High-strength extracellular matrix membrane and preparation method and application thereof

Abstract

The application discloses a high-strength extracellular matrix film and a preparation method and application thereof, and belongs to the field of biomedical materials. The application provides a preparation method of the high-strength extracellular matrix film, which comprises the following steps: decellularization treatment and freeze-drying, grinding the freeze-dried decellularized pig organs into powder and adding the powder into a pepsin-hydrochloric acid solution for digestion, forming a pre-gel, forming an extracellular matrix hydrogel, obtaining an extracellular matrix film, obtaining a high-strength extracellular matrix film wet film, and obtaining a high-strength extracellular matrix film. The extracellular matrix film prepared by the foregoing method has the characteristics of high strength, and the strength far exceeds that of the currently reported natural polymer gel film, and even exceeds that of most synthetic high-molecular polymer gel films and composite films added with a crosslinking agent.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, and more specifically, to a high-strength extracellular matrix film, its preparation method, and its application. Background Technology

[0002] Extracellular matrix (ECM) bioscaffolds, obtained by decellularizing tissues and organs, retain the biochemical complexity, nanostructure, and bio-inducible properties of the natural extracellular matrix, promoting cell growth, migration, proliferation, and differentiation. Therefore, they are widely used in tissue repair, reconstruction, and regeneration research. Commercially available decellularized scaffolds are already common and have long been used clinically, including decellularized allogeneic skin and decellularized small intestinal submucosa. To expand the applications of ECM bioscaffolds, researchers have developed ECM hydrogels for use in in vitro cell culture, 3D bioprinting, and stem cell tissue engineering. However, the poor biomechanical properties of hydrogels limit the application scope of ECM; ECM hydrogels cannot withstand large loads, making them unsuitable for many scenarios requiring high biomechanical performance. While adding cross-linking agents or compounding with other polymers can enhance mechanical properties in ECM hydrogels, this undoubtedly introduces biocompatibility issues.

[0003] Currently, the only pure ECM materials available are the original ECM scaffolds and ECM hydrogels; other forms of pure ECM materials have not been developed, and there is a lack of ECM materials with high mechanical properties. Given current technology, developing a high-strength, high-mechanical-performance ECM material is of great significance for fields such as material coating, wound repair, nerve conduit fabrication, artificial blood vessel fabrication, and drug delivery. Summary of the Invention

[0004] The problem to be solved by this invention is how to improve the strength of ECM materials.

[0005] To address the above problems, the first aspect of this invention provides a method for preparing a high-strength extracellular matrix film, comprising the following steps:

[0006] S1: Take fresh pig organs, remove hair and fat, chop them up, decellularize them, and freeze-dry them for preservation.

[0007] S2: Grind the freeze-dried decellularized pig organs into powder, and add the decellularized pig organ powder to a pepsin-hydrochloric acid solution for digestion and dissolution;

[0008] S3: Add PBS and NaOH to the digested decellularized porcine organ powder-pepsin-HCl mixture on ice. The concentration of PBS in the mixture is 0.01-0.15M. Store the mixture at 4°C to form a pregel.

[0009] S4: Pour the pregel obtained in step S3 into the model and place it in an environment of 37°C to form an extracellular matrix hydrogel;

[0010] S5: Place the extracellular matrix hydrogel on an antistatic plastic film and dry it at 37°C for 40-50 hours to obtain the extracellular matrix film.

[0011] S6: Immerse the extracellular matrix membrane obtained in step S5 in water for 10-20 minutes, then change the water. After changing the water, continue to immerse for 3-10 minutes, then change the water again. After changing the water, continue to immerse for 3-10 minutes to obtain a high-strength extracellular matrix membrane wet film.

[0012] S7: Place the high-strength extracellular matrix membrane wet film obtained in step S6 on an antistatic plastic film and dry it at 37°C for 40-80 minutes to obtain a high-strength extracellular matrix membrane.

[0013] The formation of ECM films is based on physical cross-linking principles, including van der Waals forces, hydrogen bonds, electrostatic attraction, and intermolecular assembly. Pepsin-digested porcine organs are converted into a solution composed of protein monomers (mainly collagen), polysaccharide chains, and polypeptide molecules. Under controlled temperature, salt solution, and pH neutralization, the intramolecular bonds of the monomer components spontaneously assemble into a homogeneous gel. During the drying of the ECM hydrogel into a film, hydrogen bonds between amides and water break, hydrogen bonds between amides rebuild, and proteins and polypeptides further self-assemble, resulting in tighter fiber aggregation and the formation of more β-sheet structures. The increased β-sheet content is associated with higher membrane strength and toughness.

[0014] Preferably, the pig organ is pig skin and / or tendon.

[0015] Preferably, in step S1, the decellularization of pig organs is performed using chemical and / or mechanical and / or enzymatic methods, including the following steps:

[0016] S11: Wash with a 2% (v / v) Triton X-100 solution for 24 hours;

[0017] S12: Wash with 0.1% (w / v) SDS solution for 48 hours;

[0018] S13: Wash with 0.1M NaCl solution for 48 hours;

[0019] S14: Wash with ultrapure water for 24 hours, freeze-dry the sample, and complete the decellularization process.

[0020] Preferably, in step S2, the content of decellularized organ powder in the pepsin-hydrochloric acid solution is 8-15 mg / ml.

[0021] Preferably, in step S2, the mass ratio of the decellularized porcine organ powder to pepsin is 10-15:1.

[0022] Preferably, in step S2, the concentration of hydrochloric acid in the hydrochloric acid solution is 0.01M to 0.1M.

[0023] Preferably, in step S3, the pH of the mixture after adding NaOH is 7 to 7.5.

[0024] A second aspect of the present invention provides a high-strength extracellular matrix film, wherein the high-strength extracellular matrix film is prepared by any of the aforementioned preparation methods.

[0025] A third aspect of the present invention provides an application of the aforementioned high-strength extracellular matrix membrane, wherein the high-strength extracellular matrix membrane is applied to any one of material coating, wound repair, nerve conduit preparation, artificial blood vessel preparation, and drug delivery.

[0026] The beneficial effects of this invention are as follows: This invention aims to create a simple ECM film that has the transparent appearance of a plastic film, excellent toughness, and will not break when folded and crumpled. It also has high mechanical strength, with a maximum strength of 115 MPa. Its mechanical properties and toughness far exceed those of currently reported natural polymer gel films (collagen, chitosan, gelatin, etc.), and even exceed most synthetic polymer gel films and composite films with added crosslinking agents. Attached Figure Description

[0027] Figure 1 The image shown is a scanning electron microscope (SEM) image of the ECM thin film prepared in Example 1 of this invention.

[0028] Figure 2 The photographs show the ECM film prepared in Example 2 of this invention and after being kneaded and unfolded.

[0029] Figure 3 The graph shows the strength test results of the ECM film prepared in Example 1 of this invention.

[0030] Figure 4 The figure shows the strength test results of the anticoagulant film prepared by the prior art in a specific embodiment of the present invention. Detailed Implementation

[0031] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the claims of the present invention.

[0032] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0033] This invention provides a high-strength extracellular matrix (ECM) film, its preparation method, and its applications. Currently, the extracellular matrix is ​​a perfect regenerative material, widely used in regenerative medicine and tissue engineering. Its applications are increasingly broad due to its versatility, including sheets, powders, and hydrogels. After processing with biomanufacturing processes, decellularized ECM can form injectable, inducible hydrogels, offering opportunities for new minimally invasive surgeries. ECM hydrogels are already available from various organ / tissue sources and are used to treat various disease models, such as ischemic injuries and organ regeneration or replacement. However, extracellular matrix in sheet, powder, and hydrogel forms has limitations: implantation requires a surgical channel, making it unsuitable for minimally invasive delivery, including injection and catheter-based surgeries. Decellularized ECM can be further processed using two main biomanufacturing processes to prepare injectable materials, but most decellularized ECMs remain unsuitable for catheter technology, drug delivery, and other fields due to their low strength. Therefore, this invention provides an extracellular matrix film that can be used for material coating, wound repair, neural conduit fabrication, artificial blood vessel fabrication, and drug delivery.

[0034] The extracellular matrix membrane provided by this invention is prepared through the following steps:

[0035] S11: Wash pig organs with a 2% (v / v) Triton X-100 solution for 24 hours;

[0036] S12: Wash pig organs with a 0.1% (w / v) SDS solution for 48 hours;

[0037] S13: Wash pig organs with 0.1M NaCl solution for 48 hours;

[0038] S14: Wash with ultrapure water for 24 hours, freeze-dry the sample to complete the decellularization process;

[0039] S2: Grind the frozen decellularized pig organs into powder, add the decellularized pig organ powder and pepsin to hydrochloric acid solution for digestion, and the content of the decellularized organ powder in the pepsin-hydrochloric acid solution is 8-15 mg / ml;

[0040] S3: Add PBS and NaOH to the digested decellularized porcine organ powder-pepsin-HCl mixture to form a pregel. The mass ratio of decellularized porcine organ powder to pepsin is 10-15:1.

[0041] S4: Pour the pregel obtained in step S3 into a model and place it in an oven to form an extracellular matrix hydrogel;

[0042] S5: Place the extracellular matrix hydrogel on an antistatic plastic film and dry it at 37°C for 40-50 hours to obtain the extracellular matrix film.

[0043] S6: Immerse the extracellular matrix membrane obtained in step S5 in water for 10-20 minutes, then change the water. After changing the water, continue to immerse for 3-10 minutes, then change the water again. After changing the water, continue to immerse for 3-10 minutes to obtain a high-strength extracellular matrix membrane wet film.

[0044] S7: Place the high-strength extracellular matrix membrane wet film obtained in step S6 on an antistatic plastic film and dry it at 37°C for 40-80 minutes to obtain a high-strength extracellular matrix membrane.

[0045] In some preferred embodiments, the strength of the extracellular matrix membrane provided by the specific embodiments of the present invention can reach 80-115 MPa.

[0046] In specific embodiments of the present invention, ECM film and extracellular matrix film have the same meaning.

[0047] Example 1

[0048] Take fresh pig skin, remove the lower layer of fat tissue, remove all hair, chop it, and then decellularize it. Wash with 2% (v / v) Triton X-100 solution for 24 h, 0.1% (w / v) SDS for 48 h, a mixed solution of 0.1 mol NaCl for 48 h, and ddH2O for 24 h. Then freeze-dry and store.

[0049] The freeze-dried decellularized pigskin was ground into powder and then digested overnight in 0.01M HCl solution at a ratio of decellularized pigskin to pepsin of 10:1, with a decellularized digestion concentration of 8 mg / ml.

[0050] The digested decellularized pig skin was adjusted to pH 7 with 10×PBS and NaOH. 112.6 μl of 10×PBS and 12 μl of NaOH were added per milliliter of digestion solution.

[0051] Pour the pH-adjusted pregel solution into the model and place it in a 37°C oven for 30 minutes to allow the hydrogel to form.

[0052] Pour the ECM hydrogel out of the mold, place it on an anti-static plastic film, and dry it in a 37°C oven for 48 hours.

[0053] Remove the dried film and immerse it in ddH2O for 15 minutes. Replace the ddH2O and immerse for 5 minutes. Replace the ddH2O again and immerse for another 5 minutes.

[0054] The soaked film was placed on an antistatic plastic film and dried in an oven at 37°C for 1 hour to obtain the ECM film. A scanning electron microscope image of the ECM film is shown below. Figure 1 As shown.

[0055] Example 2

[0056] Take fresh pig skin, remove the lower layer of fat tissue, remove all hair, chop it, and then decellularize it. Wash with 2% (v / v) Triton X-100 solution for 24 h, 0.1% (w / v) SDS for 48 h, a mixed solution of 0.1 mol NaCl for 48 h, and ddH2O for 24 h. Then freeze-dry and store.

[0057] The freeze-dried decellularized pigskin was ground into powder and then digested overnight in 0.01M HCl solution at a ratio of decellularized pigskin to pepsin of 10:1, with a decellularized digestion concentration of 10 mg / ml.

[0058] The digested decellularized pig skin was adjusted to pH 7.5 with 10×PBS and NaOH. 112.6 μl of 10×PBS and 13 μl of NaOH were added per milliliter of digestion solution.

[0059] Pour the pH-adjusted pregel solution into the model and place it in a 37°C oven for 30 minutes to allow the hydrogel to form.

[0060] Pour the ECM hydrogel out of the mold, place it on an anti-static plastic film, and dry it in a 37°C oven for 48 hours.

[0061] Remove the dried film and immerse it in ddH2O for 15 minutes. Replace the ddH2O and immerse for 5 minutes. Replace the ddH2O again and immerse for another 5 minutes.

[0062] The soaked film was placed on an antistatic plastic film and dried in an oven at 37°C for 1 hour to obtain the ECM film.

[0063] Example 3

[0064] Take fresh pig skin, remove the lower layer of fat tissue, remove all hair, chop it, and then decellularize it. Wash with 2% (v / v) Triton X-100 solution for 24 h, 0.1% (w / v) SDS for 48 h, a mixed solution of 0.1 mol NaCl for 48 h, and ddH2O for 24 h. Then freeze-dry and store.

[0065] The freeze-dried decellularized pigskin was ground into powder and then digested overnight in 0.01M HCl solution at a ratio of decellularized pigskin to pepsin of 10:1, with a decellularized digestion concentration of 15 mg / ml.

[0066] The digested decellularized porcine skin was adjusted to pH 7.5 with 10×PBS and NaOH. 112.6 μl of 10×PBS and 14 μl of NaOH were added per milliliter of digestion solution.

[0067] Pour the pH-adjusted pregel solution into the model and place it in a 37°C oven for 30 minutes to allow the hydrogel to form.

[0068] Pour the ECM hydrogel out of the mold, place it on an anti-static plastic film, and dry it in a 37°C oven for 48 hours.

[0069] Remove the dried film and immerse it in ddH2O for 15 minutes. Replace the ddH2O and immerse for 5 minutes. Replace the ddH2O again and immerse for another 5 minutes.

[0070] The soaked film was placed on an antistatic plastic film and dried in an oven at 37°C for 1 hour to obtain the ECM film.

[0071] Example 4

[0072] Figure 2 The images show photographs of the ECM film prepared in Example 1, as well as photographs after being rubbed and unfolded. The test results are as follows. Figure 2 As shown, the ECM film provided in Example 1 has the transparent appearance of a plastic film and has very good toughness; it will not break even if folded and crumpled at will.

[0073] The ECM film prepared in Example 1 of this invention was cut into strips 5 mm wide and fixed on the fixtures of a universal testing machine (ElectroForce Testbench, TA Instruments, USA). The tensile speed was set to 2 mm / min, and the distance between the two fixtures was set to 6 mm. The strength of the extracellular matrix film was measured, and the experimental results are as follows: Figure 3As shown, the ECM film prepared in Example 1 exhibits high mechanical strength, with a maximum strength reaching 115 MPa. The maximum strength of the pure collagen film is 7.63 ± 0.55 MPa, the pure methyl cellulose film is 15.78 ± 1.57 MPa, the pure gelatin film is 4.4 ± 0.26 MPa, the collagen / hydroxypropyl methyl cellulose (HPMC) blend film is 49.2 ± 7.1 MPa, and the polylactic acid (PLA) film has a maximum strength of [missing information]. The maximum strength of the polyvinyl alcohol (PVA)-gelatin composite film is 29.3±2.4 MPa, the maximum strength of the poly(hydroxyalkanoate) / polylactic acid (PLA) blend film is 36±1.5 MPa, and the maximum strength of the poly(hydroxyalkanoate) / polylactic acid (PLA) blend film is 18.98 MPa. It can be seen that the mechanical properties and toughness of the ECM film prepared in Example 1 far exceed those of the natural polymer gel films (collagen, chitosan, gelatin, etc.) that have been reported so far, and even most of the synthetic polymer gel films and composite films with added crosslinking agents.

[0074] Comparative Example 1

[0075] Chinese invention patent application number 202110589339.X discloses an anticoagulant material, wherein the anticoagulant material is an anticoagulant film. The ECM anticoagulant film prepared according to Chinese invention patent application number 202110589339.X was subjected to strength testing. The specific testing steps are as follows:

[0076] The ECM anticoagulant membrane was cut into strips 5 mm wide and fixed on the fixtures of a universal testing machine (ElectroForce Testbench, TA Instruments, USA). The tensile speed was set to 2 mm / min, and the distance between the two fixtures was set to 6 mm. The strength of the extracellular matrix membrane was measured, and the test results are as follows: Figure 4 As shown.

[0077] contrast Figure 3 and Figure 4 It can be seen that the tensile strength of the ECM film prepared in Example 1 is 86±17.25MPa, with a maximum of 115MPa and a strain of 24±0.06%. The tensile strength of the ECM anticoagulation film prepared by Chinese invention patent application number 202110589339.X is 39±3.21MPa, with a maximum of 42MPa and a strain of 13±3.13%. Compared with the two, the mechanical properties of the ECM film provided by the present invention are doubled, which is far superior to the original ECM anticoagulation film.

[0078] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having a conventional meaning are defined herein for clarification or ease of reference, and such definitions should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and employed by conventional methods. Unless otherwise stated, the use of commercially available reagents and instruments shall be performed according to the manufacturer's instructions and parameters.

[0079] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A method for preparing a high-strength extracellular matrix film, characterized in that, Includes the following steps: S1: Take fresh pig organs, remove hair and fat, chop them, decellularize them, and freeze-dry them for preservation. The pig organs are pig skin and / or tendons. S2: Grind the freeze-dried decellularized pig organs into powder, add the decellularized pig organ powder to a pepsin-hydrochloric acid solution for digestion and dissolution, wherein the content of the decellularized pig organ powder in the pepsin-hydrochloric acid solution is 8~15 mg / ml, the mass ratio of the decellularized pig organ powder to pepsin is 10~15:1, and the concentration of hydrochloric acid in the hydrochloric acid solution is 0.01 M~0.1 M; S3: Add PBS and NaOH to the digested decellularized porcine organ powder-pepsin-HCl mixture on ice. After adding NaOH, the pH of the mixture is 7~7.5, and the concentration of PBS in the mixture is 0.01~0.15M. Store the mixture at 4℃ to form a pregel. S4: Pour the pregel obtained in step S3 into the model and place it in an environment of 37°C to form an extracellular matrix hydrogel; S5: Place the extracellular matrix hydrogel on an antistatic plastic film and dry it at 37°C for 40-50 hours to obtain the extracellular matrix film. S6: Immerse the extracellular matrix membrane obtained in step S5 in water for 10-20 minutes, then change the water. After changing the water, continue to immerse for 3-10 minutes, then change the water again. After changing the water, continue to immerse for 3-10 minutes to obtain a high-strength extracellular matrix membrane wet film. S7: Place the high-strength extracellular matrix membrane wet film obtained in step S6 on an antistatic plastic film and dry it at 37°C for 40-80 minutes to obtain a high-strength extracellular matrix membrane.

2. The method for preparing a high-strength extracellular matrix film as described in claim 1, characterized in that, In step S1, the pig organs are decellularized using chemical and / or mechanical and / or enzymatic methods, including the following steps: S11: Wash with a 2% v / v Triton X-100 solution for 24 hours; S12: Wash with 0.1% w / v SDS solution for 48 hours; S13: Wash with 0.1 M NaCl solution for 48 hours; S14: Wash with ultrapure water for 24 hours, freeze-dry the sample, and complete the decellularization process.

3. A high-strength extracellular matrix membrane, characterized in that, The high-strength extracellular matrix film is prepared by the preparation method described in claim 1 or 2.

4. An application of the high-strength extracellular matrix membrane as described in claim 3, characterized in that, High-strength extracellular matrix films are used in the preparation of wound repair materials, nerve conduits, drug-loaded materials, or material coatings.

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