Fish skin-based biological patch, preparation method thereof, and medical material

Abstract

The present invention provides a fish skin-based biopatch, a preparation method, and a medical material. The fish skin-based biopatch comprises a sheet-like body containing a collagen matrix. The fish skin-based biopatch is prepared from fish skin through a depigmentation and decellularization process. The biopatch has a degradation cycle of 7 to 180 days, a fat content of 1% to 19%, and a residual DNA content of less than 50 ng / mg. Neither the fish skin structure nor its properties are affected in the biopatch. Furthermore, the biopatch has a wide degradation cycle, suitable for repairing different tissues.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a fish skin-based biological patch, a preparation method thereof, and a medical material. Background Art

[0002] In recent years, marine fisheries have experienced rapid development, and marine resources have been extensively developed and utilized. Consequently, a large amount of aquatic product processing waste, such as fish skin, fish bones, and fish scales, is generated. While a small portion is used to make feed and fertilizer, the vast majority of the waste is fish skin. However, since these aquatic product processing wastes contain a large amount of collagen, their full utilization could significantly increase the economic value of aquatic waste, further promoting the high-value utilization of aquatic products.

[0003] To fully utilize fish skin, especially as a biomedical material, whether for collagen extraction or implants, it is necessary to process the skin's pigments and skin cells. Because fish skin pigments are contaminated within the skin's inner layer, conventional methods for removing the pigments inevitably damage the skin's structure and properties. Summary of the Invention

[0004] Based on this, it is necessary to provide a fish skin-based biological patch and its preparation method and medical material that will not damage the structure and performance of fish skin.

[0005] A first aspect of the present invention provides a fish skin-based biological patch, comprising a sheet-like body containing a collagen matrix; the fish skin-based biological patch is prepared from fish skin through a treatment process including depigmentation and decellularization; the fish skin-based biological patch has a degradation cycle of 7d-180d, a fat content of 1%-19%, and a residual DNA content of less than 50ng / mg.

[0006] In some embodiments, the fish skin-based biological patch has a tensile strength of 29.24 MPa-43.56 MPa, a breaking strength of 379.9 N-430.5 N, and a tear strength of 4.31 N-6.09 N.

[0007] A second aspect of the present invention provides a method for preparing a fish skin-based biological patch, comprising the following steps:

[0008] performing a degreasing pretreatment on the fish skin to prepare a pretreated fish skin;

[0009] placing the pretreated fish skin in an aqueous solution, performing a depigmentation treatment and an optional partial decellularization treatment on the pretreated fish skin to prepare a depigmented fish skin;

[0010] performing a cell-free treatment on the depigmented fish skin to prepare the fish skin-based biological patch;

[0011] Wherein, the liquid replacement cycle of the depigmentation treatment is 1h-4h.

[0012] In some embodiments, the fluid replacement cycle of the depigmentation treatment is 2h-3h.

[0013] In some embodiments, the depigmentation treatment comprises at least one of the following conditions:

[0014] (1) The temperature of the depigmentation treatment is 0°C-60°C;

[0015] (2) The aqueous solution further comprises one or more additives selected from the group consisting of a penetration enhancer, an adsorption medium, an acid-base regulator, and a bleaching agent.

[0016] In some embodiments, the additive contained in the aqueous solution includes a penetration enhancer, and the penetration enhancer has at least one of the following characteristics:

[0017] (1) The penetration enhancer includes one or more of ethanol, ethylene glycol, sodium lauryl sulfate, hexadecyltrimethylammonium bromide, azone and Triton X-100;

[0018] (2) The mass proportion of the penetration enhancer in the aqueous solution is 0.1%-10%, and can be optionally 0.3%-0.5%.

[0019] In some embodiments, the additive contained in the aqueous solution includes an adsorption medium having at least one of the following characteristics:

[0020] (1) The adsorption medium is selected from one or more of activated carbon, corn starch, masterbatch and calcium chloride;

[0021] (2) The mass proportion of the adsorption medium in the aqueous solution is 5%-20%, and can be optionally 8%-10%.

[0022] In some embodiments, the additive contained in the aqueous solution includes an acid-base regulator, and the acid-base regulator includes one or more of an acid regulator and an alkaline regulator; the acid regulator includes one or more of citric acid and acetic acid, and the mass proportion of the acid regulator in the aqueous solution is 0.001%-0.02%, and optionally 0.002%-0.005%;

[0023] The alkaline regulator includes one or more of ammonia water and potassium carbonate. The mass proportion of the alkaline regulator in the aqueous solution is 0.2%-10%, and can be optionally 0.5%-5%.

[0024] In some embodiments, the additive contained in the aqueous solution includes a bleaching agent, and the bleaching agent includes one or more of hydrogen peroxide and sodium hydroxide; the mass proportion of the hydrogen peroxide in the aqueous solution is 0.5%-10%, and can be optionally 3%-5%; the mass proportion of the sodium hydroxide in the aqueous solution is 0.05%-0.3%, and can be optionally 0.1%-0.2%.

[0025] In some embodiments, the step of pre-treating the fish skin by degreasing comprises: rinsing the fish skin with running water for 5-10 minutes; placing the fish skin in a starch solution for treatment, wherein the concentration of the starch solution is 1%-10%, optionally 3%-5%.

[0026] In some embodiments, the step of performing a cell-free treatment on the depigmented fish skin comprises:

[0027] The depigmented fish skin is subjected to acellular treatment by using a Triton solution containing 0.1% to 10% of Triton X-100 by weight.

[0028] The third aspect of the present invention provides a medical material, comprising the fish skin-based biological patch of the first aspect of the present invention or the fish skin-based biological patch prepared by the preparation method of the second aspect of the present invention.

[0029] The above-mentioned fish skin-based biological patch, its preparation method and medical material, wherein the fish skin-based biological patch is prepared through a treatment process including depigmentation and decellularization, the fish skin structure and fish skin performance are not affected, and the finally prepared fish skin-based biological patch has a wide degradation cycle and can meet the repair needs of different tissues.

[0030] The above-mentioned method for preparing a fish skin-based biological patch uses a water bath method to depigment the pretreated fish skin, with a controlled liquid exchange cycle of 1-4 hours. This mild depigmentation condition leaves the fish skin structure and properties intact, facilitating tissue repair. Furthermore, the preparation method is simple, involves few steps, requires a short processing time, and is environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is an appearance diagram of the fish skin-based biological patch in Example 8;

[0032] Figure 2 This is the appearance of the fish skin-based biological patch in Comparative Example 2;

[0033] Figure 3 This is the appearance of the fish skin-based biological patch in Comparative Example 1;

[0034] Figure 4 The state of melanin precipitation of fish skin in solution after water bath treatment for 4 hours for Example 1 and Example 2;

[0035] Figure 5 The state of melanin precipitation of fish skin in the solution after the water bath treatment for 4 hours in Example 1-5;

[0036] Figure 6 The state of melanin precipitation of fish skin in solution after water bath treatment for 4 hours for Example 2 and Example 6;

[0037] Figure 7 The front state of the fish skin after being treated in a water bath for 4 hours for Example 2 and Examples 6-8;

[0038] Figure 8 The reverse side of the fish skin after the water bath treatment for 4 hours for Example 2 and Example 6-8;

[0039] Figure 9 The state of melanin precipitation of fish skin in solution after water bath treatment for 4 hours for Example 1 and Example 13;

[0040] Figure 10 The state of melanin precipitation of fish skin in the solution after water bath treatment for 4 hours for Examples 13-16;

[0041] Figure 11 This is a sample tissue diagram of the fish skin-based biological patch of Example 17 degrading in rats for 2 weeks. DETAILED DESCRIPTION

[0042] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the accompanying drawings. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0044] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0045] In the present invention, when referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Furthermore, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0046] Only certain numerical ranges are specifically disclosed herein. However, any lower limit may be combined with any upper limit to form an unspecified range; and any lower limit may be combined with other lower limits to form an unspecified range, and similarly, any upper limit may be combined with any other upper limit to form an unspecified range. In addition, each individually disclosed point or single value may itself serve as a lower limit or upper limit and be combined with any other point or single value, or with other lower limits or upper limits, to form an unspecified range.

[0047] The temperature parameters in the present invention, unless otherwise specified, allow for either constant temperature treatment or treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range controlled by the instrument.

[0048] In the description of the invention, “plurality” means at least two, for example, two, three, etc., unless otherwise clearly and specifically defined.

[0049] Unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form a new technical solution. Unless otherwise specified, all technical features and optional technical features of the present invention can be combined with each other to form a new technical solution.

[0050] Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, but are preferably performed sequentially.

[0051] When using fish skin as a biomedical material, it needs to be depigmented and decellularized; however, when removing the pigment from the fish skin in related technologies, it is inevitable that the structure and properties of the fish skin will be damaged.

[0052] Furthermore, in clinical applications, degradation performance is a key indicator of biomedical materials. In common gynecological repair applications, anti-adhesion membranes need to degrade within 1-2 weeks, depending on the woman's menstrual cycle. In oral guided tissue regeneration membranes, tissue ingrowth takes more than 2 weeks, and the membranes degrade within a month or even longer. Furthermore, in sports medicine, rotator cuff patches require a longer degradation period of 3-6 months to meet the needs of tendon tissue repair and regeneration.

[0053] Therefore, when fish skin is used to repair different tissues, the fish skin is required to have different degradation cycles.

[0054] In response to the aforementioned issues, the first aspect of the present invention provides a fish skin-based biopatch comprising a sheet-like body containing a collagen matrix. The fish skin-based biopatch is prepared from fish skin through a process that includes depigmentation and decellularization. The fish skin-based biopatch has a degradation cycle of 7 to 180 days, a fat content of 1% to 19%, and a residual DNA content of less than 50 ng / mg. The depigmentation and decellularization processes preserve the fish skin's structure and properties, and the resulting fish skin-based biopatch has a wide range of degradation cycles, meeting the repair needs of different tissues.

[0055] It should be noted that the "d" mentioned above stands for day.

[0056] The degradation cycle of the fish skin-based biopatch is 7 days to 180 days; for example, it can be 7 days, 14 days, 21 days, 30 days, 60 days, 90 days, 120 days, 150 days, 180 days, or a range between any two of the aforementioned values. The length of the degradation cycle of the fish skin-based biopatch is related to the depigmentation method used during the preparation process. Changing the depigmentation method may also change the degradation cycle of the resulting fish skin-based biopatch.

[0057] The fat content of the fish skin-based biopatch is 1%-19%. For example, it can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or any range between any two of these values. When the fat solids content of the fish skin-based biopatch is within the above range, tissue repair is facilitated.

[0058] The fat content of fish skin-based biological patches is less than 50ng / mg, which can avoid potential risks such as infection, tumorigenesis, and immune rejection.

[0059] In some embodiments, the fish skin-based biological patch has a tensile strength of 29.24 MPa-43.56 MPa, a breaking strength of 379.9 N-430.5 N, and a tear strength of 4.31 N-6.09 N. When the tensile strength, breaking strength, and suture strength of the fish skin-based biological patch are within the above ranges, the mechanical strength of the biomaterial is improved.

[0060] The second aspect of the present invention provides a method for preparing the fish skin-based biological patch of the first aspect, comprising the following steps: performing a degreasing pretreatment on the fish skin to prepare pretreated fish skin; placing the pretreated fish skin in an aqueous solution, performing a depigmentation treatment and an optional partial decellularization treatment on the pretreated fish skin to prepare depigmented fish skin; performing a decellularization treatment on the depigmented fish skin to prepare a fish skin-based biological patch; wherein the liquid change cycle of the depigmentation treatment is 1 hour to 4 hours.

[0061] It should be noted that the liquid change cycle refers to the time interval between two liquid changes when the pretreated fish skin is placed in the aqueous solution for depigmentation treatment; that is, the aqueous solution used for depigmentation treatment of the pretreated fish skin is replaced once every liquid change cycle.

[0062] The depigmentation treatment liquid change cycle is 1 hour to 4 hours. When the liquid change cycle is set within the above range, the fish skin structure and fish skin properties are not affected, and the depigmentation effect is significant over time. As an example, the liquid change cycle can be 1 hour, 1.2 hours, 1.5 hours, 1.8 hours, 2 hours, 2.3 hours, 2.5 hours, 2.8 hours, 3 hours, 3.2 hours, 3.5 hours, 3.8 hours, 4 hours, or a range between any two of the above values.

[0063] It should be noted that the "optional partial decellularization treatment" mentioned above refers to the partial depigmentation method used during the depigmentation treatment, and the fish skin may be decellularized at the same time.

[0064] As can be understood, the method for preparing the fish skin-based biological patch of the present invention utilizes a water bath method to depigment the pretreated fish skin, with a controlled liquid exchange cycle of 1-4 hours. This mild depigmentation condition leaves the fish skin structure and properties unaffected, facilitating tissue repair. Furthermore, the preparation method is simple, involves few steps, requires a short processing time, and is environmentally friendly.

[0065] After the pretreated fish skin is depigmented by a water bath method, the fish skin needs to be vigorously stirred; the stirring rate is 10 rpm-50 rpm; for example, it can be but is not limited to 10 rpm, 15 rpm, 20 rpm, 25 rpm, 30 rpm, 35 rpm, 40 rpm, 45 rpm, 50 rpm or a range between any two of the above values.

[0066] In some embodiments, the liquid replacement cycle of the depigmentation treatment is 2 hours to 3 hours.

[0067] In some embodiments, the depigmentation treatment temperature is 0°C-60°C; for example, it can be but is not limited to 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C or a range between any two of the above values.

[0068] As a possible embodiment, the depigmentation aqueous solution further contains additives to enhance the depigmentation effect of fish skin. The additives are selected from one or more of a penetration enhancer, an adsorption medium, an acid-base regulator, and a bleaching agent. By adding different additives, the degradation cycle of fish skin can be effectively regulated while maintaining its excellent biological properties, allowing for the repair of different tissues.

[0069] As a possible implementation, the additives contained in the aqueous solution include a penetration enhancer; the addition of the penetration enhancer helps the surface pigment to dissolve in the solution, thereby accelerating the depigmentation process.

[0070] In some optional embodiments, the penetration enhancer includes one or more of ethanol, ethylene glycol, sodium dodecyl sulfate (abbreviated as SDS)-, cetyltrimethylammonium bromide (abbreviated as CTAB), azone and Triton X-100 (abbreviated as Triton).

[0071] In some optional embodiments, the mass proportion of the penetration enhancer in the aqueous solution is 0.1%-10%; when the mass proportion of the penetration enhancer in the aqueous solution is within the above range, it can effectively promote the precipitation of the pigment in the solution. As an example, the mass proportion of the penetration enhancer in the aqueous solution can be but is not limited to 0.1%, 0.3%, 0.5%, 0.7%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10% or a range between any two of the above values. Optionally, the mass proportion of the penetration enhancer in the aqueous solution is 0.3%-0.5%.

[0072] In some embodiments, the additive contained in the aqueous solution is an adsorption medium. The addition of the adsorption medium helps the pigment to be adsorbed on the adsorption medium, avoids the accumulation of the pigment layer in the solution, and prevents the pigment from adhering to the rough surface of the fish skin.

[0073] In some optional embodiments, the adsorption medium includes one or more of activated carbon, corn starch, masterbatch and calcium chloride.

[0074] In some optional embodiments, the mass proportion of the adsorption medium in the aqueous solution is 5%-20%; when the mass proportion of the adsorption medium in the aqueous solution is within the above range, it helps to timely adsorb the pigment and avoid pigment deposition. As an example, the mass proportion of the adsorption medium in the aqueous solution can be, but is not limited to, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or a range between any two of the above values. Optionally, the mass proportion of the adsorption medium in the aqueous solution is 8%-10%.

[0075] In some embodiments, the additive contained in the aqueous solution is a acid-base regulator, which includes one or more of an acidic regulator and an alkaline regulator; adding the acid-base regulator can promote the precipitation of pigments on the surface of the fish skin.

[0076] In some optional embodiments, the acidity regulator includes one or more of citric acid and acetic acid.

[0077] In some optional embodiments, the mass proportion of the acidic regulator in the aqueous solution is 0.001%-0.02%; when the mass proportion of the acidic regulator in the aqueous solution is within the above range, it can further promote the precipitation of the pigment in the solution. As an example, the mass proportion of the acidic regulator in the aqueous solution can be, but is not limited to, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.011%, 0.012%, 0.013%, 0.014%, 0.015%, 0.016%, 0.017%, 0.018%, 0.019%, 0.02% or a range between any two of the above values. Optionally, the mass proportion of the acidic regulator in the aqueous solution is 0.002%-0.005%.

[0078] In some optional embodiments, the alkaline regulator includes one or more of aqueous ammonia and potassium carbonate.

[0079] In some optional embodiments, the mass proportion of the alkaline regulator in the aqueous solution is 0.2%-10%; when the mass proportion of the alkaline regulator in the aqueous solution is within the above range, the pigment can be further promoted to precipitate in the solution. As an example, the mass proportion of the alkaline regulator in the aqueous solution can be, but is not limited to, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10% or a range between any two of the above values. Optionally, the mass proportion of the alkaline regulator in the aqueous solution is 0.5%-5%.

[0080] As a possible implementation manner, the additive contained in the aqueous solution is a bleaching agent, which can bleach the surface of the fish skin.

[0081] In some alternative embodiments, the bleaching agent includes one or more of hydrogen peroxide and sodium hydroxide.

[0082] In some optional embodiments, the mass proportion of hydrogen peroxide in the aqueous solution is 0.5%-10%; when the mass proportion of hydrogen peroxide in the aqueous solution is within the above range, it can further promote the yellowing and whitening of the pigment on the surface of the fish skin. As an example, the mass proportion of hydrogen peroxide in the aqueous solution can be, but is not limited to, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, or a range between any two of the above values. Optionally, the mass proportion of hydrogen peroxide in the aqueous solution is 3%-5%.

[0083] In some optional embodiments, the mass proportion of sodium hydroxide in the aqueous solution is 0.05%-0.3%; when the mass proportion of sodium hydroxide in the aqueous solution is within the above range, it can further promote the yellowing and whitening of the pigment on the surface of the fish skin. As an example, the mass proportion of sodium hydroxide in the aqueous solution can be, but is not limited to, 0.05%, 0.08%, 0.1%, 0.13%, 0.15%, 0.17%, 0.2%, 0.22%, 0.25%, 0.28%, 0.3% or a range between any two of the above values. Optionally, the mass proportion of sodium hydroxide in the aqueous solution is 0.1%-0.2%.

[0084] In some embodiments, after the fish skin is depigmented using sodium hydroxide and / or hydrogen peroxide, the depigmented fish skin can be placed in a hypertonic or hypotonic solution for further decolorization.

[0085] It should be noted that during the above-mentioned depigmentation process, one or more additives may be added simultaneously. For example, a penetration enhancer and an adsorption medium may be used simultaneously, a penetration enhancer and an acid-base modifier may be used simultaneously, an adsorption medium and an acid-base modifier may be used simultaneously, or a penetration enhancer, an adsorption medium, and an acid-base modifier may be used simultaneously. It should be noted that after depigmenting the fish skin with a penetration enhancer and / or an adsorption medium and / or an acid-base modifier, the fish skin may further be depigmented with hydrogen peroxide and sodium hydroxide.

[0086] For example, when the additive contained in the depigmentation aqueous solution is a bleach, the degradation period of the obtained biological patch is about 1 week; when the additive contained in the depigmentation aqueous solution is a bleach and a penetration enhancer, the degradation period of the obtained biological patch is about 2 weeks; when the additive contained in the depigmentation aqueous solution is an acid-base regulator and no bleach is present, the degradation period of the obtained biological patch is about 1 month; when the additive contained in the depigmentation aqueous solution is a penetration enhancer and no acid-base regulator or bleach is present, the degradation period of the obtained biological patch is 3-6 months.

[0087] When a penetration enhancer (eg, Triton X-100) is used, depigmentation is accompanied by incomplete decellularization.

[0088] In some embodiments, the step of pre-treating the fish skin for fat removal includes: rinsing the fish skin with running water; and treating the fish skin in a starch solution. Rinsing the fish skin with running water can remove visible yellow grease on the surface of the fish skin. Treating the fish skin with the starch solution further removes fat and impurities, such as oil, from the surface of the fish skin without damaging the surface of the fish skin; it also removes oil and dirt.

[0089] Optionally, the flushing time is 5 min-10 min; for example, it can be but not limited to 5 min, 5.5 min, 6 min, 6.5 min, 7 min, 7.5 min, 8 min, 8.5 min, 9 min, 9.5 min, 10 min or a range between any two of the above values.

[0090] It should be noted that since fish skin is animal-derived tissue, storage at room temperature or above -4°C can easily lead to rotting. Therefore, fish skin is typically stored in a freezer at -20°C or -80°C. Because fish skin is frequently used in experiments, it is temporarily stored in a -20°C freezer after being aliquoted. Therefore, before defatting the fish skin, it is removed from the -20°C freezer and placed in a -4°C freezer for slow thawing overnight.

[0091] In some optional embodiments, the concentration of the starch solution is 1%-10%; for example, but not limited to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the above values. Optionally, the concentration of the starch solution is 3%-5%.

[0092] As a possible implementation method, the fish skin is repeatedly cleaned three times with a starch solution.

[0093] In some embodiments, the pretreated fish skin obtained after defatting pretreatment is immersed in a cryoprotectant (such as sucrose or trehalose) for 10-20 minutes, the surface moisture is removed, and then the pretreated fish skin is placed in a -20°C refrigerator and repeatedly frozen and thawed, and the freezing and thawing is repeated three times.

[0094] In some optional embodiments, the step of performing a cell-free treatment on the depigmented fish skin includes: performing a cell-free treatment on the depigmented fish skin using Triton X-100.

[0095] Optionally, the depigmented fish skin is decellularized using a Triton solution containing 0.1-10% by mass of Triton X-100.

[0096] The third aspect of the present invention provides a medical material, comprising the fish skin-based biological patch of the first aspect of the present invention or the fish skin-based biological patch prepared by the preparation method of the second aspect of the present invention.

[0097] The technical solution of the present invention is described in detail below with reference to specific embodiments.

[0098] It should be noted that if specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions were used. Reagents or instruments used without specifying the manufacturer are all commercially available conventional products.

[0099] Example 1

[0100] Remove the fish skin from the -20°C freezer and place it in a -4°C refrigerator for slow overnight thawing. Rinse the thawed fish skin under running tap water to remove any visible yellow grease. Maintain a constant water flow rate during the rinse process, and rinse the fish skin for 10 minutes.

[0101] The fish skin was cleaned with a 3% starch solution to remove surface fat, and the cleaning was repeated three times. The sample that had undergone non-damage degreasing was placed in a 15% sucrose solution containing a cryoprotectant solution and immersed for 30 minutes, after which the surface moisture was removed. The fish skin was then placed in a -20°C refrigerator and subjected to repeated freeze-thaw cycles, with freeze-thaw cycles repeated three times to obtain the pretreated fish skin.

[0102] The pretreated fish skin is immersed in an aqueous solution and depigmented, and after immersion and stirring for 2 hours, it is taken out, washed, and further treated with the aqueous solution to remove surface melanin to obtain a depigmented fish skin;

[0103] The depigmented fish skin is placed in a solution of 0.5% by mass of Triton X-100 for decellularization, and then freeze-dried to obtain a fish skin-based biological patch.

[0104] Example 2

[0105] Remove the fish skin from the -20°C freezer and place it in a -4°C refrigerator for slow overnight thawing. Rinse the thawed fish skin under running tap water to remove any visible yellow grease. Maintain a constant water flow rate during the rinse process, and rinse the fish skin for 10 minutes.

[0106] The fish skin was cleaned with a 3% starch solution to remove surface fat, and the cleaning was repeated three times. The sample that had undergone non-damage degreasing was placed in a 15% sucrose solution containing a cryoprotectant solution and immersed for 30 minutes, after which the surface moisture was removed. The fish skin was then placed in a -20°C refrigerator and subjected to repeated freeze-thaw cycles, with freeze-thaw cycles repeated three times to obtain the pretreated fish skin.

[0107] The pretreated fish skin is immersed in an aqueous solution containing sodium dodecyl sulfate (SDS) to perform a depigmentation treatment on it, stirred, and taken out after the immersion and stirring treatment for 2 hours, washed, and the solution is changed, and the aqueous solution treatment is continued for a total of 4 hours to remove surface melanin and obtain a depigmented fish skin; wherein the mass proportion of sodium dodecyl sulfate (SDS) in the aqueous solution is 0.5%;

[0108] The depigmented fish skin was placed in a Triton X-100 solution for decellularization. After 24 hours, the fish skin was washed with distilled water to completely remove the Triton X-100 remaining on the surface. The fish skin was then freeze-dried to obtain a fish skin-based biological patch. The mass proportion of Triton X-100 in the Triton X-100 solution was 0.5%.

[0109] Example 3-17

[0110] The preparation methods of the fish skin-based biological patches in Examples 3-17 are basically similar to the preparation method of the fish skin-based biological patch in Example 1, with the main difference being that at least one of the type and amount of the additive is different, as shown in Table 1.

[0111] Comparative Example 1

[0112] The difference between Comparative Example 1 and Example 1 is that the fish skin is only subjected to a degreasing pretreatment to prepare the pretreated fish skin, and no depigmentation and decellularization treatment is performed.

[0113] Comparative Example 2

[0114] The difference between Comparative Example 2 and Example 1 is that the pretreated fish skin is manually depigmented, and all other aspects are the same. The process of manual depigmentation is as follows:

[0115] The pretreated fish skin is immersed in a dilute acetic acid solution with a mass percentage concentration of 0.05% for 2 minutes. After the fish skin initially swells, it is taken out and the pigment layers on the inner and outer surfaces of the fish skin are removed with a scraper to obtain depigmented fish skin.

[0116] The parameter settings of the above embodiments are shown in Table 1.

[0117] Table 1

[0118]

[0119]

[0120] In Table 1, SDS represents sodium dodecyl sulfate, and CTAB represents cetyltrimethylammonium bromide.

[0121] The appearance of the fish skin-based biological patch prepared in Example 8 is shown in FIG. Figure 1As shown, the appearance of the fish skin-based biological patch prepared in Comparative Example 2 is as follows Figure 2 As shown, the appearance of the fish skin-based biological patch prepared in Comparative Example 1 is as follows Figure 3 shown.

[0122] Depend on Figure 1-2 and Figure 3 It can be seen from the comparison that after depigmentation and decellularization, the melanin on the surface of the fish skin is completely removed; Figure 1 and Figure 2 The comparison shows that the depigmentation effect of adding additives to the aqueous solution is better than that of manual depigmentation, and the surface of the biological patch is uniform without defects such as holes and scratches, which will affect the use effect.

[0123] It should be noted that the depigmentation methods described in the above examples can be combined to enhance the depigmentation effect. For example, thawed fish skin is subjected to a fat-free pretreatment to obtain pretreated fish skin. This pretreated fish skin is then depigmented using the depigmentation method described in Example 1, followed by further depigmentation according to the depigmentation method described in Example 16, and then decellularized to produce a fish skin-based biological patch. Compared to Examples 1 and 16, the resulting fish skin-based biological patch exhibits improved depigmentation and further reduces its fat content and residual DNA.

[0124] 2. Comparison of fish skin conditions

[0125] In Example 1 and Example 2, the state of melanin precipitation of fish skin in the solution after water bath treatment for 4 hours is as follows: Figure 4 As shown, Figure 4 From left to right, they correspond to Example 1 and Example 2 respectively. Figure 4 It can be seen that after the same treatment time, the addition of the penetration enhancer in Example 2 can significantly promote the precipitation of melanin, indicating that the decolorization of the fish skin is accelerated after the addition of the penetration enhancer.

[0126] The state of melanin precipitation of fish skin in the solution after 4 hours of water bath treatment in Example 1-5 is shown in the figure. Figure 5 From left to right are Example 2, Example 5, Example 4, Example 3 and Example 1. The decolorization results are shown in Table 2.

[0127] Table 2

[0128]

[0129] Depend on Figure 5As shown in Table 2, after a relatively short treatment time, the effects of a pure water bath, ethanol, CTAB, and Triton X-ray diffraction were similar, while the addition of SDS was more effective, essentially removing the surface pigment from the fish skin. Over time, the surface pigment was completely removed from all five groups of samples.

[0130] After Example 2 and Example 6 were treated in a water bath for 4 hours, the melanin precipitation state of the fish skin in the solution was as follows: Figure 6 As shown; Figure 6 From left to right are Example 2 and Example 6. Figure 6 It can be seen that after the same treatment time, the simultaneous addition of the adsorbent in Example 6 can absorb the melanin in the solution, and the upper layer solution is clarified, making it difficult for the melanin to adhere to the rough surface of the fish skin.

[0131] The front state of the fish skin after the water bath treatment for 4 hours in Example 2 and Example 6-8 is as follows Figure 7 As shown; Figure 7 From left to right are Example 7, Example 6, Example 8 and Example 2. The reverse side of the fish skin after water bath treatment for 4 hours in Example 2 and Examples 6-8 is as follows Figure 8 As shown; Figure 8 From left to right are Example 7, Example 6, Example 8 and Example 2. Figure 7 and Figure 8 The results show that the pigment layer deposition on the reverse side of the fish skin after adding the adsorbent in Examples 6-8 is significantly less than the pigment layer deposition on the reverse side of the fish skin without adding the adsorbent in Example 1, indicating that the addition of the adsorbent can effectively prevent the deposition of pigment on the rough surface.

[0132] The decolorization results of Example 2 and Examples 6-8 are shown in Table 3.

[0133] Table 3

[0134]

[0135] The decolorization results in Example 1 and Examples 9-12 are shown in Table 4.

[0136] Table 4

[0137]

[0138] From the results in Table 3, it can be seen that under the same action time, the addition of a penetration enhancer can significantly promote the precipitation of pigments; and the decolorization effect of ammonia water is significantly better than that of potassium carbonate. Potassium carbonate can reduce the color of the pigment layer, and ammonia water can dissolve the pigment.

[0139] After Example 1 and Example 13 were treated in a water bath for 4 hours, the state of melanin precipitation of fish skin in the solution was as follows: Figure 9 As shown; Figure 9From left to right are Example 13 and Example 1. Figure 9 It can be seen that after the same treatment time, the melanin in the solution with acid added in Example 13 is significantly more than the melanin in the pure water solution, indicating that the addition of acid can promote the precipitation of pigment on the surface of fish skin.

[0140] The state of melanin precipitation of fish skin in solution after the water bath treatment for 4 hours in Example 13-16 is as follows: Figure 10 As shown; Figure 10 From left to right are Example 13, Example 15, Example 14 and Example 16.

[0141] The decolorization status of Examples 13-16 is shown in Table 5.

[0142] Table 5

[0143]

[0144] Depend on Figure 10 As shown in Table 5, after the same treatment time, the simultaneous addition of a penetration enhancer and an acid can greatly promote the precipitation of melanin; at the same time, the decolorization effect of citric acid at the same concentration is better than that of acetic acid, but neither of them completely decolorizes, and acetic acid has less damage to the stability of fish skin.

[0145] 3. Comparison of fish skin degradation performance

[0146] The fish skins of Examples 1, 13, 14, and 17 were each completely immersed in PBS at a concentration of 0.1 g / 10 mL and placed in a shaking incubator at 37 ± 1°C for degradation. The time required for the fish skin to completely degrade (i.e., to become fragmented) was observed, and the results are shown in Table 6.

[0147] Table 6

[0148]

[0149] The results in Table 6 show that the fish skin-based biopatches obtained by different depigmentation methods have different degradation rates and degradation times, and can be applied to the repair of different tissues. This indicates that through different processes, the degradation performance of the samples can be controlled, thereby expanding the application of fish skin.

[0150] 4. Degradation performance of fish skin in vivo

[0151] The fish skin-based biological patch of Example 17 was implanted into rats for experimental observation. The tissue diagram of the sample after degradation for 2 weeks in rats is as follows: Figure 11 It was found that after 2 weeks, the sample had lost 91.1% of its weight, which was basically consistent with the in vitro degradation test period.

[0152] 5. Fat content of fish skin

[0153] Fat Content Determination Method: Fish skin was treated with supercritical CO2 using the Soxhlet extraction method according to national standard GB5009.6-2016. The lipid content of the fish skin before and after treatment was determined. Specifically, the mass of the test sample was first determined. The sample was placed in an extraction cylinder for extraction. After 6-10 hours, lipid extraction was completed. The receiving bottle was weighed to determine the lipid content. The fat content of the sample was calculated according to the following formula:

[0154]

[0155] Where: X is the fat content in the sample (g / 100g); m1 is the content of the receiving bottle and fat after constant weight (g); m0 is the mass of the receiving bottle (g); m2 is the mass of the sample (g).

[0156] Table 7 Fat content of fish skin after different treatments

[0157] parameter Fat content (%) Comparative Example 1 24.1±1.2 Comparative Example 2 20.2±0.6 Example 1 18.2±0.8 Example 2 12.3±0.4 Example 6 10.2±2.1 Example 7 12.3±0.5 Example 8 10.8±0.7 Example 11 4.6±1.1 Example 12 5.2±0.6 Example 15 4.1±0.3 Example 16 3.6±0.8

[0158] As shown in Table 7, the fat content of the fish skin after different treatments differed significantly from that of the original fish skin. The fat content of the fish skin gradually decreased after the different treatments. Furthermore, compared to the comparative example, the fish skin-based biological patch treated using the technical solution of this application had a significantly lower fat content.

[0159] 6. DNA Residue

[0160] Residual DNA is a key metric for evaluating animal-derived tissue materials. The procedure is as follows: 10 mg of scaffold is weighed and pulverized in liquid nitrogen. The scaffold is then placed in a DNA-free tube and DNA is extracted according to the DNA extraction kit's instructions. The total DNA amount is measured using an Eppendorf BioPhotometer D30, Germany, and the DNA content is calculated based on the previously weighed sample weight.

[0161] Table 8 DNA residues after different treatments

[0162]

[0163] Table 8 shows the DNA residue in fish skin after different treatments. Untreated fish skin contained over 50 ng / mg of DNA. After initial treatment, the DNA residue decreased. With increasing treatment levels, the DNA residue in fish skin fell below 50 ng / mg, meeting regulatory requirements.

[0164] 7. Cytotoxicity

[0165] Test method: Each group of 4 parallel samples, after sterilization, uses L929 cell line for sample toxicity test, and prepares L929 DMEM complete medium extract.

[0166] Table 9 Cytotoxicity after different treatments

[0167] parameter Cell viability Blank control group 1 Comparative Example 2 0.98±0.08 Example 1 0.87±0.12 Example 16 1.03±0.31 - -

[0168] Table 9 shows the cytotoxicity of the samples. As shown in Table 9, the cell viability of the treated samples is greater than 0.70, indicating that they are non-cytotoxic. Furthermore, the samples treated with Example 16 have the effect of promoting cell proliferation.

[0169] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0170] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A fish skin-based biological patch, characterized in that: The fish skin-based biological patch comprises a sheet-like body containing a collagen matrix; the fish skin-based biological patch is prepared from fish skin through a process including depigmentation and decellularization; the fish skin-based biological patch has a degradation period of 7 days to 180 days, a fat content of 1% to 19%, and a residual DNA content of less than 50 ng / mg; When performing the depigmentation treatment, the aqueous solution used further contains a penetration enhancer and an acid-base regulator, and the temperature of the depigmentation treatment is 0°C-60°C; the penetration enhancer is sodium lauryl sulfate, the acid-base regulator includes one or more of an acidic regulator and an alkaline regulator, the acidic regulator includes one or more of citric acid and acetic acid, and the alkaline regulator includes one or more of ammonia water and potassium carbonate; the degradation cycle of the fish skin-based biological patch can be adjusted according to the different additives contained in the aqueous solution used for the depigmentation treatment.

2. The fish skin-based biological patch according to claim 1, characterized in that: The fish skin-based biological patch has a tensile strength of 29.24 MPa-43.56 MPa, a breaking strength of 379.9 N-430.5 N, and a tear strength of 4.31 N-6.09 N.

3. A method for preparing a fish skin-based biological patch according to any one of claims 1 to 2, characterized in that: The steps include: performing a degreasing pretreatment on the fish skin to prepare a pretreated fish skin; The pretreated fish skin is placed in an aqueous solution, and the pretreated fish skin is subjected to a depigmentation treatment and an optional partial decellularization treatment to prepare a depigmented fish skin; the aqueous solution further comprises a penetration enhancer and an acid-base regulator; the penetration enhancer is sodium lauryl sulfate, the acid-base regulator comprises one or more of an acid regulator and an alkaline regulator, the acid regulator comprises one or more of citric acid and acetic acid, and the alkaline regulator comprises one or more of ammonia water and potassium carbonate; performing a cell-free treatment on the depigmented fish skin to prepare the fish skin-based biological patch; The liquid replacement cycle of the depigmentation treatment is 1 hour to 4 hours; the temperature of the depigmentation treatment is 0°C to 60°C.

4. The method for preparing the fish skin-based biological patch according to claim 3, wherein: The liquid replacement cycle of the depigmentation treatment is 2h-3h.

5. The method for preparing the fish skin-based biological patch according to claim 3, wherein: The mass proportion of the penetration enhancer in the aqueous solution is 0.1%-10%.

6. The method for preparing the fish skin-based biological patch according to claim 5, wherein: The mass proportion of the penetration enhancer in the aqueous solution is 0.3%-0.5%.

7. The method for preparing the fish skin-based biological patch according to claim 3, wherein: The acidic regulator accounts for 0.001% to 0.02% by mass in the aqueous solution.

8. The method for preparing the fish skin-based biological patch according to claim 7, wherein: The acidic regulator accounts for 0.002% to 0.005% by mass in the aqueous solution.

9. The method for preparing a fish skin-based biological patch according to claim 3, wherein: The mass proportion of the alkaline regulator in the aqueous solution is 0.2%-10%.

10. The method for preparing a fish skin-based biological patch according to claim 9, wherein: The mass proportion of the alkaline regulator in the aqueous solution is 0.5%-5%.

11. The method for preparing a fish skin-based biological patch according to claim 3, wherein: The step of degreasing the fish skin comprises: Rinse the fish skin with running water for 5-10 minutes; The fish skin is placed in a starch solution for treatment, wherein the concentration of the starch solution is 1%-10%.

12. The method for preparing a fish skin-based biological patch according to claim 11, wherein: The concentration of the starch solution is 3%-5%.

13. The method for preparing a fish skin-based biological patch according to claim 3, wherein: The step of performing acellular treatment on the depigmented fish skin comprises: The depigmented fish skin is subjected to a cell-removing treatment using a Triton solution containing 0.1% to 10% Triton X-100 by weight.

14. A medical material, characterized in that: The invention comprises the fish skin-based biological patch according to any one of claims 1 to 2 or the fish skin-based biological patch prepared by the preparation method according to any one of claims 3 to 13.

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