of 8 MPa, and 1 cycle to obtain a bio-dry film. The thickness of the bio-dry film was measured to be 0.19 mm.

[0173] S4: Sterilize the biofilm by nitrogen sealing and ethylene oxide sterilization.

[0174] The biofilm prepared in this example is white, and the weight loss rate of the biofilm is 40%. 后 V 前 The length, width and thickness of the biological dry film prepared in step (4) are 88%, 88% and 90% of the length, width and thickness of the dry film prepared in step (1), respectively.

[0175] The biofilm prepared in this example had a moisture content of 15.0 wt.%, an organic solvent content of 0.5 wt.%, and a glutaraldehyde content of 2.5 mg / g. The rehydration time was 5 minutes, and the softness after rehydration was 90%. The length, width, and thickness of the dry film after rehydration were 95%, 95%, and 95% of the length, width, and thickness of the dry film before rehydration, respectively.

[0176] The biological dry film prepared in this embodiment is applied to a heart valve, which includes an iron-based valve frame. The heart valve does not absorb water and does not corrode after being stored for 2 years. After being implanted in a goat, the heart valve can be quickly rehydrated and restored to its original size without the occurrence of thrombosis, hyperplasia, and calcification. It can meet the anti-fatigue requirements for more than 10 years, and the valve functions normally, with good biocompatibility, biosafety, and mechanical properties.

[0177] Example 9

[0178] The method for preparing the biological dry film of this embodiment comprises the following steps:

[0179] S1: Fresh porcine pericardium was cross-linked in a glutaraldehyde solution at 25°C, pH 7.4, and 0.5% volume concentration for 72 h, and then cut into leaflets with a thickness of 0.20 mm using a laser cutter.

[0180] S2: Soak the leaflets in a mixture of 90% ethanol and 10% glycerol for 24 hours.

[0181] S3: The dehydrated leaflets were subjected to supercritical drying for 0.5 h, wherein the supercritical drying parameters were: completely dry carbon dioxide as the drying medium, temperature of 32° C., pressure of 8 MPa, and number of cycles of 2 to obtain a bio-dry film. The thickness of the bio-dry film was measured to be 0.18 mm.

[0182] S4: Sterilize the biofilm by nitrogen sealing and ethylene oxide sterilization.

[0183] The biofilm prepared in this example is white, and the weight loss rate of the biofilm is 41%. 后 V 前 70.5%; the length, width and thickness of the biological dry film prepared in step (4) are 89%, 89% and 89% of the length, width and thickness of the dry film prepared in step (1), respectively.

[0184] The biofilm prepared in this example had a moisture content of 12.0 wt.%, an organic solvent content of 0.48 wt.%, and a glutaraldehyde content of 2.2 mg / g. The rehydration time was 4 minutes, and the softness after rehydration was 90%. The length, width, and thickness of the dry film after rehydration were 95%, 95%, and 95% of the length, width, and thickness of the dry film before rehydration, respectively.

[0185] The biological dry film prepared in this embodiment is applied to a heart valve, which includes an iron-based valve frame. The heart valve does not absorb water and does not corrode after being stored for 2 years. After being implanted in a goat, the heart valve can be quickly rehydrated and restored to its original size without the occurrence of thrombosis, hyperplasia, and calcification. It can meet the anti-fatigue requirements for more than 10 years, and the valve functions normally, with good biocompatibility, biosafety, and mechanical properties.

[0186] Example 10

[0187] The method for preparing the biological dry film of this embodiment comprises the following steps:

[0188] S1: Fresh porcine pericardium was cross-linked in a glutaraldehyde solution at 25°C, pH 7.4, and 0.5% volume concentration for 72 h, and then cut into leaflets with a thickness of 0.22 mm using a laser cutter.

[0189] S2: Dehydrate with 100% ethanol for 20 min.

[0190] S3: The dehydrated leaflets were subjected to supercritical drying for 1 hour. The supercritical drying parameters were: completely dry carbon dioxide as the drying medium, temperature of 32°C, pressure of 9 MPa, and 1 cycle to obtain a bio-dry film. The thickness of the bio-dry film was measured to be 0.19 mm.

[0191] S5: The biofilm is sealed by nitrogen filling and sterilized by ethylene oxide.

[0192] The biofilm prepared in this example is white, and the weight loss rate of the biofilm is 44%. 后 V 前 The length, width and thickness of the biological dry film prepared in step (4) are 87%, 87% and 88% of the length, width and thickness of the dry film prepared in step (1), respectively.

[0193] The biofilm prepared in this example had a moisture content of 7.5 wt.%, an organic solvent content of 0.35 wt.%, and a glutaraldehyde content of 1.5 mg / g. The rehydration time was 4 minutes, and the softness after rehydration was 93%. The length, width, and thickness of the dry film after rehydration were 95%, 95%, and 96% of the length, width, and thickness of the dry film before rehydration, respectively.

[0194] The biological dry film prepared in this embodiment is applied to a heart valve, which includes an iron-based valve frame. The heart valve does not absorb water and does not corrode after being stored for 2 years. After being implanted in a goat, the heart valve can be quickly rehydrated and restored to its original size without the occurrence of thrombosis, hyperplasia, and calcification. It can meet the anti-fatigue requirements for more than 10 years, and the valve functions normally, with good biocompatibility, biosafety, and mechanical properties.

[0195] Comparative Example 1

[0196] The preparation method of the biological dry film of this comparative example comprises the following steps:

[0197] (1) Fresh porcine pericardium was cross-linked in a glutaraldehyde solution at 25°C, pH 7.4, and 0.5% volume concentration for 72 h, and then cut into leaflets with a thickness of 0.2 mm using a laser cutter.

[0198] (2) The leaflets were sequentially immersed in a complex alcohol solution of 25% ethanol, 25% isopropanol, 25% polyethylene glycol and 25% glycerol for 24 h; a complex alcohol solution of 30% ethanol, 30% isopropanol, 20% polyethylene glycol and 20% glycerol for 24 h; and a mixture of 45% ethanol, 45% isopropanol and 10% glycerol for 24 h.

[0199] (3) The biofilm was sealed with nitrogen and sterilized with ethylene oxide, and the thickness of the biofilm was measured to be 0.18 mm.

[0200] The biological dry film prepared in this comparative example is white, with a weight loss rate of 35%, V 后 V 前 The length, width and thickness of the biological dry film prepared in step (3) are 90%, 90% and 90% of the length, width and thickness of the biological patch prepared in step (1), respectively.

[0201] The biofilm prepared in this comparative example had a moisture content of 25 wt.%, an organic solvent content of 2 wt.%, and a glutaraldehyde content of 3 mg / g. The rehydration time was 20 minutes, and the softness after rehydration was 75%. The length, width, and thickness of the dry film after rehydration were 80%, 79%, and 80% of the length, width, and thickness of the dry film before rehydration, respectively.

[0202] The moisture content, organic solvent content and glutaraldehyde content of the biological dry film prepared in this comparative example are higher than those in Examples 1 to 10. At the same time, it is difficult to rehydrate, and the hardness increases after rehydration.

[0203] The biological dry film obtained in this comparative example was applied to a heart valve, which included an iron-based valve frame. After opening the package 3 months later, the iron-based valve frame was corroded, the valve frame broke during valve expansion, and the rehydration effect was poor.

[0204] Comparative Example 2

[0205] The preparation method of the biological dry film of this comparative example comprises the following steps:

[0206] (1) Fresh porcine pericardium was cross-linked in a glutaraldehyde solution at 25°C, pH 7.4, and 0.5% volume concentration for 72 h, and then cut into leaflets with a laser cutter. The leaflet thickness was 0.2 mm.

[0207] (2) The leaflets were immersed in a mixture of 50% ethanol and 50% glycerol for 24 h, and then in 90% ethanol and 10% glycerol for 24 h, and then dried at room temperature for 24 h. The thickness of the leaflets after dehydration was measured to be 0.21 mm.

[0208] (3) The leaflets were then dehydrated with 100% ethanol for 15 min.

[0209] (4) The biofilm was sealed with nitrogen and sterilized with ethylene oxide, and the thickness of the biofilm was measured to be 0.17 mm.

[0210] The biological dry film prepared in this comparative example is white, with a weight loss rate of 40%, V 后 V 前The length, width and thickness of the biological dry film prepared in step (4) are 85%, 85% and 85% of the length, width and thickness of the biological dry film prepared in step (1), respectively.

[0211] The biofilm prepared in this comparative example had a moisture content of 20 wt.%, an organic solvent content of 1.5 wt.%, and a glutaraldehyde content of 0.1 mg / g. The rehydration time was 10 minutes, and the softness after rehydration was 80%. The length, width, and thickness of the dry film after rehydration were 85%, 85%, and 86% of the length, width, and thickness of the dry film before rehydration, respectively.

[0212] The moisture content, organic solvent content and glutaraldehyde content of the biological dry film prepared in this comparative example are higher than those in Examples 1 to 10, and the hardness increases after rehydration.

[0213] In this comparative example, the leaflets shrank and became smaller with dehydration, leading to deformation and partial tearing of the thread holes sewn into the iron-based valve frame during the double alcohol dehydration process. Three months after sterilization and packaging, the valve was unpacked and found to have corroded iron-based valve frames, which fractured during valve expansion and exhibited poor rehydration performance.

[0214] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A biological dry film, characterized in that, The residual amount of aldehydes in the biological dry film < 3 mg / g.

2. The biological dry film according to claim 1, characterized in that, The residual amount of aldehydes in the biological dry film < 0.1 mg / g; the residual amount of aldehydes in the biological dry film < 10 μg / g.

3. The biological dry film according to claim 1, wherein The water content in the biological dry film ≤ 15 wt.%; the water content in the biological dry film < 10 wt.%; the water content in the biological dry film ≤ 5 wt.%.

4. The biological dry film according to claim 1, characterized in that, The total content of organic solvents in the biological dry film ≤ 0.5 wt.%.

5. The biological dry film according to claim 1, wherein The weight loss rate T of the biological dry film before and after preparation ≥ 40%; the weight loss rate T of the biological dry film before and after preparation ≥ 50%; the weight loss rate T of the biological dry film before and after preparation ≥ 58%.

6. The biological dry film according to claim 1, characterized in that, The volume V of the biological dry film after preparation 后 ≤ the volume V before preparation 前 by 90%; and / or the length / width / thickness of the biological dry film after preparation ≤ 90% of the length / width / thickness before preparation.

7. The biological dry film according to claim 1, wherein The color of the biological dry film is beige, white, wool color or ivory white.

8. The biological dry film according to any one of claims 1 to 7, characterized in that, The rehydration time of the biological dry film in physiological saline ≤ 5 minutes.

9. The biological dry film according to any one of claims 1 to 8, characterized in that, The softening rate of the biological dry film after rehydration in physiological saline ≥ 90%, and / or the length / width / thickness of the biological dry film after rehydration in physiological saline ≥ 95% of the length / width / thickness before rehydration.

10. The biological dry film according to claim 1, wherein The precursor of the biological dry film includes at least one of animal pericardium, heart valve, blood vessel, ligament, muscle, intestine, skin, peritoneum, pleura, Achilles tendon or valved venous conduit; the precursor of the biological dry film includes at least one of the pericardium, heart valve, blood vessel, ligament, muscle, intestine, skin, peritoneum, pleura, Achilles tendon or valved venous conduit of pig, cattle, horse, donkey, mule and sheep.

11. Use of the biological dry film according to any one of claims 1 to 10 in at least one of biological patches, artificial blood vessels, artificial conduits and heart valves.

12. The application of the biological dry film according to claim 11 on a heart valve, characterized in that, The heart valve includes a valve frame.

13. A method for preparing a biological dry film according to any one of claims 1 to 10, characterized in that, It includes subjecting the precursor of the biological dry film to supercritical drying treatment.

14. The preparation method according to claim 13, characterized in that, The temperature of the supercritical drying is 31.2 °C to 69 °C, and the pressure is 7.38 Mpa to 30 Mpa; the temperature of the supercritical drying is 31.2 °C to 40 °C, and the pressure is 7.38 Mpa to 10 Mpa; the temperature of the supercritical drying is 35 °C to 40 °C, and the pressure is 8 Mpa to 10 Mpa.

15. The preparation method according to claim 14, wherein, The time of the supercritical drying is 0.2 h to 24 h, and the number of cycles is 1 to 99 times; the time of the supercritical drying is 0.5 h to 5 h, and the number of cycles is 2 to 20 times.

16. The preparation method according to claim 13, characterized in that, The medium of the supercritical drying includes at least one of carbon dioxide, methane, ethane and propane.

17. The preparation method according to claim 13, characterized in that, Before the supercritical drying, the precursor of the biological dry film further includes a step of crosslinking the precursor of the biological dry film; The crosslinking agent used in the crosslinking treatment includes organic aldehyde.

18. The preparation method according to claim 13, characterized in that, Before the supercritical drying treatment, the precursor of the biological dry film further includes at least one dehydration treatment step; The dehydrating agent used in the dehydration treatment is organic alcohol.

19. A method for preparing the biological dry film according to any one of claims 1 to 10, characterized in that, It includes the following steps: Crosslink the precursor of the biological dry film; Cut the precursor of the biological dry film; Perform at least one alcohol dehydration treatment on the precursor of the biological dry film; Subject the precursor of the biological dry film to supercritical drying treatment; Sterilize the precursor of the biological dry film.