Acellular fish skin matrix biological scaffold as well as preparation method and application thereof

Abstract

The preparation method comprises the following steps: (1) fish skin pretreatment: (2) gas foaming treatment: weighing sodium borohydride with a certain molar concentration, dissolving the sodium borohydride in a phosphate buffer solution with a certain concentration, stirring until bubbles in the solution are obviously generated and most of solids are dissolved, and then, carrying out gas foaming treatment; putting the cleaned fish skin tissues in the step (2), and performing foaming treatment; (3) cleaning treatment; and (4) protection and storage. The gas foaming technology is innovatively adopted for decellularization treatment, the interface stripping effect between cells and a matrix is remarkably enhanced on the premise that the collagen three-dimensional structure and the scaffold morphology integrity are not damaged, and the method is suitable for the decellularization pretreatment process of soft tissue (such as fish skin and placenta) with a compact structure or cell-intensive type. The development of a biomedical material, especially a tissue engineering scaffold technology, is promoted, and the preparation method has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and particularly relates to a decellularized fish skin matrix bioscaffold, its preparation method, and its application. Background Art

[0002] With the continuous development of biomedical technology, the repair of damaged tissues such as skin, tendons, ligaments, nerves, blood vessels, and bones has become a major long-standing challenge in clinical and regenerative medicine. Existing scaffold materials mainly include polymeric materials, biomaterials, and synthetic materials. Among them, decellularized matrix materials (dECM), as a natural three-dimensional scaffold material, are the closest to biological tissues. Due to their complex composition, they have shown broad application prospects in tissue repair and regenerative medicine in recent years. These materials can retain the natural extracellular matrix components and structure of tissues, possessing good biocompatibility, biodegradability, and tissue inducibility, making them an important foundation for constructing tissue engineering scaffolds and grafts.

[0003] Currently, the preparation methods for decellularized matrix materials mainly include physical methods (such as repeated freeze-thaw cycles and high-pressure treatment), chemical methods (such as detergent treatment and acid-base treatment), and enzymatic methods (such as treatment with trypsin, DNase, and RNase). In recent years, some studies have attempted to improve decellularization efficiency through special treatment methods. The existing related patented technologies are as follows:

[0004] CN112755247A discloses an acellular dermal matrix and its preparation method. The acellular dermal matrix is ​​prepared from tilapia skin as raw material through processes including scale removal, decellularization, solution preservation, and irradiation sterilization. The preparation method includes: 1) scraping off fish scales, residual meat, and fat; 2) chemically swelling followed by scraping off the scale layer using a skin-splitting machine; 3) washing with surfactants and enzyme solutions; 4) bleaching and disinfection; 5) soaking in a high-salt solution containing antioxidants for preservation solution replacement; and 6) low-dose irradiation sterilization at low temperature.

[0005] CN118059290A discloses a decellularized fish skin matrix, its preparation method, and its application. The preparation method of the decellularized fish skin matrix includes the following steps: wetting the decellularized fish skin with a moisture-retaining agent; and subjecting the wetted fish skin to microwave treatment to cause the fish skin to expand; wherein the power of the microwave treatment is 500W to 900W, and the microwave treatment time is 5s to 60s.

[0006] CN108187140A discloses a method for preparing a decellularized dermal matrix derived from fish skin, comprising the following steps: (1) fish skin pretreatment; (2) sterilization and disinfection treatment; (3) degreasing treatment; (4) decolorization treatment; (5) high and low osmotic treatment; (6) strong alkali etching treatment; (7) repeated freeze-thaw treatment; and (8) freeze-drying and shaping treatment.

[0007] As can be seen from the aforementioned patented technologies, while the methods currently used to prepare decellularized fish skin matrix materials have achieved certain results in practical applications, they also have many problems. For example, existing technologies such as repeated freeze-thaw cycles, high-pressure treatment, acid-base treatment, and treatment with trypsin, DNase, and RNase can lead to immune rejection reactions due to residual DNA, cell debris, or immunogenic substances such as α-Gal antigens after decellularization; damage to the matrix structure affecting material properties; and cumbersome processes that are difficult to standardize. Especially for dense connective tissues such as skin, whose structure is dense and decellularization fluid is difficult to penetrate, conventional methods are unable to achieve deep and effective decellularization.

[0008] In summary, how to enhance the permeability of decellularization fluid, improve decellularization efficiency, reduce residual DNA content, minimize damage to the matrix structure, improve the quality and application performance of decellularized materials from dense tissues (such as fish skin and mammalian skin), simplify the process, effectively reduce production costs, and achieve industrial application has become a pressing problem for technicians in the field of biomedical materials. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the technical problem to be solved by this invention is to provide a decellularized fish skin matrix biological scaffold with a simple and convenient process, flexible and controllable parameters, effectively improving decellularization efficiency, efficiently reducing DNA residue, and more completely preserving the original extracellular matrix structure, as well as its preparation method and application.

[0010] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing a decellularized fish skin matrix biological scaffold, characterized by comprising the following steps:

[0011] (I) Fish skin pretreatment:

[0012] (1) Take the skin tissue of tilapia skin that has been processed, frozen and thawed; or select fresh tilapia skin, scrape off the remaining scales and remove redundant tissue and impurities, and trim the skin tissue into the required shape for later use.

[0013] (2) The fish skin tissue obtained in step (1) is cleaned with deionized water and a certain concentration of phosphate buffer solution.

[0014] (II) Gas foaming treatment:

[0015] (3) Weigh out a certain molar concentration of sodium borohydride, dissolve it in a certain concentration of phosphate buffer solution, stir until bubbles are clearly generated in the solution and most of the solid dissolves, then put it into the fish skin tissue cleaned in step (2) for foaming treatment.

[0016] (4) After the foaming process is completed, remove the fish skin tissue;

[0017] (III) Cleaning treatment:

[0018] (5) Use a certain concentration of phosphate buffer solution to repeatedly shake and wash the foamed fish skin tissue until no obvious bubbles are produced after shaking the soaked fish skin tissue.

[0019] (iv) Protection and storage:

[0020] (6) Store the cleaned fish skin tissue in a phosphate buffer solution containing a mixture of penicillin and streptomycin and let it stand at low temperature for 12-24 hours.

[0021] (7) After being taken out, freeze-dried, sterilized and packaged to make a dry finished product, thus obtaining a decellularized fish skin matrix biological scaffold.

[0022] The above-mentioned method for preparing decellularized fish skin matrix bioscaffold, in step (1), the processing, freezing and thawing process is as follows: Thaw fresh frozen tilapia skin in deionized water at 3-5℃, scrape off residual scales and remove redundant tissue and impurities, trim the fish skin tissue into the required shape, rinse or wash it multiple times with deionized water and phosphate buffer solution at 3-5℃, divide the processed fish skin tissue into portions and quickly freeze it at -80℃, before use, take out the frozen fish skin tissue and soak it in phosphate buffer solution at 3-5℃ to thaw.

[0023] In the above-mentioned method for preparing decellularized fish skin matrix bioscaffold, in step (3), the molar concentration of sodium borohydride is 1-3M, the foaming time is 30min or 60min, and the foaming treatment temperature is 20-25℃.

[0024] In the above-mentioned method for preparing decellularized fish skin matrix bioscaffold, in steps (2), (3), (5), and (6), the pH of the phosphate buffer solution is 7.2-7.4 and the concentration is 0.01 mol / L.

[0025] In the above-mentioned method for preparing decellularized fish skin matrix bioscaffolds, the pH of the phosphate buffer solution used in rinsing or washing and soaking / thawing processes is 7.2-7.4, and the concentration is 0.01 mol / L.

[0026] In the above-described method for preparing decellularized fish skin matrix bioscaffolds, the phosphate buffer solution is any one of PBS buffer, Hank's buffer, or D-Hank's buffer.

[0027] In the above-mentioned method for preparing decellularized fish skin matrix bioscaffold, the volume concentration of penicillin-streptomycin mixture in the phosphate buffer solution in step (6) is 1% (v / v), the concentration of bioactive substances in penicillin-streptomycin mixture is 5000 U / mL, the low temperature environment is set at 3-5℃, and the standing time is 18 hours.

[0028] A decellularized fish skin matrix bioscaffold is prepared by the above-described preparation method.

[0029] The aforementioned decellularized fish skin matrix bioscaffold has a DNA content of 29.6 ± 8.2 ng / mg.

[0030] Application of a decellularized fish skin matrix bioscaffold in the repair of injuries to tissues such as skin, tendons, ligaments, nerves, blood vessels, and bones.

[0031] The advantages of the decellularized fish skin matrix bioscaffold, its preparation method, and its application in this invention are:

[0032] (1) Improve decellularization efficiency: Through gas foaming treatment, the fish skin tissue expands due to the generation of gas, creating a porous structure that increases the porosity of the tissue and significantly enhances the permeability of the decellularization solution, thereby improving the decellularization efficiency.

[0033] (2) Reducing DNA residue: Gas foaming treatment can open up dense connective tissues (such as skin, bone, cartilage, and tendon tissues), allowing the solution to penetrate the tissue better and achieve more comprehensive DNA removal. The amount of DNA residue in the treated tissue is significantly reduced during the decellularization process, which reduces immunogenicity and improves biocompatibility.

[0034] (3) Protecting the matrix structure: This method improves the efficiency of decellularization while better preserving the three-dimensional structure and bioactive components of the extracellular matrix, which is beneficial to tissue repair and regeneration.

[0035] (4) Easy to operate: The gas foaming technology used is simple to operate, easy to standardize, and suitable for large-scale production applications.

[0036] This invention innovatively employs gas foaming technology for decellularization, which is characterized by its gentle and non-thermal-damaging properties. Without damaging the three-dimensional structure of collagen and the integrity of the scaffold morphology, it significantly enhances the interfacial separation between cells and the matrix. It is suitable for the decellularization pretreatment of dense or cell-dense soft tissues (such as fish skin and placenta), promoting the development of biomedical materials, especially tissue engineering scaffold technology, and has broad application prospects. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the preparation method of the decellularized fish skin matrix biological scaffold of the present invention;

[0038] Figure 2 This is a photograph of the decellularized fish skin matrix bioscaffold of the present invention.

[0039] Figure 3 Scanning electron micrographs of cross-sections of fish skin matrix before and after foaming decellularization treatment;

[0040] Figure 4 Images of HE-stained sections of fish skin matrix before and after foaming decellularization treatment;

[0041] Figure 5 Morphological characteristics of rat tendon cells (RTs) on the surface of decellularized fish skin matrix bioscaffold after 1 day and 5 days of culture;

[0042] Figure 6 The graph shows the mechanical properties of fish skin matrix before and after foaming and decellularization treatment under wet conditions. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0044] like Figure 1-2 As shown, a method for preparing a decellularized fish skin matrix bioscaffold includes the following steps:

[0045] (I) Fish skin pretreatment:

[0046] (1) Take the skin tissue of tilapia skin that has been processed, frozen and thawed; or select fresh tilapia skin, scrape off the remaining scales and remove redundant tissue and impurities, and trim the skin tissue into the required shape for later use.

[0047] (2) The fish skin tissue obtained in step (1) is cleaned with deionized water and a certain concentration of phosphate buffer solution.

[0048] (II) Gas foaming treatment:

[0049] (3) Weigh out a certain molar concentration of sodium borohydride, dissolve it in a certain concentration of phosphate buffer solution, stir until bubbles are clearly generated in the solution and most of the solid dissolves, then put it into the fish skin tissue cleaned in step (2) for foaming treatment.

[0050] (4) After the foaming process is completed, remove the fish skin tissue;

[0051] (III) Cleaning treatment:

[0052] (5) Use a certain concentration of phosphate buffer solution to repeatedly shake and wash the foamed fish skin tissue until no obvious bubbles are produced after shaking the soaked fish skin tissue.

[0053] (iv) Protection and storage:

[0054] (6) Store the cleaned fish skin tissue in a phosphate buffer solution containing a mixture of penicillin and streptomycin and let it stand at low temperature for 12-24 hours.

[0055] (7) After being taken out, freeze-dried, sterilized and packaged to make a dry finished product, thus obtaining a decellularized fish skin matrix biological scaffold.

[0056] The fish skin material selection for this invention is quite flexible. Fresh fish skin can be processed on-site to obtain fish skin tissue. However, for industrial-scale production, large quantities of fish skin raw materials need to be purchased. Since the industrial-scale decellularization preparation process requires a large amount of fish skin tissue that meets the requirements, temporary processing of fresh fish skin is often unsuitable for industrial-scale production. To avoid the deterioration of fish skin raw materials and to facilitate large-scale production, the fish skin raw materials can be pre-treated and frozen. When it is necessary to prepare decellularized fish skin matrix bioscaffolds, the required fish skin tissue can be directly obtained by thawing. The specific processing, freezing, and thawing process is as follows: The purchased fresh tilapia skin frozen product is thawed in deionized water at 3-5℃. After thawing, the residual scales of the tilapia skin raw material are scraped off, and redundant tissues and impurities are removed. The redundant and uneven parts at the edges are cut off, and the fish belly is the main part to be retained for cutting. The fish skin tissue is trimmed into the required shape and rinsed or washed multiple times with deionized water and phosphate buffer solution at 3-5℃ until the tissue surface is clean. After processing, the fish skin tissue is aliquoted and quickly frozen at -80°C. Before use, the frozen fish skin tissue is removed and thawed in a phosphate buffer solution at 3-5°C. The tilapia skin raw material used in this invention should be freshly peeled and frozen, have a traceable source, and meet relevant quarantine standards.

[0057] In step (3), the molar concentration of sodium borohydride is 1-3M, the foaming time is 30 min or 60 min, and the foaming temperature is 20-25℃. In steps (2), (3), (5), and (6), and during rinsing, washing, and soaking / thawing, the pH of the phosphate buffer solution used is 7.2-7.4, and the concentration is 0.01 mol / L. The phosphate buffer solution can be any one of PBS buffer, Hank's buffer, or D-Hank's buffer. Since different tissues and organs vary, the required concentration and time are not unique; parameters can be adjusted as needed before decellularization.

[0058] The gas foaming technology in step (3) of this invention refers to any technology that can generate gas or cause porous materials to expand due to their own gaseous state, or cause the paste melt to form a porous structure, such as, but not limited to, the sodium borohydride foaming technology in step (3). Given that in the existing technology, in actual production and in domestic and internationally disclosed methods for treating decellularized materials, no prior technology using gas foaming technology for decellularization has been found, this invention aims to provide a new process for preparing decellularized matrix materials, simplifying process steps, reducing production costs, improving material quality, and being suitable for industrial production. During the cleaning process after gas foaming treatment in step (II), physiological saline containing antibiotics or other suitable buffer solutions can be used for further cleaning as needed to ensure the sterility and purity of the tissue. In the phosphate buffer solution of step (6), the volume concentration of the penicillin-streptomycin mixture is 1% (v / v), the concentration of the bioactive substance in the penicillin-streptomycin mixture is 5000 U / mL, the low temperature environment is set at 3-5℃, and the standing time is 18 hours. The DNA content of the decellularized fish skin matrix bioscaffold prepared by the method of this invention is 29.6 ± 8.2 ng / mg. This decellularized fish skin matrix bioscaffold can be widely used for the repair of injuries to tissues such as skin, tendons, ligaments, nerves, blood vessels, and bones.

[0059] The mechanism of action of the foaming treatment used in the preparation method of this invention is as follows:

[0060] Sodium borohydride (NaBH4) undergoes hydrolysis in water or buffer solutions, resulting in a violent gas release reaction. The core reaction formula is as follows:

[0061] NaBH4 + 2H2O → NaBO2 + 4H2↑

[0062] This invention utilizes the hydrolysis reaction of sodium borohydride (NaBH4) in aqueous solution to release hydrogen gas (H2), which generates numerous micro- and nano-bubbles in situ, creating a physical foaming effect within the tissue. This foaming effect establishes instantaneous expansion pressure within the tissue's micropores, thereby achieving physical separation between cells and the extracellular matrix, assisting in cell membrane rupture and the expulsion of cellular contents, and improving decellularization efficiency. Simultaneously, the alkaline environment (pH approximately 9-10) generated during the reaction denatures cell membrane lipids and cytoplasmic proteins, also facilitating the penetration and washing of other decellularization solutions. Furthermore, the foaming treatment is characterized by its mildness and non-thermal damage, significantly enhancing the interfacial separation between cells and the matrix without disrupting the three-dimensional structure and scaffold morphology of collagen. It is suitable for the pretreatment of dense or cellular soft tissues (such as fish skin and placenta) for decellularization.

[0063] In the raw material preparation stage, pretreatment of tilapia skin, including thawing, removal of scales and adipose tissue, edge trimming, and pre-rinsing, removes surface impurities, foreign matter, and planktonic cells, reducing contamination sources and providing homogeneous tissue samples with intact structure and low contamination load for subsequent decellularization. Simultaneously, multiple rinses using low-temperature PBS and deionized water effectively maintain the original three-dimensional structure and collagen integrity of the tissue, reducing pre-damage caused by enzymatic hydrolysis or drastic pH fluctuations, and ensuring the consistency and controllability of the decellularization process.

[0064] In the gas foaming stage, hydrogen gas (H2) generated by the hydrolysis of sodium borohydride (NaBH4) in PBS solution is used to achieve in-situ foaming within the tissue. The released micro- and nano-bubbles can create localized instantaneous expansion pressure within the tissue structure, physically stripping the cell membrane and cell-matrix interface, thereby achieving rapid cell removal (i.e., decellularization). This method effectively reduces the risk of enzymatic damage to structural proteins (such as collagen) and achieves physical desorption of deep cellular components under relatively mild conditions. Furthermore, the alkaline environment (pH approximately 9-10) formed during the reaction facilitates the denaturation of cell membrane lipids and cytoplasmic proteins, enhancing the permeability and efficiency of subsequent washing steps, and overall improving the depth and integrity of the decellularization process.

[0065] During the washing stage, the foamed fish skin tissue is subjected to multiple rounds of agitation washing using PBS buffer at a speed of 110-120 rpm. This step aims to thoroughly remove residual NaBH4, alkaline intermediates, and cell debris, preventing them from affecting subsequent tissue structure or cell experiments. Simultaneously, slow stirring combined with multiple changes of the washing solution maximizes the removal of foam residue, avoiding secondary tissue damage or continued hydrolysis. Manual agitation can also be used. Multiple rounds of physical agitation washing also serve as a decellularization method, simultaneously removing residual chemicals and providing mild decellularization. Furthermore, agitation washing minimizes damage to the three-dimensional structure and scaffold morphology of the fish skin tissue's protease, ensuring high-quality fish skin tissue. Of course, depending on actual production needs and specific requirements of the fish skin tissue, further washing with antibiotic-containing saline can be used to improve the sterility and storage stability of the treated tissue, providing a clean and non-toxic sample basis for subsequent biological performance testing and in vitro / in vivo experiments.

[0066] During the protection and storage phase, placing decellularized fish skin tissue in a PBS solution containing antibiotics and storing it at 3-5°C effectively inhibits microbial growth and maintains the structural stability and biological activity of the tissue in a non-frozen state. Simultaneously, mild storage conditions prevent tissue drying, deformation, or collagen degradation, providing a time window and technical support for subsequent mechanical property testing, cell inoculation, or animal experiments.

[0067] The present application will be specifically described below through specific embodiments. The following embodiments are only some embodiments of the present application and are not intended to limit the present application.

[0068] Example 1:

[0069] A method for preparing a decellularized fish skin matrix bioscaffold includes the following steps:

[0070] (I) Fish skin pretreatment:

[0071] (1) Take the skin tissue of tilapia skin that has been processed, frozen and thawed; or select fresh tilapia skin, scrape off the remaining scales and remove redundant tissue and impurities, and trim the skin tissue into the required shape for later use; the processing, freezing and thawing process is as follows: Thaw the fresh frozen tilapia skin in deionized water at 3℃, scrape off the remaining scales and remove redundant tissue and impurities, trim the skin tissue into the required shape, rinse or wash it multiple times with deionized water and PBS buffer solution at 3℃, divide the processed skin tissue into portions and quickly freeze it at -80℃, and before use, take out the frozen skin tissue and soak it in PBS buffer solution at 3℃ to thaw;

[0072] (2) The fish skin tissue obtained in step (1) was washed with deionized water and PBS buffer solution with pH 7.2 and concentration of 0.01 mol / L.

[0073] (II) Gas foaming treatment:

[0074] (3) Weigh out sodium borohydride with a molar concentration of 1M, dissolve it in PBS buffer solution with a pH of 7.2 and a concentration of 0.01mol / L, stir until bubbles are clearly generated in the solution and most of the solid is dissolved, then put it into the fish skin tissue cleaned in step (2) for foaming treatment. The foaming time is 30min and the foaming temperature is 20℃.

[0075] (4) After the foaming process is completed, remove the fish skin tissue;

[0076] (III) Cleaning treatment:

[0077] (5) Use PBS buffer solution with pH 7.2 and concentration of 0.01 mol / L to repeatedly shake and wash the foamed fish skin tissue until no obvious bubbles are produced after shaking the soaked fish skin tissue.

[0078] (iv) Protection and storage:

[0079] (6) Store the cleaned fish skin tissue in a PBS buffer solution containing 1% (v / v) penicillin-streptomycin mixture (5000 U / mL). The pH of the PBS buffer solution is 7.2 and the concentration is 0.01 mol / L. Let it stand at 3°C ​​for 12 hours.

[0080] (7) After being taken out, freeze-dried, sterilized and packaged to make a dry finished product, thus obtaining a decellularized fish skin matrix biological scaffold.

[0081] Example 2:

[0082] A method for preparing a decellularized fish skin matrix bioscaffold includes the following steps:

[0083] (I) Fish skin pretreatment:

[0084] (1) Take the skin tissue of tilapia skin that has been processed, frozen and thawed; or select fresh tilapia skin, scrape off the remaining scales and remove redundant tissue and impurities, and trim the skin tissue into the required shape for later use; the processing, freezing and thawing process is as follows: Thaw the fresh frozen tilapia skin in deionized water at 4℃, scrape off the remaining scales and remove redundant tissue and impurities, trim the skin tissue into the required shape, rinse or wash it multiple times with deionized water and Hank's buffer solution at 4℃, divide the processed skin tissue into portions and quickly freeze it at -80℃, and before use, take out the frozen skin tissue and soak it in Hank's buffer solution at 4℃ to thaw;

[0085] (2) The fish skin tissue obtained in step (1) is cleaned with deionized water and Hank's buffer solution with pH 7.3 and a concentration of 0.01 mol / L.

[0086] (II) Gas foaming treatment:

[0087] (3) Weigh out sodium borohydride with a molar concentration of 2M, dissolve it in Hank's buffer solution with a pH of 7.3 and a concentration of 0.01mol / L, stir until bubbles are clearly generated in the solution and most of the solid dissolves, then put it into the fish skin tissue cleaned in step (2) for foaming treatment. The foaming time is 30min and the foaming treatment temperature is 22℃.

[0088] (4) After the foaming process is completed, remove the fish skin tissue;

[0089] (III) Cleaning treatment:

[0090] (5) Use Hank's buffer solution with pH 7.3 and concentration of 0.01 mol / L to repeatedly shake and wash the foamed fish skin tissue until no obvious bubbles are produced after shaking the soaked fish skin tissue.

[0091] (iv) Protection and storage:

[0092] (6) Store the cleaned fish skin tissue in Hank's buffer solution containing 1% (v / v) penicillin-streptomycin mixture (5000 U / mL). The pH of Hank's buffer solution is 7.3 and the concentration is 0.01 mol / L. Let it stand at 4°C for 18 hours.

[0093] (7) After being taken out, freeze-dried, sterilized and packaged to make a dry finished product, thus obtaining a decellularized fish skin matrix biological scaffold.

[0094] Example 3:

[0095] A method for preparing a decellularized fish skin matrix bioscaffold includes the following steps:

[0096] (I) Fish skin pretreatment:

[0097] (1) Take the skin tissue of tilapia skin that has been processed, frozen and thawed; or select fresh tilapia skin, scrape off the remaining scales and remove redundant tissue and impurities, and trim the skin tissue into the required shape for later use; the processing, freezing and thawing process is as follows: Thaw the fresh frozen tilapia skin in deionized water at 4℃, scrape off the remaining scales and remove redundant tissue and impurities, trim the skin tissue into the required shape, rinse or wash it multiple times with deionized water and D-Hank's buffer solution at 4℃, divide the processed skin tissue into portions and quickly freeze it at -80℃, and before use, take out the frozen skin tissue and soak it in D-Hank's buffer solution at 4℃ to thaw;

[0098] (2) The fish skin tissue obtained in step (1) is cleaned with deionized water and D-Hank's buffer solution with pH 7.4 and a concentration of 0.01 mol / L.

[0099] (II) Gas foaming treatment:

[0100] (3) Weigh out sodium borohydride with a molar concentration of 3M, dissolve it in D-Hank's buffer solution with a pH of 7.4 and a concentration of 0.01mol / L, stir until bubbles are clearly generated in the solution and most of the solid is dissolved, then put it into the fish skin tissue cleaned in step (2) for foaming treatment. The foaming time is 30min and the foaming treatment temperature is 25℃.

[0101] (4) After the foaming process is completed, remove the fish skin tissue;

[0102] (III) Cleaning treatment:

[0103] (5) Use D-Hank's buffer solution with pH 7.4 and concentration of 0.01 mol / L to repeatedly shake and wash the foamed fish skin tissue until no obvious bubbles are produced after shaking the soaked fish skin tissue.

[0104] (iv) Protection and storage:

[0105] (6) Store the cleaned fish skin tissue in D-Hank's buffer solution containing 1% (v / v) penicillin-streptomycin mixture (5000 U / mL). The pH of D-Hank's buffer solution is 7.4 and the concentration is 0.01 mol / L. Let it stand at 5°C for 24 hours.

[0106] (7) After being taken out, freeze-dried, sterilized and packaged to make a dry finished product, thus obtaining a decellularized fish skin matrix biological scaffold.

[0107] Example 4:

[0108] A method for preparing a decellularized fish skin matrix bioscaffold includes the following steps:

[0109] (I) Fish skin pretreatment:

[0110] (1) Take the skin tissue of tilapia skin that has been processed, frozen and thawed; or select fresh tilapia skin, scrape off the remaining scales and remove redundant tissue and impurities, and trim the skin tissue into the required shape for later use; the processing, freezing and thawing process is as follows: Thaw the fresh frozen tilapia skin in deionized water at 4℃, scrape off the remaining scales and remove redundant tissue and impurities, trim the skin tissue into the required shape, rinse or wash it multiple times with deionized water and PBS buffer solution at 4℃, divide the processed skin tissue into portions and quickly freeze it at -80℃, and before use, take out the frozen skin tissue and soak it in PBS buffer solution at 4℃ to thaw;

[0111] (2) The fish skin tissue obtained in step (1) is washed with deionized water and PBS buffer solution with pH 7.3 and concentration of 0.01 mol / L.

[0112] (II) Gas foaming treatment:

[0113] (3) Weigh out sodium borohydride with a molar concentration of 1M, dissolve it in PBS buffer solution with a pH of 7.3 and a concentration of 0.01mol / L, stir until bubbles are clearly generated in the solution and most of the solid is dissolved, then put it into the fish skin tissue cleaned in step (2) for foaming treatment. The foaming time is 60min and the foaming temperature is 22℃.

[0114] (4) After the foaming process is completed, remove the fish skin tissue;

[0115] (III) Cleaning treatment:

[0116] (5) Use PBS buffer solution with pH 7.3 and concentration of 0.01 mol / L to repeatedly shake and wash the foamed fish skin tissue until no obvious bubbles are produced after shaking the soaked fish skin tissue.

[0117] (iv) Protection and storage:

[0118] (6) Store the cleaned fish skin tissue in a PBS buffer solution containing 1% (v / v) penicillin-streptomycin mixture (5000 U / mL). The pH of the PBS buffer solution is 7.3 and the concentration is 0.01 mol / L. Let it stand at 4°C for 18 hours.

[0119] (7) After being taken out, freeze-dried, sterilized and packaged to make a dry finished product, thus obtaining a decellularized fish skin matrix biological scaffold.

[0120] Example 5:

[0121] A method for preparing a decellularized fish skin matrix bioscaffold includes the following steps:

[0122] (I) Fish skin pretreatment:

[0123] (1) Take the skin tissue of tilapia skin that has been processed, frozen and thawed; or select fresh tilapia skin, scrape off the remaining scales and remove redundant tissue and impurities, and trim the skin tissue into the required shape for later use; the processing, freezing and thawing process is as follows: Thaw the fresh frozen tilapia skin in deionized water at 4℃, scrape off the remaining scales and remove redundant tissue and impurities, trim the skin tissue into the required shape, rinse or wash it multiple times with deionized water and PBS buffer solution at 4℃, divide the processed skin tissue into portions and quickly freeze it at -80℃, and before use, take out the frozen skin tissue and soak it in PBS buffer solution at 4℃ to thaw;

[0124] (2) The fish skin tissue obtained in step (1) is washed with deionized water and PBS buffer solution with pH 7.3 and concentration of 0.01 mol / L.

[0125] (II) Gas foaming treatment:

[0126] (3) Weigh out sodium borohydride with a molar concentration of 3M, dissolve it in PBS buffer solution with a pH of 7.3 and a concentration of 0.01mol / L, stir until bubbles are clearly generated in the solution and most of the solid is dissolved, then put it into the fish skin tissue cleaned in step (2) for foaming treatment. The foaming time is 60min and the foaming treatment temperature is 22℃.

[0127] (4) After the foaming process is completed, remove the fish skin tissue;

[0128] (III) Cleaning treatment:

[0129] (5) Use PBS buffer solution with pH 7.3 and concentration of 0.01 mol / L to repeatedly shake and wash the foamed fish skin tissue until no obvious bubbles are produced after shaking the soaked fish skin tissue.

[0130] (iv) Protection and storage:

[0131] (6) Store the cleaned fish skin tissue in a PBS buffer solution containing 1% (v / v) penicillin-streptomycin mixture (5000 U / mL). The pH of the PBS buffer solution is 7.3 and the concentration is 0.01 mol / L. Let it stand at 4°C for 18 hours.

[0132] (7) After being taken out, freeze-dried, sterilized and packaged to make a dry finished product, thus obtaining a decellularized fish skin matrix biological scaffold.

[0133] The performance test results of the decellularized fish skin matrix bioscaffold prepared using the method of the present invention are as follows:

[0134] 1. Morphological analysis.

[0135] like Figure 3 As shown, the morphology and structure of the fish skin samples after gas foaming treatment in the examples were observed using a scanning electron microscope (TESCAN VEGA3, Czech Republic). To improve the conductivity of the samples, gold was sputtered onto their surface for 160 seconds. Figure 3 (a) Electron micrograph of a cross-section of pure fish skin matrix; Figure 3 (bd) are cross-sectional electron micrographs of decellularized fish skin matrix after foaming with different sodium borohydride concentrations (1-3M) and different foaming times (30 min or 60 min). Morphological analysis comparing the cross-sectional electron micrographs of pure fish skin and foamed fish skin shows that the foamed fish skin has a greater degree of pore opening and a significantly increased thickness. The average thickness of pure fish skin was 464.8 ± 25.3 μm, while the average thickness of foamed fish skin was 1123.1 ± 265.7 μm, significantly higher than the original thickness. This increased thickness confirms that the foaming experiment effectively opens the pores, enhances liquid permeability, and results in more thorough and uniform decellularization. Using ImageJ software (National Institutes of Health, USA), the collagen fiber porosity in the obtained cross-sectional SEM images was measured to be 78%. This demonstrates that the present invention can significantly improve decellularization efficiency.

[0136] from Figure 3 A comparison of the electron micrographs of the cross-sections of pure fish skin and foamed fish skin shows that the cross-section of the foamed fish skin still retains a porous structure, which theoretically can effectively promote cell adhesion, proliferation, and differentiation. Figure 4 As shown, to more clearly observe the integrity of the tissue structure and the presence of residual cells, hematoxylin and eosin (HE) staining was used. Figure 4 (a) is an image of a section stained with HE from pure fish skin matrix; Figure 4(b) is an HE-stained section of the decellularized fish skin matrix after foaming. The comparison of the HE-stained sections shows that the pink collagen / extracellular matrix structure is clear, maintaining the complete three-dimensional collagen scaffold of pure fish skin. This proves that the preparation method of this invention completely preserves the original extracellular matrix structure (performance), thus ensuring good biocompatibility and mechanical properties.

[0137] 2. Determination of DNA residue and assessment of tissue structure.

[0138] Highly Effective Reduction of DNA Residue: According to the standard document ASTM F3354-19, "Standard Guidelines for Evaluating the Decellularization Process of Extracellular Matrix," decellularized products require evaluation of cellular components, nucleic acid residues (including DNA), and the preservation of the extracellular matrix (ECM). This invention tested the DNA content of pure fish skin and foamed fish skin using a foaming technique. The DNA content of pure fish skin was 157.0 ± 9.5 ng / mg, and the DNA content of foamed fish skin was 29.6 ± 8.2 ng / mg, far below the clinical transplant safety upper limit of 50 ng / mg (the most commonly cited safety limit for "low immunogenicity" of decellularized materials internationally), which is recognized by many studies.

[0139] 3. Biocompatibility.

[0140] To observe cell adhesion and morphology on the scaffold surface, dual fluorescent staining (red: F-actin; blue: DAPI) was performed using phalloidin and DAPI. Phalloidin selectively binds to F-actin to label the cytoskeleton, while DAPI stained the cell nuclei to assess cell distribution, nuclear integrity, and morphology. After staining, images were captured using laser confocal microscopy (LSCM) to analyze cell spreading, cytoskeleton arrangement, and material cytocompatibility. Figure 5 As shown, where, Figure 5 (a) A photograph of actin (green) and cell nucleus (blue) staining after day 1; Figure 5 (b) Photographs of actin (green) and cell nuclei (blue) staining after day 5. Immunofluorescence assays of cell morphology show that the cells on the scaffold surface are spindle-shaped and spread out, with a clear skeletal structure, exhibiting good cell adhesion and morphological maintenance. On day 5, the cell number is significantly increased compared to day 1, the overall density is significantly higher, the intercellular spacing is reduced, and the cells are more tightly packed. Slight cell stacking or compression can be observed in some areas. The cell morphology gradually changes from an initial flattened shape to a long spindle or stretched shape, showing a certain directional distribution. This morphological change reflects spatial competition among cells in a localized high-density environment, suggesting that the cells on the scaffold surface have entered an active proliferative phase and maintain strong growth capacity.

[0141] 4. Mechanical properties.

[0142] To evaluate the mechanical properties of the prepared decellularized matrix materials, the fracture load, fracture strength, initial modulus, and elongation at break of natural tilapia skin matrix (TS-ECM) and decellularized matrix materials obtained under different foaming conditions were tested in both dry and wet states. The results are shown in Table 1.

[0143] Table 1 Results of Mechanical Data Analysis

[0144]

[0145] The mechanical properties of the original fish skin and the fish skin samples after gas foaming treatment in the examples were determined using a universal tensile testing machine (Instron 5965, USA). All fish skin samples were cut into strips of 5mm × 80mm, with a clamping distance of 10mm, a pre-tension of 0.02N, and a fixed tensile speed of 200mm / min. Load-elongation curves and mechanical property data were plotted.

[0146] Test results show that TS-ECM has a high initial modulus (121.9±19.7MPa) in the dry state, but the modulus is significantly reduced (77.1±28.3MPa) in the wet state, and the elongation at break (48.3±12%) is low, exhibiting a certain degree of brittleness.

[0147] In contrast, TS-dECM samples foamed with sodium borohydride (NaBH4) solutions of different concentrations significantly improved the material's flexibility and tensile ductility while maintaining a certain fracture strength. The sample treated with 3 mol / L NaBH4 solution for 30 minutes exhibited an initial modulus of 50.4 ± 3.4 MPa in the wet state and an elongation at break of 152.5 ± 34.1%, while its modulus in the dry state was 47.4 ± 12.1 MPa, demonstrating excellent tensile properties and a certain degree of structural stability, along with good biomimetic flexibility. Figure 6 ,in, Figure 6 (a) is the load-elongation curve. Figure 6 (b) represents the fracture load. Figure 6 (c) represents the fracture strength. Figure 6 (d) is the initial modulus. Figure 6 (e) represents the elongation at break. From Figure 6 As can be seen from the comparison of results under different treatment conditions, the fracture load and fracture strength of the material increase with the increase of NaBH4 concentration, while the initial modulus changes relatively steadily. This indicates that the foaming and decellularization method effectively improves the toughness and ductility of the material without significantly sacrificing its rigidity, thus enhancing the material's service adaptability in tissue engineering scaffolds.

[0148] In other words, compared with unfoamed fish skin tissue, the decellularized fish skin matrix bioscaffold of this invention, under simulated wet conditions, exhibits significantly improved fracture load, fracture strength, and elongation at break regardless of the concentration of sodium borohydride (NaBH4) solution used for foaming treatment. This demonstrates the superior tissue repair performance of the tilapia skin decellularized matrix bioscaffold prepared by this invention. In terms of mechanical properties, it exhibits superior overall performance compared to natural matrices, making it particularly suitable for artificial ligaments or soft tissue repair materials that require both flexibility and a certain level of mechanical strength.

[0149] In summary, the process of this invention is simple and convenient. It only requires weighing sodium borohydride, dissolving it, and then immersing the tissue or organ in it to achieve the foaming effect. Of course, the specific method also includes subsequent treatment of the tissue or organ and post-foaming elution of the foaming solution, but these operations are essential procedures for decellularization experiments. This patent simplifies the main decellularization process. The only parameters that need to be changed in the preparation method are the molar concentration of sodium borohydride and the foaming time. Higher concentrations can be used to reduce the reaction time, or lower concentrations can be used to increase the reaction time, making the parameters flexible and controllable. This invention solves the problems of complex preparation processes, significant material damage, and limited applicability in existing technologies, achieving deep and effective decellularization and improving product quality. It fundamentally solves the problem of large-scale industrial production being difficult due to complex processes and high costs, promoting the development of decellularized fish skin matrix bioscaffolds.

[0150] Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention should be protected by the present invention.

Claims

1. A method for preparing a decellularized fish skin matrix bioscaffold, characterized in that, Includes the following steps: (I) Fish skin pretreatment: (1) Take the skin tissue of tilapia skin that has been processed, frozen and thawed; or select fresh tilapia skin, scrape off the remaining scales and remove redundant tissue and impurities, and trim the skin tissue into the required shape for later use. (2) The fish skin tissue obtained in step (1) is cleaned with deionized water and a certain concentration of phosphate buffer solution. (II) Gas foaming treatment: (3) Weigh out a certain molar concentration of sodium borohydride, dissolve it in a certain concentration of phosphate buffer solution, stir until bubbles are clearly generated in the solution and most of the solid dissolves, then put it into the fish skin tissue cleaned in step (2) for foaming treatment. (4) After the foaming process is completed, remove the fish skin tissue; (III) Cleaning treatment: (5) Use a certain concentration of phosphate buffer solution to repeatedly shake and wash the foamed fish skin tissue until no obvious bubbles are produced after shaking the soaked fish skin tissue. (iv) Protection and storage: (6) Store the cleaned fish skin tissue in a phosphate buffer solution containing a mixture of penicillin and streptomycin and let it stand at low temperature for 12-24 hours. (7) After being taken out, freeze-dried, sterilized and packaged to make a dry finished product, thus obtaining a decellularized fish skin matrix biological scaffold.

2. The method for preparing the decellularized fish skin matrix bioscaffold according to claim 1, characterized in that, In step (1), the processing, freezing, and thawing process is as follows: Thaw the fresh frozen tilapia skin in deionized water at 3-5℃, scrape off the remaining scales and remove redundant tissues and impurities, trim the skin tissue into the required shape, rinse or wash it multiple times with deionized water and phosphate buffer solution at 3-5℃, divide the processed skin tissue into portions and quickly freeze it at -80℃, and before use, take out the frozen skin tissue and soak it in phosphate buffer solution at 3-5℃ to thaw.

3. The method for preparing the decellularized fish skin matrix bioscaffold according to claim 1, characterized in that: In step (3), the molar concentration of sodium borohydride is 1-3M, the foaming time is 30min or 60min, and the foaming treatment temperature is 20-25℃.

4. The method for preparing the decellularized fish skin matrix bioscaffold according to claim 1, characterized in that: In steps (2), (3), (5), and (6), the pH of the phosphate buffer solution is 7.2-7.4, and the concentration is 0.01 mol / L.

5. The method for preparing the decellularized fish skin matrix bioscaffold according to claim 2, characterized in that: The phosphate buffer solution used in rinsing, washing, and soaking thawing processes has a pH of 7.2-7.4 and a concentration of 0.01 mol / L.

6. The method for preparing the decellularized fish skin matrix bioscaffold according to claim 1, characterized in that: The phosphate buffer solution is any one of PBS buffer, Hank's buffer, or D-Hank's buffer.

7. The method for preparing the decellularized fish skin matrix bioscaffold according to claim 1, characterized in that: In step (6), the phosphate buffer solution contains a penicillin-streptomycin mixture with a volume concentration of 1% (v / v) and a bioactive substance concentration of 5000 U / mL. The low temperature environment is set at 3-5℃ and the standing time is 18 hours.

8. A decellularized fish skin matrix bioscaffold, characterized in that: It is prepared by the preparation method according to any one of claims 1-7.

9. The decellularized fish skin matrix bioscaffold according to claim 8, characterized in that: The DNA content of this decellularized fish skin matrix bioscaffold was 29.6 ± 8.2 ng / mg.

10. The application of a decellularized fish skin matrix bioscaffold as described in any one of claims 8-9 in the repair of injuries to tissues such as skin, tendons, ligaments, nerves, blood vessels, and bones.

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