Bone-blood vessel coupling regeneration material based on S1P modified decellularized fish scale bone meal as well as preparation method and application of bone-blood vessel coupling regeneration material

Abstract

The invention discloses a bone-blood vessel coupling regeneration material based on S1P modified decellularized fish scale bone meal as well as a preparation method and application thereof, and belongs to the technical field of biology. After the fish scales are treated by a decellularization method, sphingosine-1-phosphoric acid (S1P) is efficiently anchored on the surfaces of the fish scales by utilizing a cationic gelatin middle layer, and a novel bone-blood vessel coupling regeneration material is developed. The bone-blood vessel coupling regeneration material retains natural components and topological structures of fish scales, and can guide ordered deposition of calcium and phosphorus to improve bone matrix density and compression strength; the endogenous bone marrow mesenchymal stem cells and vascular endothelial cells are actively collected to migrate to the center of a defect cavity by loading the S1P to simulate the homing principle of natural stem cells in an organism, osteogenesis and angiogenesis signal channels are synchronously activated, and endogenous bone-blood vessel coupling regeneration is rapidly started. The invention provides an effective strategy for regulating and controlling osteogenesis-angiogenesis coupling for the field of bone injury repair.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a bone-vascular coupling regeneration material based on S1P modified decellularized fish scale bone powder and a preparation method and application thereof. BACKGROUND

[0002] Bone tissue regeneration is a highly coordinated complex process involving the recruitment of mesenchymal stem cells, the release of growth factors, the ingrowth of new blood vessels, and the proliferation and differentiation of osteoblasts, etc. Among them, the interaction between mesenchymal stem cells and vascular cells is particularly crucial - the early recruitment of vascular cells and the formation of new vascular networks help to deliver and metabolize nutrients required for bone repair, and transport stem cells to the defect site, laying the foundation for later bone mineralization. The close relationship between this bone formation and angiogenesis in bone defect reconstruction is called "osteogenesis-angiogenesis coupling". An ideal bone repair material should be able to promote both osteogenesis and angiogenesis processes, and precisely regulate the spatiotemporal coupling relationship between the two. However, the clinical treatment of large bone defects is still a major challenge in the field of orthopedics, and millions of people around the world need to receive bone repair treatment every year due to traffic accidents, sports injuries, bone diseases and tumor resection, etc. Although autologous bone grafting is considered the "gold standard", its source is limited, and it can cause secondary damage to the donor site, pain, infection and other complications; while allogeneic bone grafting has the risk of immune rejection and disease transmission. These limitations have prompted researchers to constantly explore new bone repair material alternatives.

[0003] Traditional tissue engineering scaffold materials, including hydroxyapatite (HA), beta-tricalcium phosphate (β-TCP) and their composite scaffolds, have shown certain potential in bone defect repair. However, these materials mostly have problems such as unsatisfactory biocompatibility, mismatched degradation rate, insufficient mechanical properties, and lack of ability to induce angiogenesis, etc. Especially in the repair of critical size bone defects, the lack of vascularization inside the scaffold often leads to obstacles in the delivery of nutrients and oxygen, which in turn causes cell death and bone formation failure in the core area. In recent years, although various strategies to promote vascularization have emerged, such as the addition of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), etc., these growth factors are expensive, have poor stability, and are easily degraded in vivo, making it difficult to maintain a long-term effective concentration. SUMMARY

[0004] The application aims to provide a bone-vascular coupling regeneration material based on S1P modified decellularized fish scale bone powder and a preparation method and application thereof, so as to solve the problems of the prior art. The application develops a new bone-vascular coupling regeneration material by anchoring sphingosine-1-phosphate (S1P) on the surface of fish scales treated by a decellularization method. The bone-vascular coupling regeneration material retains the natural components and topological structure of fish scales, can guide the ordered deposition of calcium and phosphorus to improve the bone matrix density and compressive strength, simulates the principle of natural stem cell homing in the body by loading S1P to actively recruit endogenous bone marrow mesenchymal stem cells and vascular endothelial cells to migrate to the center of the defect cavity, synchronously activates the bone formation and angiogenesis signal pathways, and rapidly starts the endogenous bone-vascular coupling regeneration. The application provides a new regeneration material, provides a new medical resource for bone tissue repair, and provides an effective strategy for regulating bone-angiogenesis coupling in the field of bone injury repair.

[0005] To achieve the above-mentioned purpose, the application provides the following solutions.

[0006] The application provides a preparation method of a bone-vascular coupling regeneration material based on S1P modified decellularized fish scale bone powder, comprising the following steps:

[0007] The pretreated fish scales are sequentially soaked in a decellularization solution A and a decellularization solution B, and then subjected to nuclease digestion treatment to obtain decellularized fish scale bone powder.

[0008] The decellularized fish scale bone powder is sequentially subjected to cationic gelatin surface modification and S1P modification to obtain the bone-vascular coupling regeneration material.

[0009] The decellularization solution A is 10 mM Tris-HCl+0.5wt% TritonX-100+0.6wt% benzylsulfonyl fluoride.

[0010] The decellularization solution B is 1M Tris-HCl+0.5wt% TritonX-100+0.6wt% benzylsulfonyl fluoride.

[0011] Optionally, the soaking time in the decellularization solution A and the decellularization solution B is 22-28 hours.

[0012] Further, the nucleases include deoxyribonuclease and ribonuclease; the digestion treatment is performed at a temperature of 35-38℃ for 4-6 hours.

[0013] Further, the method of cationic gelatin surface modification is that the decellularized fish scale bone powder is subjected to adsorption treatment in a cationic gelatin solution and then immersed in a crosslinking agent for crosslinking treatment.

[0014] Optionally, the adsorption treatment time is 5-7 hours; and the cross-linking treatment time is 8-12 seconds.

[0015] Further, the cationic gelatin solution is obtained by mixing gelatin solution with ethylenediamine and reacting for 20-28 hours under the catalysis of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.

[0016] Further, the method for modifying S1P is that the surface-modified cationic gelatin of the decellularized fish scale bone powder is immersed in an S1P solution for adsorption treatment.

[0017] Optionally, the concentration of the S1P solution is 50 μM; the adsorption treatment temperature is 22-26 ℃, and the adsorption treatment time is 22-28 hours.

[0018] The application further provides the bone-vascular coupling regenerative material prepared by the preparation method.

[0019] The application further provides application of the bone-vascular coupling regenerative material in preparation of a product for promoting bone regeneration and / or vascular regeneration, and the product includes a medicine, a bone repair material and a medical device.

[0020] The application discloses the following technical effects:

[0021] The application uses natural aquatic processing waste fish scales as a base material, and develops a new bone-vascular coupling regenerative material, i.e., an S1P active regulation system based on a decellularized fish scale scaffold (named S1P@DC-FS), by combining a decellularization method and anchoring sphingosine-1-phosphate (S1P) on the surface of the cationic gelatin intermediate layer. The bone-vascular coupling regenerative material has the following advantages in bone defect regeneration and repair: ① natural component and topological structure advantages of fish scales: the regularly arranged microgroove structure on the surface of the DC-FS provides a contact guidance effect, can significantly accelerate the directional migration of stem cells, and the reserved type 1 collagen, hydroxyapatite and magnesium element composite mineral phase constitute a biomimetic mineralization template to guide the ordered deposition of calcium and phosphorus and improve the bone matrix density and compressive strength. ② S1P targeted recruitment function advantage: by precisely targeting the S1P receptors (S1PR) highly expressed on the surface of bone marrow mesenchymal stem cells and vascular endothelial cells, a strong chemotactic effect is triggered. By loading S1P to simulate the natural stem cell homing principle in the body, endogenous bone marrow mesenchymal stem cells and vascular endothelial cells are actively recruited to migrate to the center of the defect cavity, and the bone formation and angiogenesis signal pathways are simultaneously activated to rapidly start the endogenous bone-vascular coupling regeneration.

[0022] The application realizes "waste into treasure", promotes resource recycling and environmental protection, reduces environmental pollution and resource waste by converting fish scales in aquatic processing waste into high-value biomedical materials. The preparation process of the bone-vascular coupling regenerated material is simple, and the clinical feasibility is high. By optimizing the material composition and structure design, the complex partition structure is reduced, the production cost and preparation difficulty are reduced, and the scale production and clinical transformation are facilitated. The application provides a new regenerated material for bone injury repair field, and provides a new medical resource for bone tissue repair. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0024] Figure 1 DAPI fluorescence image of fish scale bone powder (scale: 50 μm); A is non-decellularized fish scale bone powder; B is decellularized fish scale bone powder DC-FS;

[0025] Figure 2 EDS spectrum (EDS) of DC-FS (scale: 50 μm);

[0026] Figure 3 EDS element content detection results of DC-FS;

[0027] Figure 4 Atomic force microscope scanning spectrum of DC-FS; A is 3D image; B is topography; C is amplitude image;

[0028] Figure 5 Atomic force microscope scanning spectrum of S1P@DC-FS; A is 3D image; B is topography; C is amplitude image;

[0029] Figure 6 Surface roughness statistical results of DC-FS and S1P@DC-FS;

[0030] Figure 7 In vitro toxicity test results of S1P@DC-FS;

[0031] Figure 8 Cell alkaline phosphatase staining experiment diagram after different material treatment;

[0032] Figure 9 Statistical results of cell alkaline phosphatase activity after different material treatment;

[0033] Figure 10 Figure 8 shows the results of alizarin red staining of cells treated with different materials;

[0034] Figure 11 Figure 9 shows the results of alizarin red content statistics of cells treated with different materials;

[0035] Figure 12 Figure 10 shows the results of osteogenesis-related gene expression level and pro-angiogenic factor content statistics of cells treated with different materials. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of certain aspects, features and embodiments of the present application, but not a limitation of the scope of the present application.

[0037] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where a range of values is provided, it is understood that each intervening value, to the extent that it does not expressly exclude or otherwise undermine the intended recitation of the patent, between the values that are stated are also expressly contemplated. The same applies to any intervening numeric values as defined by both endpoints. These intervening values are to be construed as being included in the range.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art, but rather that the reference is part of the technical literature that is relevant to A person of ordinary skill in the art.

[0039] Many modifications and variations of this application specification can be made in the light of the above teachings without departing from the spirit or ambit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0040] It is to be understood that the terms "including", "comprising", "consisting" and "having" and the like, are used interchangeably and are meant to be inclusive.

[0041] The technical idea and principle of the present application are as follows:

[0042] Due to the difficulty of traditional materials to realize the cascade regulation of "stem cell homing → vascular cascade regeneration" synchronously, the existing materials have single function, so it is necessary to prepare a good biocompatibility, superior osteogenesis and angiogenesis function and highly controllable process flow of the scaffold material. However, the following difficulties still exist in this process: first, the cationic gelatin intermediate layer needs to be anchored on the surface of the decellularized fish scale bone powder with high density and uniformity, and stably combined with the negative charged S1P to form a "scaffold-S1P" dynamic regulation interface, which involves complex charge interaction and surface energy regulation. Secondly, the content and ratio of the gradient concentration of S1P is also one of the difficulties in constructing the biological material, and the concentration gradient distribution of S1P on the surface of DC-FS needs to be accurately regulated to simulate the physiological stem cell homing signal, so as to ensure that it can actively guide the directional migration of BMSCs and vascular endothelial cells to the defect center. Finally, the immunogenicity problem of fish scale is also a technical difficulty, and the immunogenic components (such as cell fragments and lipids) need to be completely removed through the decellularization process, but at the same time, the key mineralization templates such as natural type I collagen, hydroxyapatite and magnesium element also need to be retained, so as to ensure the biological activity and osteoinductive ability of the scaffold.

[0043] The application improves the decellularization process, reduces the damage to the radial scale groove and annular scale ridge structure of the fish scale, enhances the ability of guiding cell directional migration and differentiation, avoids heterogeneity risk and immunogenicity, and ensures the uniformity of function. In order to realize the efficient active recruitment of stem cells, the chemical chemotaxis of S1P is used to actively recruit mesenchymal stem cells in the surrounding tissue to the bone defect site, so as to provide sufficient cell source for bone regeneration and overcome the deficiency of traditional materials lacking active biological activity. In order to improve the biocompatibility and degradation matching of the material, the components (type I collagen and hydroxyapatite) similar to the human hard tissue of the fish scale itself are retained, so as to ensure that the material has good biocompatibility and degradation performance matching the bone regeneration rate. The application coordinates the process of angiogenesis and osteogenesis by accurately controlling the release kinetics of S1P and the spatial guiding effect of the fish scale topological structure, improves the efficiency of bone defect repair, and provides an effective strategy for regulating osteogenesis-angiogenesis coupling in the field of bone injury repair.

[0044] Example 1

[0045] 1. Preparation of decellularized fish scale bone powder

[0046] Fresh sea bass scales with a length of 4 mm were used as raw materials. 100 g of fresh sea bass scales were thoroughly washed with deionized water, and impurities such as oil, mucus, soft tissue, and fish silver on the fish scales were removed. Subsequently, the fish scales were immersed in a mixed Tris-HCl hypotonic solution (10 mM, pH = 7.4) containing 0.5% (mass fraction) Triton X-100 and 0.6% (mass fraction) benzyl sulfonyl fluoride for 24 hours, and then replaced with a mixed Tris-HCl hypertonic solution (1 M, pH = 7.4) containing 0.5% (mass fraction) Triton X-100 and 0.6% (mass fraction) benzyl sulfonyl fluoride for continued stirring for 24 hours. After the fish scales were thoroughly washed, they were digested with deoxyribonuclease (DNase, 5 μg / mL) and ribonuclease (RNase, 5 μg / mL) in a PBS buffer solution (0.01 M, pH = 7.4) at 37°C for 5 hours. After being washed with deionized water and a PBS buffer solution (0.01 M, pH = 7.4) for 3 times, respectively, the de-cellularized fish scale powder was obtained and denoted as DC-FS. The structural characteristics and composition of the de-cellularized fish scale powder (DC-FS) were detected by energy dispersive spectroscopy (EDS) and DAPI staining.

[0047] The DAPI staining results are shown in Figure 1 After the de-cellularization treatment, the fish scales still retained the natural topological ridge-shaped micro-pattern structure on the surface of the fish scales. After the de-cellularization treatment of the fresh fish scales, the cell nuclei on the surface of the fish scales were completely removed, and there were no residual cell nuclei on the surface of the de-cellularized fish scale powder (A has residual cell nuclei, and B has no residual cell nuclei).

[0048] The EDS detection results are shown in Figure 2 and Figure 3 It can be seen that the fish scales treated by de-cellularization are rich in carbon, nitrogen, oxygen, phosphorus, and calcium elements, indicating that the fish scales are rich in mineral elements required for bone formation.

[0049] 2. Cationic gelatin surface modification

[0050] 0.3 mL of ethylenediamine was added to 100 mL of 10% (w / v) gelatin solution (Type A), and the mixture was reacted at 4°C for 24 hours under the catalysis of EDC. After dialysis and purification, the cationic gelatin was obtained by freeze-drying. The DC-FS bone powder was immersed in the cationic gelatin solution, and vacuum negative pressure adsorption was performed for 6 hours. Subsequently, the bone powder was quickly immersed in a 0.05% glutaraldehyde solution for crosslinking for 10 seconds, and PBS was used for washing for 3 times to remove the free crosslinking agent.

[0051] 3. S1P loading and functionalization

[0052] The cationic gelatin modified bone powder was immersed in 50 μM S1P solution (dissolved in PBS containing 0.1% BSA, and the operation was in the dark), and was oscillated at 25°C for 24 hours. The successful loading of S1P was confirmed by Zeta potential instrument. The S1P loaded bone powder was recorded as S1P@DC-FS.

[0053] Test Example 1

[0054] Atomic force microscope was used to detect the structural characteristics of bone-vascular coupling regeneration materials (DC-FS and S1P@DC-FS). The atomic force microscope was operated in tapping mode: first, the sample was fixed on the substrate and installed on the sample stage, and the laser light path and detector position were precisely adjusted after the probe was installed; then the probe was driven to vibrate near its resonance frequency, and the tip was approached to the sample surface to a set amplitude through the approach process; during the scanning process, high-quality scanning images were obtained by real-time optimization of set point, feedback gain and scanning rate and other parameters, data were collected, and then image processing and data analysis were performed using AFM software.

[0055] The results are shown in Figure 4 and Figure 5 It can be seen that the 3D images reveal the three-dimensional structural arrangement of the surface of DC-FS and S1P@DC-FS. Compared with DC-FS, the basic micro-nano texture of fish scales did not change after adding S1P, and the fragmented modifier S1P was visible under the microscope. The roughness detection results are shown in Figure 6 It can be seen that the roughness of S1P@DC-FS increases due to the loading of surface modifiers. The AFM results directly prove that the surface modification strategy is effective, and S1P is successfully anchored on the microstructure of the decellularized fish scales, providing an ideal bioactive interface for bone-vascular coupling regeneration.

[0056] Test Example 2

[0057] The in vitro cytotoxicity experiment was used to detect the biocompatibility of S1P@DC-FS. The material extract was prepared according to ISO 10993-5 standard: S1P@DC-FS was placed in DMEM medium, and the final concentration was 0.2 g / mL, and was extracted at 37°C for 24 hours. L929 cells were inoculated at a density of 1×10 4 / well, and the extract was added for culture for 24 hours. After washing with PBS, CCK-8 reagent was added, and incubated for 2 hours. The absorbance at 450 nm was measured by microplate reader, and the relative proliferation rate was calculated.

[0058] The results are shown in Figure 7As shown, after L929 cells were co-cultured with DC-FS, S1P and S1P@DC-FS material, the cell proliferation rate had no significant change compared with the control group, and no cytotoxicity was shown, which proved that the material had excellent biocompatibility, and provided important safety basis for subsequent experiments and transformation application.

[0059] Test Example 3

[0060] Bone marrow mesenchymal stem cells (BMSCs) were used as experimental materials, and alkaline phosphatase (ALP) staining experiment was used to detect the osteogenesis promoting ability of the bone-vascular coupling regenerative material prepared in the application.

[0061] Obtaining and culturing of bone marrow mesenchymal stem cells (BMSCs): After the newborn SD rats were sacrificed and sterilized, the bilateral femurs were separated, the metaphyseal ends were cut off, 1 mL syringe was used to suck α-MEM to flush the bone marrow cavity into the centrifuge tube at 1000 rpm for 5 min, the supernatant was discarded, and the precipitate was resuspended with α-MEM containing 10% FBS, inoculated in a culture bottle, and cultured in a 37℃, 5% CO2 incubator, the non-adherent cells were removed by changing the liquid for the first time at 48h, and the liquid was changed every 3d thereafter, and when the confluence reached 70%-80%, the cells were passaged by trypsinization, and 3-5 passages of cells were used for experiments.

[0062] Detection of osteogenic differentiation ability of BMSCs: the material extract (DC-FS, S1P and S1P@DC-FS) was prepared according to the method of test example 2. BMSCs cells were inoculated at a density of 1×10 4 per well, 2mL of material extract was added, and cultured in a 37℃, 5% CO2 incubator for 7 days, and a blank culture medium control group was set up at the same time. The osteogenesis of cells in each group was detected by alkaline phosphatase (ALP) staining experiment, and the alkaline phosphatase (ALP) activity of cells in each group was detected.

[0063] The results are shown in Figure 8 and Figure 9 As shown, the DC-FS group, the S1P group and the S1P@DC-FS group all showed certain osteogenesis ability compared with the control group, and the osteogenesis ability of the S1P@DC-FS group was the strongest. It is shown that S1P and DC-FS have certain synergistic effect in promoting osteogenesis.

[0064] Test Example 4

[0065] Bone marrow mesenchymal stem cells (BMSCs) were used as experimental materials, and alizarin red staining experiment was used to detect the osteogenesis promoting ability of the bone-vascular coupling regenerative material.

[0066] Alizarin Red (ARS) staining: Material extracts (DC-FS, S1P, and S1P@DC-FS) were prepared according to the method in Example 2. BMSCs were cultured to 80-90% confluence and then digested with trypsin. Cells were sputtered at a rate of 2000 / cm². 2 BMSCs were seeded at a density of 2 mL of material extract in each well of a six-well plate and cultured for 14 days at 37°C in a 5% CO2 incubator. A control group with blank culture medium was also included. After culture, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, stained with alizarin red for 5 min, rinsed with distilled water, and dried. The differences in calcium nodule formation among different groups of BMSCs were observed, and the alizarin red (ARS) content of cells in each group was quantitatively detected.

[0067] The results are as follows Figure 10 and Figure 11 As shown, the DC-FS group exhibited some small mineralized nodules compared to the control group, the S1P group showed blocky nodules, and the S1P@DC-FS group showed extensive mineralized nodule staining. Quantitative analysis showed the same results. This demonstrates that the additive portion of fish scales increases the content of ARS, a late-stage osteogenic marker in BMSCs, and S1P showed a stronger osteogenic effect than fish scales. The synergistic treatment of both demonstrated the best osteogenic capacity.

[0068] Experimental Example 5

[0069] Using bone marrow mesenchymal stem cells (BMSCs) as experimental material, qPCR was used to detect the effects of bone-vascular coupling regenerative materials on osteogenic and angiogenic markers in cells.

[0070] qPCR detection of osteogenic markers: Following the procedure in Example 4, BMSCs were co-cultured with the extract of bone-vascular coupling regeneration material for 14 days. After culture, the culture medium was discarded, and the cells were washed three times with PBS. Cells on the sample surface were dissolved with TRIzol. 1 μg of intracellular total RNA was collected and reverse transcribed into cDNA. The expression levels of osteogenic-related genes, osteogenic-specific transcription factor (Runx2), osteocalcin (Ocn), and bone morphogenetic protein 2 (BMP2), were detected.

[0071] qPCR detection of angiogenesis markers: Following the method in Example 4, vascular endothelial cells were co-cultured with the extract of bone-vascular coupling regeneration material for 3 days. After culture, the culture medium was discarded, and the cells were washed three times with PBS. Cells on the sample surface were dissolved with TRIzol. 1 μg of intracellular total RNA was collected and reverse transcribed into cDNA. Changes in the mRNA levels of pro-angiogenic factors HIF-1α, Ang2, and vegf were detected.

[0072] The results are as follows Figure 12As shown, both DC-FS and S1P, acting alone, can increase the expression of osteogenic genes BMP2, RUNX2, and OCN in BMSCs to some extent. The osteogenic effect of S1P-modified S1P@DC-FS is stronger than that of either material alone. When these materials were applied to vascular endothelial cells, the levels of angiogenic factors showed a similar trend to the effects on osteogenic genes, with increased expression levels of pro-angiogenic factors HIF-1α, Ang2, and vegf.

[0073] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a bone-vascular coupling regenerative material based on S1P-modified decellularized fish scale bone powder, characterized in that, Includes the following steps: The pretreated fish scales were soaked in decellularization solution A and decellularization solution B in sequence, and then digested with nuclease to obtain decellularized fish scale bone powder. The decellularized fish scale bone powder was sequentially modified with cationic gelatin and then with S1P to obtain the bone-vascular coupling regenerative material. The decellularization treatment solution A is: 10mM Tris-HCl + 0.5wt% Triton X-100 + 0.6wt% benzyl sulfonyl fluoride; The decellularization solution B is: 1M Tris-HCl + 0.5wt% Triton X-100 + 0.6wt% benzyl sulfonyl fluoride.

2. The preparation method according to claim 1, characterized in that, The soaking time in both decellularization treatment solution A and decellularization treatment solution B is 22-28 hours.

3. The preparation method according to claim 1, characterized in that, The nucleases include deoxyribonuclease and ribonuclease; the digestion process is carried out at a temperature of 35-38°C for 4-6 hours.

4. The preparation method according to claim 1, characterized in that, The method for modifying the surface of the cationic gelatin is as follows: after the decellularized fish scale bone powder is soaked in a cationic gelatin solution for adsorption treatment, it is then immersed in a crosslinking agent for crosslinking treatment.

5. The preparation method according to claim 4, characterized in that, The adsorption treatment lasts for 5-7 hours; the cross-linking treatment lasts for 8-12 seconds.

6. The preparation method according to claim 4, characterized in that, The cationic gelatin solution is obtained by mixing a gelatin solution with ethylenediamine and reacting it for 20-28 hours under the catalysis of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

7. The preparation method according to claim 1, characterized in that, The method of S1P modification is as follows: the decellularized fish scale bone powder modified with cationic gelatin is immersed in S1P solution for adsorption treatment.

8. The preparation method according to claim 7, characterized in that, The concentration of the S1P solution was 50 μM; the adsorption treatment temperature was 22-26 °C, and the time was 22-28 hours.

9. The bone-vascular coupling regenerative material prepared by the preparation method according to any one of claims 1-8.

10. The use of the bone-vascular coupling regenerative material according to claim 9 in the preparation of products that promote bone regeneration and / or vascular regeneration, characterized in that, The products include drugs, bone repair materials, and medical devices.

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