A decellularized pericardium, its preparation method and uses

Abstract

This application provides a method for preparing decellularized pericardium, employing a combination of specific pretreatment, sterilization, defatting, decellularization, and freeze-drying processes. This method effectively removes antigenic substances from animal tissues while maximally preserving the integrity of bioactive components and structures in the material. Simultaneously, the preparation process effectively incorporates inorganic metal ions, polysaccharides, and drugs to prevent adhesions. The resulting decellularized pericardium possesses both natural rough and smooth surfaces. The smooth surface has a dense fibrous structure, effectively blocking the invasion of adhesion tissues without affecting tendon function. The rough surface allows tissue ingrowth, facilitating rapid vascularization and tendon repair. Furthermore, the decellularized pericardium is loaded with functional components, further promoting tendon healing and preventing adhesions.

Description

Technical Field

[0001] This application relates to the field of biomedical materials technology, and in particular to a decellularized pericardium, its preparation method, and its uses. Background Technology

[0002] Tendon adhesions following injury are a clinically challenging problem. Due to a lack of effective adhesion prevention measures, treatment often falls into a vicious cycle of "adhesion-release-re-adhesion," severely impacting limb function rehabilitation. Applying physical barriers to block extrinsic healing is the primary method for preventing tendon adhesions. Early non-biological materials such as silicone and gold foil have been phased out due to drawbacks including significant tissue reaction, obvious rejection, lack of permeability, interference with intrinsic tendon healing, and the need for secondary surgery for removal. With advancements in materials science, researchers have shifted their focus to absorbable polymers such as chitosan, hyaluronic acid and its derivatives, collagen, silk fibroin, and polylactic acid. The excellent degradability and biocompatibility of absorbable polymers allow nutrients and cytokines to pass through without affecting local tendon nutrition, achieving better results in preventing tendon adhesions. However, these traditional materials still differ from natural tissue structures and have various shortcomings.

[0003] With the development of regenerative medicine and tissue engineering technology, increasingly higher requirements are being placed on the function and structure of tendon anti-adhesion membranes. Besides good biocompatibility and anti-adhesion effects, they must also possess functions similar to the extracellular matrix, providing cells with specific physiological conditions to generate the necessary cellular responses and promote functional repair of damaged tendons. Decellularized matrix materials, due to their retention of the inherent structure and composition of the extracellular matrix, exhibit high bioactivity and low immunogenicity, and have received widespread attention in the field of tissue engineering. Currently, decellularized matrix materials obtained through decellularization technology are widely used in human tissue reconstruction, such as heart valves, skin, and dura mater. Decellularized pericardium has excellent biocompatibility and tissue adhesion. Simultaneously, it retains the dense collagen structure and mechanical properties of natural tissue, effectively blocking extrinsic healing and facilitating surgical procedures. However, most reported decellularized pericardium membranes for tendon repair and adhesion prevention have single functions and unsatisfactory effects. Therefore, there is a need to develop more effective decellularized pericardium materials for preparing tendon anti-adhesion membranes, as well as multifunctional tendon anti-adhesion products to achieve better therapeutic results. Summary of the Invention

[0004] The purpose of this application is to provide a decellularized pericardium and its preparation method to solve the problem of adhesion formation after tendon injury.

[0005] The first aspect of this application provides a method for preparing decellularized pericardium, which includes the following steps:

[0006] Pretreatment: Fresh animal pericardial tissue was taken, and the attached fat was removed to obtain pericardial tissue;

[0007] Disinfection: The pericardial tissue is mixed with disinfectant at a material-to-liquid mass-to-volume ratio of 1g:1-10mL and treated for 1-4 hours; wherein the disinfectant is selected from at least one of 0.1-0.2wt% peracetic acid solution, 0.01-0.1wt% chlorine dioxide solution, and 1-10wt% hydrogen peroxide solution;

[0008] Degreasing: The pericardial tissue is mixed with a degreasing agent at a material-to-liquid mass-to-volume ratio of 1g:1-10mL, and ultrasonically treated for 1-4 hours; wherein the degreasing agent is selected from at least one of 100wt% isopropanol solution, 100wt% acetone solution, 0.1-1mol / L sodium hydroxide solution, and 0.1-1mol / L potassium hydroxide solution;

[0009] Decellularization: The pericardial tissue was mixed with a decellularization reagent at a material-to-liquid mass-to-volume ratio of 1g:1-10mL and treated for 1-48 hours; then, the pericardial tissue treated with the decellularization reagent was mixed with a 1-10U / mL deoxyribonuclease (DNase) solution at a material-to-liquid mass-to-volume ratio of 1g:1-10mL and treated for 2-48 hours to obtain decellularized pericardium; wherein, the decellularization reagent is selected from at least one of 0.1-1mol / L sodium hydroxide solution, 0.1-1wt% trypsin solution, 0.1-2wt% Triton X-100 solution, and 0.1-1wt% sodium deoxycholate solution;

[0010] Freeze-drying process: Freeze-fix the decellularized pericardium at -20℃ to -80℃ for 1-4 hours, place it in the freeze dryer chamber, turn on the condenser to cool it down to -10℃ to -56℃, turn on the vacuum pump, and freeze-dry under vacuum.

[0011] The second aspect of this application provides a decellularized pericardium prepared by the preparation method described in the first aspect of this application.

[0012] The third aspect of this application provides the use of decellularized pericardium prepared by the preparation method described in the first aspect of this application in the prevention and / or treatment of tendon adhesions.

[0013] The method for preparing decellularized pericardium provided in this application combines specific pretreatment, sterilization, defatting, decellularization, and freeze-drying processes. This effectively removes antigenic substances from animal tissues while maximally preserving the integrity of bioactive components and structure in the material. Furthermore, the preparation process effectively incorporates inorganic metal ions, polysaccharides, and drugs to prevent adhesions, resulting in better tendon healing and adhesion prevention. Moreover, the preparation conditions of this application are simple, the quality is controllable, and the cost is low.

[0014] The decellularized pericardium of this application is a single-layer double-sided material with a natural rough and smooth surface. The smooth surface has a denser fibrous structure, which can effectively block the invasion of adhesion tissues without affecting tendon function. The rough surface allows tissue ingrowth, which is conducive to rapid vascularization and tendon repair. At the same time, the decellularized pericardium is loaded with functional components, providing a platform for the development of multifunctional tendon anti-adhesion membranes.

[0015] Of course, implementing any product or method of this application does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other embodiments can be obtained based on these drawings.

[0017] Figure 1A A scanning electron microscope (SEM) image of the smooth surface of the decellularized pericardium prepared in Example 1;

[0018] Figure 1B Here is a SEM image of the rough surface of the decellularized pericardium prepared in Example 1;

[0019] Figure 1C This is a SEM image of a cross-section of the decellularized pericardium prepared in Example 1;

[0020] Figure 2 This is a hematoxylin-eosin (HE) staining image of the decellularized pericardium prepared in Example 1;

[0021] Figure 3A HE staining image of the Achilles tendon cross section of rats in control group 1 4 weeks after repair;

[0022] Figure 3B HE staining image of the Achilles tendon cross section of rats in experimental group 1 4 weeks after repair;

[0023] Figure 3CHE staining image of the Achilles tendon cross section of rats in control group 2 4 weeks after repair;

[0024] Figure 3D HE staining image of the Achilles tendon cross section of rats in experimental group 2 4 weeks after repair. Detailed Implementation

[0025] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art based on this application are within the scope of protection of this application.

[0026] The first aspect of this application provides a method for preparing decellularized pericardium, which includes the following steps:

[0027] Pretreatment: Fresh animal pericardial tissue was taken, and the attached fat was removed to obtain pericardial tissue;

[0028] Disinfection: The pericardial tissue is mixed with disinfectant at a material-to-liquid mass-to-volume ratio of 1g:1-10mL and treated for 1-4 hours; wherein the disinfectant is selected from at least one of 0.1-0.2wt% peracetic acid solution, 0.01-0.1wt% chlorine dioxide solution, and 1-10wt% hydrogen peroxide solution;

[0029] Degreasing: The pericardial tissue is mixed with a degreasing agent at a material-to-liquid mass-to-volume ratio of 1g:1-10mL, and ultrasonically treated for 1-4 hours; wherein the degreasing agent is selected from at least one of 100wt% isopropanol solution, 100wt% acetone solution, 0.1-1mol / L sodium hydroxide solution, and 0.1-1mol / L potassium hydroxide solution;

[0030] Decellularization: The pericardial tissue was mixed with a decellularization reagent at a material-to-liquid mass-to-volume ratio of 1g:1-10mL and treated for 1-48 hours; then, the pericardial tissue treated with the decellularization reagent was mixed with 1-10U / mL DNase solution at a material-to-liquid mass-to-volume ratio of 1g:1-10mL and treated for 2-48 hours to obtain decellularized pericardium; wherein, the decellularization reagent is selected from at least one of 0.1-1mol / L sodium hydroxide solution, 0.1-1wt% trypsin solution, 0.1-2wt% Triton X-100 solution, and 0.1-1wt% sodium deoxycholate solution;

[0031] Freeze-drying process: Freeze-fix the decellularized pericardium at -20℃ to -80℃ for 1-4 hours, place it in the freeze dryer chamber, turn on the condenser to cool it down to -10℃ to -56℃, turn on the vacuum pump, and freeze-dry under vacuum.

[0032] In this application, the method of removing the attached fat is not limited; for example, physical scraping can be used.

[0033] The inventors discovered in their research that the degreasing method and degreasing reagent of this application resulted in a low fat content in the prepared decellularized pericardium; the decellularized pericardium prepared using the decellularization method and decellularization reagent of this application basically retained its complete structure, and the matrix components were also basically unaffected.

[0034] In this application, a combination of specific pretreatment, disinfection, degreasing, decellularization and freeze-drying methods is used to effectively remove antigenic substances from animal tissues while maximizing the preservation of the integrity of bioactive components and structure in the material, thus achieving a good effect in promoting tendon healing and preventing adhesions.

[0035] It should be noted that in the preparation method of this application, after the pretreatment is completed, the steps of disinfection, degreasing and decellularization do not indicate their order. They can be carried out in different orders as needed, for example, (1) pretreatment, disinfection, degreasing, decellularization and freeze-drying; (2) pretreatment, disinfection, decellularization, degreasing and freeze-drying; (3) pretreatment, degreasing, disinfection, decellularization and freeze-drying; (4) pretreatment, degreasing, decellularization, disinfection and freeze-drying; (5) pretreatment, decellularization, disinfection, degreasing and freeze-drying; (6) pretreatment, decellularization, degreasing, disinfection and freeze-drying.

[0036] In some embodiments of the first aspect of this application, after the disinfection, degreasing, and decellularization steps are completed, each step further includes cleaning with a cleaning agent; the cleaning agent is selected from at least one of pure water, 0.01-0.2 mol / L phosphate buffer solution (PBS buffer), and 0.9 wt% sodium chloride solution.

[0037] In some embodiments of the first aspect of this application, after the pericardial tissue is mixed with the decellularization reagent, it is rinsed with 0.01-0.2 mol / L PBS buffer until neutral; after the pericardial tissue is mixed with the DNase solution, residual DNase solution is removed with 0.01-0.2 mol / L PBS buffer, and then the PBS buffer is removed by washing with pure water.

[0038] In some embodiments of the first aspect of this application, it further includes:

[0039] Inorganic metal ion addition: The decellularized pericardium is mixed with an inorganic metal ion solution of 0.01-20 mmol / L at a mass-to-volume ratio of 1g:1-10mL and treated for 6-24 hours; wherein the inorganic metal ion solution is selected from at least one of magnesium ion solution, copper ion solution, zinc ion solution and silver ion solution.

[0040] In some embodiments of the first aspect of this application, it further includes:

[0041] Polysaccharide addition: The decellularized pericardium is mixed with a polysaccharide solution of 100-1000 μg / mL at a mass-to-volume ratio of 1g:1-10mL, and treated for 4-24 hours; wherein the polysaccharide solution is selected from at least one of chondroitin sulfate solution, heparin, hyaluronic acid, and chitosan.

[0042] In some embodiments of the first aspect of this application, it further includes:

[0043] Drug addition: A drug-loaded solution of 0.001-1 mg / mL is applied to the smooth surface of the decellularized pericardium at a mass-to-volume ratio of 1 g: 0.1-1 mL; wherein the drug is selected from at least one of pirfenidone, deferoxamine, curcumin, and ginsenosides.

[0044] In the preparation method of this application, after pretreatment, sterilization, defatting, and decellularization, and before freeze-drying, inorganic metal ions, polysaccharides, and drugs are added. The order of these steps does not imply a specific sequence; they can be performed in different orders as needed. Furthermore, the decellularized pericardium described in this application does not imply the simultaneous addition of inorganic metal ions, polysaccharides, and drugs. These can be added according to different needs, for example: (1) only inorganic metal ions are added; (2) only polysaccharides are added; (3) only drugs are added; (4) both inorganic metal ions and polysaccharides are added; (5) both inorganic metal ions and drugs are added; (6) both polysaccharides and drugs are added; and (7) both inorganic metal ions, polysaccharides, and drugs are added.

[0045] In some embodiments of the first aspect of this application, the inorganic metal ion solution is selected from magnesium ion solution; the polysaccharide solution is selected from chondroitin sulfate solution; and the drug is selected from pirfenidone.

[0046] The inventors discovered in their research that, by adding magnesium ions using the preparation method described in this application, the magnesium ions achieve complexation loading with the carboxyl groups in the pericardium through electronic coordination. This allows the magnesium ions in the decellularized pericardium to exert their therapeutic effects more effectively, thereby enhancing the inflammatory regulation capacity of the decellularized pericardium and promoting tendon healing and preventing adhesions. Adding chondroitin sulfate using the preparation method described in this application, through hydrophilic-hydrophobic interactions, enables the decellularized pericardium to load with chondroitin sulfate, effectively regulating the inflammatory response and preventing excessive collagen deposition and fibrosis caused by excessive inflammation. Adding pirfenidone using the preparation method described in this application, and coating the smooth surface of the decellularized pericardium with a pirfenidone-loaded solution, results in a decellularized pericardium that effectively prevents adhesions caused by fibrosis.

[0047] In some embodiments of the first aspect of this application, the decellularized pericardium is derived from an animal, which is selected from at least one of pigs, cattle, horses, sheep, dogs, mules, and non-human primates, preferably pigs, cattle, or sheep.

[0048] The second aspect of this application provides a decellularized pericardium prepared by the preparation method described in the first aspect of this application.

[0049] The inventors discovered in their research that the decellularized pericardium of this application contains fewer immunogenic substances and retains more bioactive components; when combined with specific amounts of inorganic metal ions, polysaccharides, or drugs, it can better promote tendon healing and prevent adhesions.

[0050] The third aspect of this application provides the use of decellularized pericardium prepared by the preparation method described in the first aspect of this application in the prevention and / or treatment of tendon adhesions.

[0051] The technical solution of this application will be further explained and illustrated below through specific embodiments.

[0052] Unless otherwise specified, the experimental materials and methods used in the following examples are all conventional materials and methods.

[0053] Example 1: Preparation of decellularized pericardium

[0054] (1) Pretreatment: Take fresh pig pericardial tissue and remove the attached fat;

[0055] (2) Disinfection: The pericardial tissue after removing the attached fat was mixed with 0.2wt% peracetic acid solution at a material-to-liquid mass-to-volume ratio of 1g:5mL and treated for 2 hours. The peracetic acid solution was discarded and then 0.2mol / L PBS buffer was added to rinse until clean.

[0056] (3) Degreasing: Mix the sterilized pericardial tissue with 100wt% isopropanol solution at a material-to-liquid mass-to-volume ratio of 1g:5mL, sonicate for 2 hours, discard the isopropanol solution, and then add 0.2mol / L PBS buffer to rinse until clean;

[0057] (4) Decellularization: The defatted pericardial tissue was mixed with 0.5 mol / L sodium hydroxide solution at a material-to-solution mass-to-volume ratio of 1 g: 5 mL and treated for 2 hours. The sodium hydroxide solution was discarded, and the pericardial tissue was rinsed with 0.2 mol / L PBS buffer until neutral. The pericardial tissue treated with sodium hydroxide was mixed with 5 U / mL DNase solution at a material-to-solution mass-to-volume ratio of 1 g: 5 mL and treated for 24 hours. The DNase solution was discarded, and the pericardial tissue was rinsed with 0.2 mol / L PBS buffer to remove residual DNase solution. The pericardial tissue was then rinsed with pure water to remove the PBS buffer and obtain decellularized pericardial membrane.

[0058] (5) Freeze-drying treatment: Freeze-fix at -20℃ for 2 hours. Place the freeze-fixed decellularized pericardium in the freeze dryer chamber, turn on the condenser to cool it down to -50℃, turn on the vacuum pump, and freeze-dry under vacuum.

[0059] Example 2: Preparation of decellularized pericardium with composite magnesium ions

[0060] (1) Pretreatment: Take fresh pig pericardial tissue and remove the attached fat;

[0061] (2) Disinfection: The pericardial tissue after removing the attached fat was mixed with 0.2wt% peracetic acid solution at a material-to-liquid mass-to-volume ratio of 1g:5mL and treated for 2 hours. The peracetic acid solution was discarded and then 0.2mol / L PBS buffer was added to rinse until clean.

[0062] (3) Degreasing: Mix the sterilized pericardial tissue with 100wt% isopropanol solution at a material-to-liquid mass-to-volume ratio of 1g:5mL, sonicate for 2 hours, discard the isopropanol solution, and then add 0.2mol / L PBS buffer to rinse until clean;

[0063] (4) Decellularization: The defatted pericardial tissue was mixed with 0.5 mol / L sodium hydroxide solution at a material-to-solution mass-to-volume ratio of 1 g: 5 mL and treated for 2 hours. The sodium hydroxide solution was discarded, and the pericardial tissue was rinsed with 0.2 mol / L PBS buffer until neutral. The pericardial tissue treated with sodium hydroxide was mixed with 5 U / mL DNase solution at a material-to-solution mass-to-volume ratio of 1 g: 5 mL and treated for 24 hours. The DNase solution was discarded, and the pericardial tissue was rinsed with 0.2 mol / L PBS buffer to remove residual DNase solution. The pericardial tissue was then rinsed with pure water to remove the PBS buffer and obtain decellularized pericardial membrane.

[0064] (5) Addition of inorganic metal ions: The decellularized pericardium was mixed with 10 mmol / L magnesium chloride solution at a mass-to-volume ratio of 1 g: 4 mL and treated for 8 hours to obtain decellularized pericardium with composite magnesium ions.

[0065] (6) Freeze-drying treatment: Freeze-fix at -20℃ for 2 hours. Place the decellularized pericardium of the composite magnesium ions frozen-fixed into the freeze dryer chamber, turn on the condenser to cool it down to -50℃, turn on the vacuum pump, and freeze-dry under vacuum.

[0066] Example 3: Preparation of decellularized pericardium with composite copper ions

[0067] Replace the inorganic metal ion step (5) with the following: mix the decellularized pericardium with a 0.1 mmol / L copper chloride solution at a mass-to-volume ratio of 1 g: 4 mL and treat for 8 hours to obtain a decellularized pericardium with composite copper ions. The rest is the same as in Example 2.

[0068] Example 4: Preparation of decellularized pericardium with composite zinc ions

[0069] Replace the inorganic metal ion step (5) with the following: mix the decellularized pericardium with a 0.1 mmol / L zinc chloride solution at a mass-to-volume ratio of 1 g: 4 mL and treat for 8 hours to obtain a decellularized pericardium with composite zinc ions. The rest is the same as in Example 2.

[0070] Example 5: Preparation of decellularized pericardium with composite silver ions

[0071] Replace the inorganic metal ion step (5) with the following: mix the decellularized pericardium with 0.01 mmol / L silver nitrate solution at a mass-to-volume ratio of 1 g: 4 mL and treat for 8 hours to obtain a decellularized pericardium with composite silver ions. The rest is the same as in Example 2.

[0072] Example 6: Preparation of decellularized pericardial membrane composed of chondroitin sulfate

[0073] (1) Pretreatment: Take fresh pig pericardial tissue and remove the attached fat;

[0074] (2) Disinfection: The pericardial tissue after removing the attached fat was mixed with 0.2wt% peracetic acid solution at a material-to-liquid mass-to-volume ratio of 1g:5mL and treated for 2 hours. The peracetic acid solution was discarded and then 0.2mol / L PBS buffer was added to rinse until clean.

[0075] (3) Degreasing: Mix the sterilized pericardial tissue with 100wt% isopropanol solution at a material-to-liquid mass-to-volume ratio of 1g:5mL, sonicate for 2 hours, discard the isopropanol solution, and then add 0.2mol / L PBS buffer to rinse until clean;

[0076] (4) Decellularization: The defatted pericardial tissue was mixed with 0.5 mol / L sodium hydroxide solution at a material-to-solution mass-to-volume ratio of 1 g: 5 mL and treated for 2 hours. The sodium hydroxide solution was discarded, and the pericardial tissue was rinsed with 0.2 mol / L PBS buffer until neutral. The pericardial tissue treated with sodium hydroxide was mixed with 5 U / mL DNase solution at a material-to-solution mass-to-volume ratio of 1 g: 5 mL and treated for 24 hours. The DNase solution was discarded, and the pericardial tissue was rinsed with 0.2 mol / L PBS buffer to remove residual DNase solution. The pericardial tissue was then rinsed with pure water to remove the PBS buffer and obtain decellularized pericardial membrane.

[0077] (5) Polysaccharide addition: The decellularized pericardium was mixed with 500 μg / mL chondroitin sulfate solution at a mass-to-volume ratio of 1 g: 5 mL and treated for 24 hours to obtain decellularized pericardium with compound chondroitin sulfate.

[0078] (6) Freeze-drying treatment: Freeze-fix at -20℃ for 2 hours. Place the decellularized pericardium of the freeze-fixed compound chondroitin sulfate in the freeze dryer chamber, turn on the condenser to cool it down to -50℃, turn on the vacuum pump, and freeze-dry under vacuum.

[0079] Example 7: Preparation of decellularized pericardial membrane of heparin compound

[0080] Replace step (5) with the following steps: mix decellularized pericardium with 100 μg / mL heparin solution at a mass-to-volume ratio of 1 g: 5 mL and treat for 24 hours to obtain decellularized pericardium with compound heparin. The rest is the same as in Example 6.

[0081] Example 8: Preparation of decellularized pericardial membrane with composite hyaluronic acid

[0082] Replace step (5) with the following steps: mix decellularized pericardium with 1000 μg / mL hyaluronic acid solution at a mass-to-volume ratio of 1 g: 5 mL and treat for 24 hours to obtain decellularized pericardium with composite hyaluronic acid. The rest is the same as in Example 6.

[0083] Example 9: Preparation of decellularized pericardial membrane composed of chitosan

[0084] Replace step (5) with the following steps: mix decellularized pericardium with a 1000 μg / mL chitosan solution at a mass-to-volume ratio of 1 g: 5 mL and treat for 24 hours to obtain decellularized pericardium with composite chitosan. The rest is the same as in Example 6.

[0085] Example 10: Preparation of decellularized pericardial membrane with pirfenidone

[0086] (1) Pretreatment: Take fresh pig pericardial tissue and remove the attached fat;

[0087] (2) Disinfection: The pericardial tissue after removing the attached fat was mixed with 0.2wt% peracetic acid solution at a material-to-liquid mass-to-volume ratio of 1g:5mL and treated for 2 hours. The peracetic acid solution was discarded and then 0.2mol / L PBS buffer was added to rinse until clean.

[0088] (3) Degreasing: Mix the sterilized pericardial tissue with 100wt% isopropanol solution at a material-to-liquid mass-to-volume ratio of 1g:5mL, sonicate for 2 hours, discard the isopropanol solution, and then add 0.2mol / L PBS buffer to rinse until clean;

[0089] (4) Decellularization: The defatted pericardial tissue was mixed with 0.5 mol / L sodium hydroxide solution at a material-to-solution mass-to-volume ratio of 1 g: 5 mL and treated for 2 hours. The sodium hydroxide solution was discarded, and the pericardial tissue was rinsed with 0.2 mol / L PBS buffer until neutral. The pericardial tissue treated with sodium hydroxide was mixed with 5 U / mL DNase solution at a material-to-solution mass-to-volume ratio of 1 g: 5 mL and treated for 24 hours. The DNase solution was discarded, and the pericardial tissue was rinsed with 0.2 mol / L PBS buffer to remove residual DNase solution. The pericardial tissue was then rinsed with pure water to remove the PBS buffer and obtain decellularized pericardial membrane.

[0090] (5) Drug addition: At a material-to-liquid mass ratio of 1g:0.5mL, a 0.5mg / mL chitosan solution loaded with pirfenidone was used to coat the smooth surface of the decellularized pericardium to obtain a decellularized pericardium with pirfenidone composite.

[0091] (6) Freeze-drying treatment: freeze-fix at -20℃ for 2 hours, place the decellularized pericardium of the freeze-fixed compound pirfenidone in the freeze dryer chamber, turn on the condenser to cool it down to -50℃, turn on the vacuum pump, and freeze-dry under vacuum.

[0092] Example 11 Preparation of decellularized pericardium with compound deferoxamine

[0093] Replace the drug addition step (5) with: applying a 0.001 mg / mL chitosan solution loaded with deferoxamine to the smooth surface of the decellularized pericardium at a material-to-liquid mass ratio of 1 g: 0.2 mL to obtain a decellularized pericardium with deferoxamine composite, and the rest is the same as in Example 10.

[0094] Example 12 Preparation of decellularized pericardial membrane with compound curcumin

[0095] Replace the drug addition step (5) with: applying a 0.001 mg / mL chitosan solution loaded with curcumin to the smooth surface of the decellularized pericardium at a material-to-liquid mass ratio of 1 g: 0.1 mL to obtain a decellularized pericardium with composite curcumin. The rest is the same as in Example 10.

[0096] Example 13: Preparation of decellularized pericardium of compound ginsenosides

[0097] Replace step (5) with the following steps: apply a 0.002 mg / mL chitosan solution loaded with ginsenosides to the smooth surface of the decellularized pericardium at a mass ratio of 1 g: 0.2 mL to obtain a decellularized pericardium with compound ginsenosides. The rest is the same as in Example 10.

[0098] Histological analysis

[0099] Organizational structure

[0100] The decellularized pericardium prepared in Example 1 was photographed using an electron microscope, and the results are as follows: Figures 1A-1C As shown. From Figures 1A-1C It can be seen that the decellularized pericardium has a smooth surface ( Figure 1A ) and rough surface ( Figure 1B The pericardium is divided into two types, and scanning electron microscopy shows that it is formed by interwoven bundles of collagen fibers of different sizes, with a thickness of approximately 0.1 mm. Figure 1C ).

[0101] Examples 2-13 are based on the decellularized pericardium of Example 1, but with different inorganic metal ions, polysaccharides or drugs added, without affecting the tissue structure of the decellularized pericardium.

[0102] Immunogenic substance analysis

[0103] As a heterogeneous material, animal-derived materials contain a large number of immunogenic substances in their cells. Therefore, the first step is to remove the heterogeneous cells contained in the material. The more thorough the removal, the lower its immunogenicity.

[0104] Host cell residue: The decellularized pericardium prepared in Example 1 was fixed with 10% neutral formalin, embedded in paraffin, cut into 0.4 μm thin slices, dewaxed with xylene, dehydrated with a series of alcohols, stained with hematoxylin and eosin (HE), and observed under a microscope. The results are as follows: Figure 2 As shown.

[0105] from Figure 2 As can be seen, the decellularized pericardium prepared in Example 1 showed no blue-stained cells or cell nuclear debris, and the basement membrane structure was clear with no donor cell residue, indicating that the decellularized pericardium obtained by the preparation method of this application is thoroughly decellularized and has good biosafety.

[0106] Examples 2-13 are based on the decellularized pericardium of Example 1, and are compounded with specific amounts of inorganic metal ions, polysaccharides and drugs. These inorganic metal ions, polysaccharides and drugs will not affect biosafety.

[0107] Animal experiments

[0108] laboratory animals

[0109] Twelve healthy 7-week-old male SPF-grade SD rats, weighing 150-200g, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. They were housed in an environment with a temperature of (23±2)℃ and a relative humidity of 55%-65%.

[0110] Achilles tendon repair experiment

[0111] Twelve healthy 7-week-old SD rats were randomly divided into four groups: experimental group 1, experimental group 2, control group 1, and control group 2. After anesthesia with sodium pentobarbital, the rats in each group underwent hair removal and disinfection using depilatory cream. A 316L stainless steel threaded fixation pin (0.4 mm diameter) was used to fix the rats to the tibia (one pin) and metatarsals (one pin). The right Achilles tendon was severed and sutured using a modified Kessler suture. Control groups 1 and 2 were sutured normally. In experimental group 1, the suture site was wrapped with the decellularized pericardium prepared in Example 1 (rough surface covering the tendon). In experimental group 2, the suture site was wrapped with the decellularized pericardium prepared in Example 2 (rough surface covering the tendon). The wound was then sutured. The rats were sacrificed four weeks post-surgery for histological analysis.

[0112] from Figures 3A-3D As can be seen, the black-boxed areas in control groups 1 and 2 show disordered arrangement of the tissue around the tendon, rich blood vessels, and adhesions; in experimental group 1, the tissue around the tendon is orderly arranged with no adhesions; in experimental group 2, the tissue around the tendon is orderly arranged, with no adhesions and a lower inflammatory response. This demonstrates that the decellularized pericardium prepared in this application promotes tendon healing and prevents adhesions. In particular, the decellularized pericardium of this application, as a carrier loaded with magnesium ions, also possesses inflammatory regulation capabilities, resulting in a better effect in promoting tendon healing and preventing adhesions.

[0113] Inorganic metal ions possess strong antibacterial and anti-inflammatory regulatory capabilities. For example, copper, zinc, and silver ions can all play antibacterial and anti-inflammatory roles in tissue repair. In addition to the decellularized pericardium with magnesium ions, Examples 3-5 also prepared decellularized pericardiums with specific amounts of copper, zinc, and silver ions, all exhibiting good anti-inflammatory and antibacterial capabilities, thus better promoting tendon healing and preventing adhesions.

[0114] Chondroitin sulfate, heparin, hyaluronic acid, and chitosan are functional polysaccharides that regulate inflammatory responses. Examples 6-9 describe decellularized pericardial membranes prepared with chondroitin sulfate, heparin, hyaluronic acid, and chitosan, respectively. By introducing specific amounts of chondroitin sulfate, heparin, hyaluronic acid, and chitosan onto the decellularized pericardial membranes, these membranes can effectively regulate inflammatory responses and prevent excessive collagen deposition and fibrosis caused by excessive inflammation.

[0115] Pirfenidone is a broad-spectrum anti-fibrotic drug that inhibits fibroblast proliferation and collagen synthesis, preventing tissue adhesions caused by fibrosis. In Example 10, a chitosan solution loaded with pirfenidone was used as a coating on the smooth surface of decellularized pericardium. During Achilles tendon repair, the smooth surface of the pirfenidone-coated decellularized pericardium contacted the peritendinous tissue, effectively preventing adhesions caused by fibrosis. Deferoxamine plays an inflammatory regulatory role in tissue repair. In Example 11, a chitosan solution loaded with deferoxamine was used as a coating on the smooth surface of decellularized pericardium. During Achilles tendon repair, the smooth surface of the deferoxamine-coated decellularized pericardium contacted the peritendinous tissue, regulating the inflammatory response and preventing adhesions. Curcumin and ginsenosides both have inflammatory regulatory and fibrosis-preventing effects. Examples 12 and 13, by introducing specific amounts of curcumin and ginsenosides onto decellularized pericardium, effectively regulated the inflammatory response in practical applications, avoiding adhesions caused by excessive inflammation and fibrosis.

[0116] This application uses animal pericardium as raw material and combines specific pretreatment, sterilization, degreasing, decellularization, and freeze-drying methods to prepare a novel decellularized matrix material that is non-immunogenic, high in bioactive components, and structurally intact. It also incorporates inorganic metal ions, polysaccharides, and drugs to prevent adhesion formation, thus achieving better results in promoting tendon healing and preventing adhesions. The decellularized pericardium of this application is a single-layer, double-sided material with naturally rough and smooth surfaces. The smooth surface has a denser fibrous structure, effectively blocking the invasion of adhesion tissues without affecting tendon function; the rough surface allows tissue ingrowth, facilitating rapid vascularization and tendon repair. Simultaneously, the decellularized pericardium is loaded with functional components, providing a platform for developing multifunctional tendon anti-adhesion membranes.

[0117] The above description is merely a preferred embodiment of this application and is not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application are included within the scope of protection of this application.

Claims

1. A method for preparing decellularized pericardium, comprising the following steps: Pretreatment: Fresh animal pericardial tissue was taken, and the attached fat was removed to obtain pericardial tissue; Disinfection: The pericardial tissue is mixed with disinfectant at a mass-to-volume ratio of 1g:1-10mL and treated for 1-4 hours; wherein the disinfectant is selected from at least one of 0.1-0.2 wt% peracetic acid solution, 0.01-0.1 wt% chlorine dioxide solution, and 1-10 wt% hydrogen peroxide solution; Degreasing: The pericardial tissue was mixed with a degreasing agent at a material-to-liquid mass-to-volume ratio of 1g:1-10mL, and ultrasonically treated for 1-4 hours; wherein the degreasing agent was selected from at least one of 100 wt% isopropanol solution, 100 wt% acetone solution, 0.1-1 mol / L sodium hydroxide solution, and 0.1-1 mol / L potassium hydroxide solution; Decellularization: The pericardial tissue was mixed with a decellularization reagent at a material-to-liquid mass-to-volume ratio of 1g:1-10mL and treated for 1-48 hours; then, the pericardial tissue treated with the decellularization reagent was mixed with 1-10 U / mL DNase solution at a material-to-liquid mass-to-volume ratio of 1g:1-10mL and treated for 2-48 hours to obtain decellularized pericardium; wherein, the decellularization reagent is selected from at least one of 0.1-1 mol / L sodium hydroxide solution, 0.1-1 wt% trypsin solution, 0.1-2 wt% Triton X-100 solution, and 0.1-1 wt% sodium deoxycholate solution; Inorganic metal ion addition: The decellularized pericardium was mixed with a 0.01-20 mmol / L inorganic metal ion solution at a material-to-liquid mass-to-volume ratio of 1 g: 1-10 mL, and treated for 6-24 hours; wherein the inorganic metal ion solution was selected from magnesium ion solution; Freeze-drying process: Freeze-fix the decellularized pericardium at -20℃ to -80℃ for 1-4 hours, place it in the freeze dryer chamber, turn on the condenser to cool it down to -10℃ to -56℃, turn on the vacuum pump, and freeze-dry under vacuum.

2. The preparation method according to claim 1, characterized in that, After the disinfection, degreasing, and decellularization steps are completed, each step further includes cleaning with a cleaning agent; the cleaning agent is selected from at least one of pure water, 0.01-0.2 mol / L PBS buffer, and 0.9 wt% sodium chloride solution.

3. The preparation method according to claim 2, characterized in that, After the pericardial tissue was mixed with the decellularization reagent, it was rinsed with 0.01-0.2 mol / L PBS buffer until neutral. After the pericardial tissue was mixed with DNase solution, residual DNase solution was removed with 0.01-0.2 mol / L PBS buffer, and then the PBS buffer was removed by washing with pure water.

4. The preparation method according to claim 1, characterized in that, Also includes: Polysaccharide addition: The decellularized pericardium is mixed with a polysaccharide solution of 100-1000 μg / mL at a mass-to-volume ratio of 1g:1-10 mL, and treated for 4-24 hours; wherein the polysaccharide solution is selected from at least one of chondroitin sulfate solution, heparin, hyaluronic acid, and chitosan.

5. The preparation method according to claim 1, characterized in that, Also includes: Drug addition: A drug-loaded solution of 0.001-1 mg / mL is applied to the smooth surface of the decellularized pericardium at a mass-to-volume ratio of 1g:0.1-1 mL; wherein the drug is selected from at least one of pirfenidone, deferoxamine, curcumin, and ginsenosides.

6. The preparation method according to claim 4, characterized in that, The polysaccharide solution is selected from chondroitin sulfate solution.

7. The preparation method according to claim 5, characterized in that, The drug is selected from pirfenidone.

8. The preparation method according to any one of claims 1-7, characterized in that, The decellularized pericardium is derived from an animal, selected from at least one of pigs, cattle, horses, sheep, dogs, mules, and non-human primates.

9. The preparation method according to claim 8, characterized in that, The animals are selected from pigs, cattle, and sheep.

10. The decellularized pericardium prepared by the preparation method according to any one of claims 1-9.

11. Use of the decellularized pericardium prepared by the method according to any one of claims 1-9 in the preparation of materials for the prevention and / or treatment of tendon adhesions.

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