A whole fish scale-based bone repair material and a preparation method thereof

Abstract

The present application relates to the technical field of biomaterials and regenerative medicine, and particularly relates to a preparation method of a whole-fish-scale-based bone repair material, comprising the following steps: S1, fish scale fiber preparation: collected fish scales are washed and subjected to decellularization treatment, and then ground into fibrous form; S2, fish scale collagen adhesive preparation: collagen is extracted from the decellularized fish scales by using at least two different extraction methods, the obtained collagen products are mixed, freeze-dried, and then dissolved in deionized water to prepare collagen adhesives with different concentrations; S3, composite material preparation: the collagen adhesive prepared in step S2 is mixed with the fish scale fibers obtained in step S1, and then extruded into a shape; the whole-fish-scale-based bone tissue engineering material can be prepared by combining fish scale collagen and fish scale fibers, the material is not only safer and more reliable in composition, but also allows accurate regulation of the composition, thereby providing more innovative possibilities for the application of fish scale collagen adhesive.

Description

Technical Field

[0001] This invention relates to the field of biomaterials and regenerative medicine, specifically to a whole fish scale-based bone repair material and its preparation method. Background Technology

[0002] With the continuous development of regenerative medicine, the demand for efficient and safe bone repair materials is becoming increasingly prominent. Although traditional bone repair materials have promoted the repair of bone defects to a certain extent, their inherent defects, such as poor biocompatibility, insufficient mechanical strength, or degradation rates that do not match the human physiological environment, limit their widespread clinical application. Therefore, naturally derived biomaterials, due to their superior biocompatibility and controllable degradation, have gradually become a hot research direction in bone repair materials. Fish scales, as an abundant marine biomass resource, are rich in collagen and hydroxyapatite in chemical composition, which is highly similar to the composition of human bones. This unique chemical composition endows them with good biocompatibility and biodegradability, making them uniquely advantageous in promoting osteoblast proliferation and accelerating bone tissue repair. Therefore, fish scales, as a new type of bone repair material, have great development potential and broad application prospects.

[0003] Currently, bone tissue engineering materials prepared from hydroxyapatite alone or in combination with collagen and polysaccharides have successfully simulated the chemical composition of human bones and promoted the repair and regeneration of bone defects to a certain extent. However, these materials still face problems such as insufficient mechanical strength, difficulty in controlling the degradation rate, and potential immune rejection caused by xenogeneic collagen, failing to overcome the shortcomings of natural macromolecules in bone repair applications. In view of this, we propose a whole fish scale-based bone repair material and its preparation method. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing bone repair materials in terms of biocompatibility, mechanical strength, and degradation rate matching, and to provide a whole fish scale-based bone repair material and its preparation method.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] A method for preparing a whole fish scale bone repair material includes the following steps:

[0007] S1. Preparation of fish scale fibers: After cleaning and decellularizing the collected fish scales, they are ground into fibers.

[0008] S2. Preparation of fish scale collagen adhesive: Collagen is extracted from decellularized fish scales using at least two different extraction methods. The obtained collagen products are mixed, freeze-dried, and then dissolved in deionized water to prepare collagen adhesives of different concentrations.

[0009] S3. Preparation of composite materials: The collagen adhesive prepared in step S2 is mixed with the fish scale fibers obtained in step S1 and then extruded into shape.

[0010] S4. Drying and shaping: The extruded material is dried to obtain the whole fish scale bone repair material.

[0011] In the preparation of fish scale collagen adhesive, different extraction strategies are integrated to fully utilize the unique advantages of each method in terms of molecular weight distribution, structural characteristics, physicochemical properties and extraction efficiency, forming a complementary effect to enhance the overall performance of the adhesive. The prepared whole fish scale natural composite bone tissue engineering material shows significant advantages in biocompatibility, degradability and mechanical properties, and can effectively promote osteoblast proliferation, accelerate bone tissue repair and improve the efficiency of bone defect repair and regeneration.

[0012] Preferably, the decellularization process in step S1 includes the following steps: first, hypotonic and hypertonic treatment is performed using a Tris-HCl solution containing Triton X-100 and benzyl sulfonyl fluoride, then enzymatic hydrolysis is performed using DNase and RNase, and finally, the solution is washed until neutral.

[0013] Preferably, the fish scale fibers described in step S1 are ground using a grinding mill, with each grinding session lasting 15 seconds, for a total of 3 grinding sessions.

[0014] Preferably, the extraction method in step S2 is selected from at least two of the following: enzyme-acid combined extraction, alkali-hydrothermal combined extraction, enzyme-hydrothermal combined extraction, alkali-enzyme-hydrothermal three-step extraction, and acid-enzyme-hydrothermal three-step extraction.

[0015] Preferably, the concentration of the collagen adhesive in step S2 is 0.5 mg / ml, 1 mg / ml, 1.5 mg / ml, 2 mg / ml or 3 mg / ml.

[0016] Preferably, the extrusion molding in step S3 is performed under a pressure of 2 MPa.

[0017] Preferably, the drying process in step S4 is carried out in a 50°C forced-air drying oven for 2 hours.

[0018] A whole-fish-scale bone repair material, which is suitable for the repair and regeneration of bone defects.

[0019] Preferably, the whole fish scale bone repair material is composed of fish scale fibers and fish scale collagen binder, and does not contain additives from non-fish scale sources.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] 1. This invention integrates different extraction strategies to obtain fish scale collagen, fully utilizing the unique advantages of each method in terms of molecular weight distribution, structural characteristics, physicochemical properties, and extraction efficiency. By combining the strengths of each method, a complementary effect is achieved, thereby enhancing the overall performance of the adhesive. The mixed product can obtain collagen of different molecular weights to meet the needs of different application scenarios. For example, large-molecule collagen with strong structural support is needed when treating large-area bone defects; while small-molecule collagen, which is more easily absorbed and utilized by cells, is needed to promote fracture healing and regulate bone density. The main type of collagen in fish scales is type I collagen, which has a unique triple-helix structure and is a major component of bone, giving it strength and toughness. The mixed product can maximize... To preserve this structure and product purity, the quality and stability of the product are improved, enabling it to better perform its mechanical support function in bone tissue engineering. Furthermore, while fish scale collagen is poorly soluble under conventional conditions such as acidic, alkaline, and dilute salt solutions, its solubility can be improved through enzyme preparations, temperature control, and pH alteration. Therefore, a mixed extraction strategy can increase the extraction rate of collagen. Secondly, compared to traditional polysaccharide adhesives (such as chitosan and gelatin), fish scale collagen has advantages due to its natural source and superior biocompatibility and biodegradability. The degradation characteristics of polysaccharide adhesives are limited by their composition and preparation process, thus affecting their use. In addition, collagen has superior mechanical properties, including high strength and high toughness, while polysaccharide adhesives have relatively weaker mechanical properties.

[0022] 2. This invention combines fish scale collagen with fish scale fibers to prepare whole fish scale bone tissue engineering materials. This material is not only safer and more reliable in terms of composition, but also allows for precise control of its composition, thus providing more innovative possibilities for the application of fish scale collagen adhesives. In addition, using fish scales to prepare adhesives can not only promote the effective recycling of marine biological resources and reduce environmental pollution caused by fish scale processing, but also replace traditional polysaccharide adhesives, reduce resource and energy consumption in the production process, and achieve green and sustainable development. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The present invention will describe the above technical solution in detail through the following embodiments:

[0025] Example 1

[0026] A method for preparing a whole fish scale bone repair material includes the following steps:

[0027] S1. Preparation of fish scale fibers:

[0028] Cleaning fish scales: First, wash the collected fish scales three times with deionized water, then soak them in deionized water for 12 hours, and finally wash them repeatedly with deionized water three times until there is no visible dirt or impurities on the surface of the fish scales.

[0029] Decellularization: Next, the cleaned fish scales undergo decellularization to remove cellular components and ensure the safety and purity of the final product. 1000 grams of cleaned fish scales are soaked in 10 L of a mixed TrisHCl hypotonic solution (10 mM, pH 7.4) containing 0.5% Triton X-100 and 0.6% benzyl sulfonyl fluoride, and stirred for 24 hours. Afterward, the solution is replaced with a solution containing 0.5% Triton X-100 and... The fish scales were stirred for 24 hours in a mixed Tris-HCl hypertonic solution (1M, pH=7.4) containing 0.6% benzyl sulfonyl fluoride. Then, the fish scales were thoroughly washed and digested in 10L PBS buffer (0.01M, pH=7.4) with DNase (5μg / mL) and RNase (5μg / mL) at 37°C for 5 hours. After washing three times each with deionized water and PBS buffer (0.01M, pH=7.4), decellularized fish scales were obtained.

[0030] Grinding Process: A grinding mill (SUPOER brand, model SMF01, item number SJ3592, frequency 50Hz, power 180W) was used to pulverize the decellularized fish scales into fibers (15 seconds per grinding cycle, 3 cycles in total). This yields decellularized fish scale fibers, the core active component of this scaffold. Pulverizing the scales into fibers alters their shape but not their composition. Fish scale morphology varies significantly, with differences in size and thickness, requiring labor-intensive classification by species and body region. This complicates industrial compatibility and greatly hinders its development. While processing fish scales increases difficulty and cost, breaking them into fibers increases their processability and adapts to more industrial needs. Secondly, whole fish scales become brittle and have poor mechanical properties after drying, while fish scale fibers have higher tensile strength, better flexibility, and more uniform mechanical properties than whole fish scales. At the same time, breaking whole fish scales into fibers exposes more active ingredients, and the fiber structure increases the absorption of other chemical substances. Finally, fish scale fibers can also improve the utilization rate of fish scales, and even broken scales can be effectively recycled, improving the waste conversion rate.

[0031] S2. Preparation of fish scale collagen adhesive:

[0032] Collagen extraction: Collagen was extracted from decellularized fish scales using acid-enzyme combined extraction and alkali-hydrothermal combined extraction methods. Each method yielded collagen products with different characteristics and molecular weights.

[0033] Acid-enzyme combined extraction method: 1) Prepare 1.5L of 0.5M acetic acid solution (containing 5mM EDTA) and add 100g of decellularized fish scales. Treat at 4℃ with shaking (120rpm) for 24h. Centrifuge at 8000×g for 20min to collect the precipitate and wash with PBS buffer until neutral (pH 7.0±0.2); 2) Suspend the above precipitate in HCl solution with pH of approximately 2.5, add 1% pepsin (activity ≥2500U / mg), and react in a 37℃ water bath for 12h (stirring for the first 4h, then standing). Adjust the pH to 7.0 with 1M NaOH to terminate the reaction; 3) Centrifuge the reaction solution at 10000×g for 30min, take the supernatant, add 0.9M NaCl to precipitate for 4h, then collect the precipitate and dialyze (MWCO 8-14kDa) for 48h. Finally, freeze-dry to obtain the collagen product.

[0034] Alkali-hydrothermal combined extraction method: 1) Prepare 1.2 L of 0.1 M NaOH solution (containing 0.02 M Na2CO3), add 100 g of decellularized fish scales, and treat at 25 °C with constant shaking (150 rpm) for 90 minutes. Then, centrifuge at 5000 × g for 15 minutes to collect the precipitate and wash with pH 7.4 PBS buffer until neutral; 2) Resuspend the above precipitate in 0.05 M Tris-HCl buffer (pH 8.5) and place it in a high-pressure reactor at 110 °C for 60 minutes (pressure 0.15 MPa). Then, immediately cool it to 4 °C in an ice bath and centrifuge at 10000 × g for 20 minutes to collect the supernatant; 3) Filter the supernatant through a 0.22 μm microporous membrane, add 0.8 M NaCl to salt out overnight, centrifuge at 15000 × g for 30 minutes to collect the precipitate, dialyze (MWCO 10 kDa) for 48 hours, and finally freeze-dry to obtain the collagen product.

[0035] Preparation of collagen binders at different concentrations: Collagen products obtained by different extraction methods (in no particular order) were added to beakers in equal proportions, along with deionized water, to prepare a 2 mg / ml collagen binder. By integrating different extraction strategies to obtain fish scale collagen, the unique advantages of each method in terms of molecular weight distribution, structural characteristics, physicochemical properties, and extraction efficiency can be fully utilized. The combined advantages of each method create a complementary effect, thereby enhancing the overall performance of the binder. This binder not only integrates fish scale fibers but also allows for better cell attachment to the scaffold due to its excellent mechanical properties. Furthermore, the scaffold performance, such as compressive strength, porosity, and pore size, can be controlled by changing the concentration, further optimizing performance. The pure fish scale-based material prepared by this method has 100% biocompatibility and natural biodegradability, enabling the cross-linking of a collagen-hydroxyapatite composite structure. The mechanical properties of the scaffold can be altered by adjusting the concentration of the collagen binder.

[0036] S3. Composite material preparation:

[0037] Mixing fish scale fibers with adhesive: The collagen adhesive prepared above is mixed with the decellularized fish scale fibers obtained in step S1. During mixing, 300ul of collagen adhesive and 150mg of fish scale fibers are placed in a mold of fixed size to ensure that the fish scale fibers are completely soaked in collagen to achieve adhesion.

[0038] S4. Drying and shaping:

[0039] The collagen-infused fish scale fibers were transferred to a powder press (BJ-5, Tianjin Bojun Technology Co., Ltd.), using a 10mm mold, and pressed at 2MPa (500kgf / cm²). 2 The material is compressed under pressure to form a tablet. The resulting material is then dried in a 50°C forced-air drying oven (Lichen brand, model LC-101-2B, temperature control range RT+5~300°C) for 2 hours to achieve final shaping. The bone repair material prepared in this way can meet the requirements of clinical bone defect shape and size according to molds of different shapes and sizes. Furthermore, the pressure can further adjust the mechanical properties of the scaffold. Compared with bone powder, solid scaffolds have a customizable three-dimensional structure, better mechanical support performance, and controllable degradation rate, which can better promote bone tissue regeneration and avoid the risk of particle migration.

[0040] Example 2

[0041] The only difference between this embodiment and Example 1 is that collagen was extracted from decellularized fish scales using an enzyme-hydrothermal combined extraction method and an alkali-enzyme-hydrothermal three-step method. Each method yielded collagen products with different characteristics and molecular weights. The concentration of the collagen binder was 1.5 mg / ml, and all other conditions were the same.

[0042] Enzyme-hydrothermal combined extraction method: 1) Add 100g of decellularized fish scales to 1.5L of 0.1M acetate buffer (pH 5.0), heat in a 95℃ water bath for 45 minutes, immediately cool to 4℃ in an ice bath, and collect the precipitate by centrifugation at 5000×g for 15 minutes; 2) Resuspend the precipitate in 0.5M acetate solution (pH 2.8), add 1.2% pepsin (activity ≥3000U / mg), react at 37℃ with shaking (120rpm) for 10 hours, adjust the pH to 7.0 with 2M NaOH to terminate the reaction, and collect the supernatant by centrifugation at 10000×g for 30 minutes; 3) Filter the supernatant through a 0.45μm filter membrane, dialyze (MWCO 8kDa) to remove small molecules, and freeze-dry to obtain collagen product.

[0043] Alkali-enzyme-hydrothermal three-step method: 1) Prepare 1 L of 0.1 M NaOH solution and add 100 g of decellularized fish scales. Stir at room temperature (25℃) for 2 hours, centrifuge at 5000×g for 15 minutes to collect the precipitate, and wash with PBS buffer until neutral (pH 7.0±0.2); 2) Suspend the above precipitate in deionized water and heat in a 100℃ water bath for 2 hours, then immediately cool to room temperature (25℃) in an ice bath. Centrifuge at 5000×g for 15 minutes to collect the precipitate; 3) Perform hydrothermal treatment... The precipitate was then suspended in pH 8.0 Tris-HCl buffer, and 0.5% trypsin (activity ≥ 2500 U / mg) was added. The mixture was then reacted in a constant temperature water bath at 37°C with shaking (100 rpm) for 8 hours. Then, an equal volume of 10% TCA was added to terminate the reaction. Finally, the supernatant was collected by centrifugation at 10000×g for 30 minutes. 4) The supernatant was filtered through a 0.45 μm filter membrane, dialyzed (MWCO 3.5 kDa) to remove small molecules, and finally freeze-dried to obtain the collagen product.

[0044] Example 3

[0045] The only difference between this embodiment and Example 1 is that in this embodiment, collagen was extracted from decellularized fish scales using an alkali-hydrothermal combined extraction method and an acid-enzyme-hydrothermal three-step method. Each method yielded collagen products with different characteristics and molecular weights. The concentration of the collagen binder was 3 mg / ml, and all other conditions were the same.

[0046] Alkali-hydrothermal combined extraction method: 1) Prepare 1.2 L of 0.1 M NaOH solution (containing 0.02 M Na2CO3), add 100 g of decellularized fish scales, and treat at 25 °C with constant shaking (150 rpm) for 90 minutes. Then, centrifuge at 5000 × g for 15 minutes to collect the precipitate and wash with pH 7.4 PBS buffer until neutral; 2) Resuspend the above precipitate in 0.05 M Tris-HCl buffer (pH 8.5) and place it in a high-pressure reactor at 110 °C for 60 minutes (pressure 0.15 MPa). Then, immediately cool it to 4 °C in an ice bath and centrifuge at 10000 × g for 20 minutes to collect the supernatant; 3) Filter the supernatant through a 0.22 μm microporous membrane, add 0.8 M NaCl to salt out overnight, centrifuge at 15000 × g for 30 minutes to collect the precipitate, dialyze (MWCO 10 kDa) for 48 hours, and finally freeze-dry to obtain the collagen product.

[0047] Acid-enzyme-hydrothermal three-step extraction method: 1) Prepare 2L of 0.3M citric acid-0.1M acetic acid mixed buffer (pH 3.5) and add 100g of decellularized fish scales. Extract at 4℃ with constant shaking (100rpm) for 24 hours. Centrifuge at 8000×g for 20 minutes and retain the precipitate for the next step; 2) Resuspend the above precipitate in 0.02M phosphate buffer and place it in an autoclave. Treat at 120℃ for 60 minutes (pressure 0.1MPa), then immediately cool rapidly to 4℃ in an ice bath. Centrifuge at 10000×g for 30 minutes to collect the precipitate. 3) The hydrothermal treatment product was resuspended in 0.5M acetic acid (pH 2.5), 1.5% pepsin (activity ≥ 3500 U / mg) was added, and the reaction was carried out at 37℃ with shaking (150 rpm) for 12 hours. The pH was adjusted to 7.4 with 1M NaOH to terminate the reaction. The supernatant was collected by centrifugation at 15000×g for 40 minutes. 4) The supernatant was filtered through a 0.22μm microporous membrane, concentrated with PEG20000, dialyzed (MWCO 12kDa) for 72 hours, and finally freeze-dried to obtain the white collagen product.

[0048] Example 4

[0049] The only difference between this embodiment and Example 1 is that in this embodiment, five methods were used in equal proportions to extract collagen from decellularized fish scales: acid-enzyme combined extraction, alkali-hydrothermal combined extraction, enzyme-hydrothermal combined extraction, alkali-enzyme-hydrothermal three-step method, and acid-enzyme-hydrothermal three-step method. Each method can obtain collagen products with different characteristics and molecular weights. The concentration of collagen binder is 2 mg / ml, and all other conditions are the same.

[0050] Comparative Example 1

[0051] The only difference between this comparative example and Example 1 is that this comparative example uses a single extraction method (acid-enzyme combined extraction method), the concentration of collagen binder is 2 mg / ml, and all other conditions are the same.

[0052] Comparative Example 2

[0053] The only difference between this comparative example and Example 1 is that an exogenous adhesive (2% chitosan solution) is used in this comparative example, while all other conditions are the same.

[0054] Comparative Example 3

[0055] The only difference between this comparative example and Example 1 is that no adhesive is used in this comparative example; only fish scale fibers are pressed, while all other conditions are the same.

[0056] Comparative Example 4

[0057] The only difference between this comparative example and Example 1 is that the extraction method in this comparative example is the same as in Example 1, but the extrusion pressure is 0.5 MPa, and all other conditions are the same.

[0058] Test case

[0059] Based on the above embodiments and comparative examples, samples of whole fish scale bone repair materials were prepared and their performance was tested:

[0060] 1. Cell compatibility test (CCK-8 assay)

[0061] Test process:

[0062] a. After sterilizing the materials prepared in Examples 1-4 and Comparative Examples 1-4, they were co-cultured with mouse pre-osteoblasts MC3T3-E1.

[0063] b. CCK-8 reagent was added after 24h, 48h and 72h respectively, and the absorbance (OD value) at 450nm was measured after 2 hours of incubation.

[0064] c. Calculate the cell proliferation rate (%) = (OD value of experimental group / OD value of blank control group) × 100%.

[0065] The cell compatibility of Examples 1-4 and Comparative Examples 1-4 was tested, and the specific test results are shown in Table 1 below.

[0066] Table 1: Cell Proliferation Rate Test Data

[0067]

[0068] From the data in Table 1 above, we can see that:

[0069] The cell proliferation rates of Examples 1-4 were significantly higher than those of Comparative Examples 1-4 after 72 hours (p<0.05). Among them, the combination of five methods in Example 4 showed the best performance, indicating that the combined use of multiple extraction methods can significantly improve the bioactivity of the material.

[0070] The low proliferation rates observed in Comparative Example 1 (single extraction method) and Comparative Example 2 (using chitosan adhesive) indicate poor cell compatibility with single collagen source or exogenous adhesive.

[0071] In Comparative Example 3, the absence of an adhesive resulted in an extremely low cell proliferation rate, indicating that fish scale fibers themselves do not possess osteogenic activity and must rely on collagen adhesives.

[0072] 2. Compression strength test

[0073] Test process:

[0074] a. Use a universal testing machine to perform a compression test on the material at a rate of 1 mm / min;

[0075] b. Record the maximum compressive strength (MPa) and compressive modulus (MPa).

[0076] The compressive strength properties of Examples 1-4 and Comparative Examples 1-4 were tested, and the specific test results are shown in Table 2 below.

[0077] Table 2: Compressive Strength Test Data

[0078]

[0079] From the data in Table 2 above, we can see that:

[0080] The compressive strength and modulus of Examples 1-4 were significantly higher than those of Comparative Examples 1-4, especially Examples 3 and 4, indicating that the combination of high-concentration binder and multiple extraction methods can significantly enhance the mechanical properties of the material.

[0081] In Comparative Example 4, low-pressure molding leads to a decrease in mechanical properties, indicating that extrusion pressure has a key impact on the final mechanical properties of the material.

[0082] 3. In vitro degradation performance test

[0083] Test process:

[0084] a. Place the material in a PBS buffer (pH=7.4) solution containing lysozyme (1 mg / mL) and shake at 37°C.

[0085] b. Take out the sample every 7 days, wash and dry it, weigh it, and calculate the residual mass rate (%) = (current mass / initial mass) × 100%.

[0086] The in vitro degradation performance of Examples 1-4 and Comparative Examples 1-4 was tested, and the specific test results are shown in Table 3 below.

[0087] Table 3: In vitro degradation performance test data (28-day mass residue rate, %)

[0088]

[0089] From the data in Table 3 above, we can see that:

[0090] Examples 1-4 show relatively stable degradation rates, retaining more than 60% of their mass after 28 days, indicating that the material has controllable degradation properties that can match the bone regeneration rate.

[0091] Comparative Examples 1, 2, and 3 degraded too quickly, especially Comparative Example 3, which contained no adhesive and degraded almost completely within 28 days, failing to provide sufficient support time.

[0092] In summary, this invention, through the combination of multiple collagen extraction methods, the use of whole fish scale components, and precise extrusion molding, successfully prepared a whole fish scale-based bone repair material with excellent biocompatibility, mechanical properties, and controllable degradation. This material is significantly superior to materials prepared by single extraction methods or exogenous adhesives, and possesses significant innovation and practical value.

[0093] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for preparing a whole fish scale-based bone repair material, characterized in that, Includes the following steps: S1. Preparation of fish scale fibers: After cleaning and decellularizing the collected fish scales, they are ground into fibers. S2. Preparation of fish scale collagen adhesive: Collagen was extracted from decellularized fish scales using at least two different extraction methods. The obtained collagen products were mixed, freeze-dried, and then dissolved in deionized water to prepare collagen adhesives of different concentrations. S3. Preparation of composite materials: The collagen adhesive prepared in step S2 is mixed with the fish scale fibers obtained in step S1 and then extruded into shape. S4. Drying and shaping: The extruded material is dried to obtain the whole fish scale bone repair material; The extraction method described in step S2 is selected from at least two of the following: enzyme-acid combined extraction, alkali-hydrothermal combined extraction, enzyme-hydrothermal combined extraction, alkali-enzyme-hydrothermal three-step extraction, and acid-enzyme-hydrothermal three-step extraction.

2. The preparation method of the whole fish scale basal bone repair material as described in claim 1, characterized in that: The decellularization process described in step S1 includes the following steps: first, hypotonic and hypertonic treatment is performed using a Tris-HCl solution containing Triton X-100 and benzyl sulfonyl fluoride, then enzymatic hydrolysis is performed using DNase and RNase, and finally, the solution is washed until neutral.

3. The preparation method of the whole fish scale bone repair material as described in claim 1, characterized in that: The fish scale fibers described in step S1 are ground using a grinding mill, with each grinding session lasting 15 seconds, for a total of 3 grinding sessions.

4. The preparation method of the whole fish scale bone repair material as described in claim 1, characterized in that: The concentration of the collagen adhesive in step S2 is 0.5 mg / ml, 1 mg / ml, 1.5 mg / ml, 2 mg / ml or 3 mg / ml.

5. The preparation method of the whole fish scale basal bone repair material as described in claim 1, characterized in that: The extrusion molding described in step S3 is carried out under a pressure of 2 MPa.

6. The method for preparing the whole fish scale bone repair material as described in claim 1, characterized in that: The drying process described in step S4 is carried out in a 50°C forced-air drying oven for 2 hours.

7. A whole-scale fish-scale bone repair material prepared by the preparation method according to any one of claims 1 to 6, characterized in that, The whole fish scale-based bone repair material is suitable for the repair and regeneration of bone defects.

8. The whole fish scale bone repair material as described in claim 7, characterized in that: The whole fish scale bone repair material is composed of fish scale fibers and fish scale collagen binder, and contains no additives from non-fish scale sources.

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