Bionic active peptide, precursor thereof, modified bone meal containing bionic active peptide, bone regeneration repair material containing bionic active peptide and application of bionic active peptide

Abstract

The invention discloses a bionic active peptide, a precursor of the bionic active peptide, modified bone meal containing the bionic active peptide, a bone regeneration repair material containing the bionic active peptide and application of the bionic active peptide. The bionic active peptide comprises n 3, 4-L-dihydroxyphenylalanine (DOPA), an E7 peptide and a self-assembled polypeptide which are sequentially connected from an N segment to a C end, wherein the amino acid sequence of the E7 peptide comprises a sequence as shown in SEQ ID NO: 1; and n is an integer between 2 and 8. The bone regeneration repair material prepared from the bionic active peptide has both mechanical property and repair property.

Description

Technical Field

[0001] The present application relates to the field of bone regeneration and repair materials, and specifically to biomimetic active peptides, their precursors, modified bone powder containing them, bone regeneration and repair materials containing them, and uses thereof. Background Art

[0002] Because bone tissue contains arteries, veins and capillaries, once bone damage occurs due to trauma, infection and other reasons, the bone wound surface will have varying degrees of bleeding and defects, which seriously affect the operation process and postoperative healing. The current status of existing bone repair materials is that the source of autologous bone is limited, and the market demand for artificial bone is huge. Combined with official statistical data analysis, the number of bone grafting surgeries in the United States is about 500,000 per year, the number of bone grafting surgeries in my country is about 1.3 million per year, and the number of bone grafting surgeries worldwide is about 2.2 million per year. It is estimated that there is a potential market of about 10 billion for bone injury repair materials. Although there are many existing bone grafting materials, autologous bone is the gold standard for bone repair among natural bone materials, but its application is limited due to its limited source; xenogeneic bone has certain activity, but there are currently few products. Currently, artificial bone materials such as hydroxyapatite, calcium sulfate, and bioactive glass (for example, CN202410416971.8) are mostly used in clinical practice, but they only have osteoconductivity and low bone activity. In addition, bleeding from bone injury wounds affects bone healing, and existing bone repair products do not have bone hemostasis and bone regeneration properties.

[0003] Existing bone wound hemostatic materials are mostly non-absorbable bone wax, which inhibits osteogenesis, has no bone repairability, and has the disadvantages of poor biocompatibility and poor convenience of use. Conventional artificial bone repair products currently on the market are mostly granular or block-shaped, with poor clinical convenience and low bone induction activity. Some products that introduce bone induction factors into some active materials have poor stability of the active factors and high cost.

[0004] In addition, although there are patent documents (CN201811014216.8) reporting that the use of chemical bonds between peptides and bone repair materials can slowly release active peptides such as BMP, the source of such active peptides is relatively unstable, which limits their industrialization. At the same time, bone repair materials use materials such as hydroxyapatite, tricalcium phosphate and bioglass, and their bone repair ability is limited. There are also patent documents (CN201510155764.2) that record the use of self-curing calcium phosphate, gelatin materials, etc. as carriers of neuropeptides, but they still have the problem of low curing porosity and inability to shape and adhere to the bone repair site to achieve effective hemostasis.

[0005] Therefore, there is an urgent need for a multifunctional bone repair material that can achieve improved mechanical and repair properties, bone surface hemostasis and regeneration. Summary of the invention

[0006] Based on this, it is necessary to provide at least one biomimetic active peptide, its precursor, modified bone powder containing it, bone regeneration and repair material containing it, and its use.

[0007] In the first aspect of the present application, a biomimetic active peptide precursor is provided, which comprises m tyrosines, an E7 peptide and a self-assembling polypeptide connected in sequence from the N segment to the C terminus; wherein the amino acid sequence of the E7 peptide comprises the sequence shown in SEQ ID NO: 1; m is an integer between 2 and 8.

[0008] In a second aspect of the present application, a biomimetic active peptide is provided, which comprises n 3,4-L-dihydroxyphenylalanine (DOPA), an E7 peptide and a self-assembling polypeptide connected in sequence from the N segment to the C terminus; wherein the amino acid sequence of the E7 peptide comprises the sequence shown in SEQ ID NO: 1; n is an integer between 2 and 8.

[0009] In the third aspect of the present application, a nucleic acid molecule is provided, which encodes the biomimetic active peptide precursor as described in the first aspect.

[0010] In the fourth aspect of the present application, a gene expression cassette is provided, which contains a promoter, the nucleic acid molecule as described in the third aspect, and a terminator.

[0011] In the fifth aspect of the present application, a recombinant vector is provided, which comprises a backbone vector and one or more of the following items:

[0012] (1) The nucleic acid molecule according to the third aspect; and

[0013] (2) The gene expression cassette as described in the fourth aspect.

[0014] In the sixth aspect of the present application, a recombinant cell is provided, which comprises one or more of the nucleic acid molecule described in the third aspect, the gene expression cassette described in the fourth aspect, and the recombinant vector described in the fifth aspect.

[0015] In a seventh aspect of the present application, a method for preparing modified bone powder is provided, comprising the following steps:

[0016] The solution of the biomimetic active peptide and the biological bone deproteinized particles as described in the second aspect are uniformly dispersed in the solution and freeze-dried.

[0017] In the eighth aspect of the present application, there is provided modified bone powder prepared by the preparation method described in the seventh aspect.

[0018] In the ninth aspect of the present application, a method for preparing a bone regeneration repair material is provided, comprising the following steps: mixing the modified bone powder and hemostatic mud described in the eighth aspect; and irradiating and sterilizing to prepare the bone regeneration repair material; wherein the weight ratio of the modified bone powder to the hemostatic mud is (5~20):(35~140).

[0019] In the tenth aspect of the present application, a bone regeneration repair material prepared by the preparation method described in the ninth aspect is provided.

[0020] In the eleventh aspect of the present application, there is provided use of the biomimetic active peptide precursor described in the first aspect, the biomimetic active peptide described in the second aspect, or the modified bone powder described in the eighth aspect in the preparation of a product for treating bone injuries.

[0021] In the twelfth aspect of the present application, a method for treating bone damage is provided, the method comprising applying the bone regeneration and repair material as described in the tenth aspect to the affected area of ​​a subject.

[0022] The biomimetic active peptides in this application, such as (DOPA)n-EPLQLKM-RADA16, can form a special spatial structure by self-assembly, and have advantages in terms of stronger bonding strength and osteogenesis compared to the use of the above fragments alone. The bone regeneration and repair material prepared using the biomimetic active peptides takes into account both mechanical properties and repair properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0024] Figure 1 The plasticity of the sample prepared in Example 1 is shown.

[0025] Figure 2 The Micro CT results of the rabbit femoral defect models in Examples 1 and 2 taken 2 weeks ago are shown; the left side is Example 1, and the right side is Example 2.

[0026] Figure 3 Micro CT results of rabbit femoral defect model taken 2 weeks ago are shown. The left side is bioactive glass and the right side is self-solidifying calcium phosphate.

[0027] Figure 4The results of Micro CT analysis of rabbit femoral defect model Example 1 at 2 weeks post-implantation.

[0028] Figure 5 The Micro CT analysis results of rabbit femoral defect model Example 2 are shown 2 weeks after sampling.

[0029] Figure 6 Shown are the results of Micro CT analysis of the bioactive glass samples taken from the rabbit femoral defect model at 2 weeks.

[0030] Figure 7 Shows the results of Micro CT analysis of samples taken from a rabbit femoral defect model after 2 weeks of self-curing calcium phosphate. DETAILED DESCRIPTION

[0031] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0033] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0034] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0035] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0036] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0037] In this application, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0038] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0039] In this application, unless otherwise specified, the term "between" used in numerical ranges does not include the number itself. For example, "between 2 and 8" does not include the numbers 2 and 8.

[0040] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0041] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0042] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0043] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0044] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0045] In view of the defects of current bone surface bleeding and bone defect products such as poor plasticity and low osteogenic activity, the inventors creatively effectively connected DOPA, E7 peptide and self-assembling short peptide (such as RADA16, which has good hemostatic properties) to form (DOPA) n -E7- self-assembling polypeptides form supramolecular hydrogels, and the inventors have found that the number of DOPAs will affect the final properties of the product, such as the bonding strength, and after a large number of experiments, the number of DOPAs is set between 2 and 8. This may be because a specific spatial structure will be formed after self-assembly, and an inappropriate number of DOPAs will affect the spatial structure, thereby affecting the effectiveness of the product.

[0046] A new type of bone repair material is prepared using supramolecular hydrogel formed by the combination of (DOPA)n-E7 as raw material, which can quickly stop bleeding, promote stem cell recruitment, and promote the differentiation of osteoblasts into bone in the bone defect area, so as to solve the problems of difficult graft survival, healing, and slow healing after bone transplantation in large bone defects.

[0047] In a first aspect of the present application, a biomimetic active peptide precursor is provided, which comprises m tyrosines, an E7 peptide and a self-assembling polypeptide connected in sequence from the N segment to the C terminus; wherein m is an integer between 2 and 8.

[0048] In some embodiments, the amino acid sequence of the biomimetic active peptide precursor comprises a sequence as shown in any one of SEQ ID NOs: 5 to 8.

[0049] In some embodiments, the amino acid sequence of the biomimetic active peptide precursor is as shown in any one of SEQ ID NOs: 5 to 8.

[0050] If not otherwise specified, the term "precursor" in this application refers to a substance in the process of synthesizing or converting into a biomimetic active peptide through a reaction. The biomimetic active peptide precursor in this application can be oxidized to obtain a biomimetic active peptide. Without wishing to be bound by any theory, it is believed that tyrosine can form DOPA after oxidation under certain conditions. In some embodiments, the oxidation method includes mixing the precursor with tyrosinase to obtain a biomimetic active peptide.

[0051] In some embodiments, the biomimetic active peptide of the second aspect is obtained by oxidizing the biomimetic active peptide precursor of the first aspect.

[0052] In some embodiments, m is 3, 4, 5, 6, 7, or an integer between any two values. In some embodiments, m is an integer between 3 and 5.

[0053] The different regions of the biomimetic active peptide precursor, namely, m tyrosines, the E7 peptide and the self-assembling polypeptide, are operably linked.

[0054] Unless otherwise specified, the term "operably linked" in this application refers to a functional relationship between two or more regions of a biomimetic active peptide, such as between m tyrosines and the E7 peptide, or between the E7 peptide and a self-assembling polypeptide; wherein the two or more regions are linked to produce a biomimetic active peptide precursor.

[0055] In some embodiments, different regions are connected by a linker, such as a flexible linker, to reduce mutual interference between the regions.

[0056] The flexible linker in the present application may be, for example, GGGGS (Gly-Gly-Gly-Gly-Ser) or GGGGG (Gly-Gly-Gly-Gly-Gly).

[0057] In a second aspect of the present application, a biomimetic active peptide is provided, which comprises n 3,4-L-dihydroxyphenylalanine, E7 peptide and self-assembling polypeptide connected in sequence from the N segment to the C end; wherein n is an integer between 2 and 8.

[0058] DOPA amino acid (3,4-L-dihydroxyphenylalanine) is widely present in the adhesive proteins secreted by mussels. The catechol (i.e., catechol) in DOPA can be covalently and non-covalently bound to the surface of various materials, and is an important means for mussels to attach to surfaces such as reefs and ships. When the catechol group and the surface of bone particles are simply in contact in water, a stable coordination binding effect is quickly formed, which promotes the enrichment of calcium ions on the surface of the material, so that the concentration of calcium ions reaches a supersaturated state, provides nucleation sites for apatite, and is conducive to its formation on the surface of bone repair materials.

[0059] As a class of compounds with unique properties, peptides can not only simulate the functions of proteins, but also have lower production complexity and cost than proteins. In addition, peptides have the advantages of easy synthesis, strong stability, adjustable size, functional groups and activity. As more and more peptides with special properties are discovered and screened, peptides used for bone repair are gradually diversified, such as protein-mimicking peptides, affinity peptides, self-assembling peptides, etc. Among them, affinity peptides refer to peptides that have the ability to bind to specific cells, scaffold materials and cytokines, and their special affinity has attracted widespread attention. For example, mesenchymal stem cell affinity peptides can play a role in recruiting mesenchymal stem cells. E7 peptide is a new type of short peptide with an amino acid sequence of "EPLQLKM" (glutamic acid-proline-leucine-glutamine-leucine-lysine-methionine). It has an active factor similar to the function of SDF-1, and can improve the hydrophilicity of the material, enhance the affinity of the material to cells, and specifically enhance the adhesion and proliferation ability of MSCs, especially with high affinity to BMSCs. The E7 peptide is considered to be a good "MSCs homing device" that has the ability to selectively capture MSCs in a flow model. The E7 short peptide targets and recruits mesenchymal stem cells at the bone defect site, maintains the osteogenic differentiation ability of mesenchymal stem cells in situ in the bone defect, and promotes the repair and regeneration of the bone defect site; at the same time, it can accelerate the formation of blood vessels at the bone defect site. In some embodiments, the amino acid sequence of the E7 peptide comprises the sequence shown in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the E7 peptide is as shown in SEQ ID NO: 1.

[0060] The self-assembling short peptide has the characteristics of programmability, good viscoelasticity, high biocompatibility and low immunogenicity; it can form nanofiber hydrogels under physiological conditions, showing good biocompatibility. In different surgical wounds, the hydrogel can form a physical barrier to exert a hemostatic effect without interacting with or being affected by the coagulation cascade, and quickly and effectively control bleeding without harming surrounding tissues or cells. In some embodiments, the self-assembling polypeptide is selected from the group consisting of self-assembling KLD-12 peptides, self-assembling RADA16 peptides, self-assembling IEIK-13 peptides, self-assembling KLD-12 peptide derivatives, self-assembling RADA16 peptide derivatives and self-assembling IEIK-13 peptide derivatives. Exemplarily, the self-assembling polypeptide is a self-assembling RADA16 peptide.

[0061] RADA16 is a classic self-assembling hemostatic peptide. In aqueous solution, hydrophobic alanine forms overlapping hydrophobic forces, while positively charged arginine (R) and negatively charged aspartic acid (D) residues in adjacent short peptides stack with each other through intermolecular non-covalent bond forces, eventually forming a nanofiber body with a width of about 10 nm, which is then further polymerized to form a hydrogel scaffold. These scaffolds have the ability to self-repair, and RADA16 hydrogel scaffolds can promote cell growth, differentiation and migration.

[0062] Unless otherwise specified, the biomimetic active peptides in this application include variant forms thereof. The term "variant" refers to biomimetic active peptides obtained by amino acid substitution, deletion, insertion and modification. Any combination of substitution, deletion, insertion and modification can be performed as long as the final variant has the desired characteristics.

[0063] In some embodiments, the amino acid sequence of the self-assembling RADA16 peptide comprises the sequence shown in SEQ ID NO: 2. In some embodiments, the amino acid sequence of the self-assembling RADA16 peptide is shown in SEQ ID NO: 2.

[0064] In some embodiments, the assembly polypeptide is a self-assembling IEIK-13 peptide. In some embodiments, the amino acid sequence of the self-assembling IEIK-13 peptide comprises the sequence shown in SEQ ID NO: 3. In some embodiments, the amino acid sequence of the self-assembling IEIK-13 peptide is shown in SEQ ID NO: 3.

[0065] In some embodiments, the self-assembling polypeptide is a self-assembling KLD-12 peptide. In some embodiments, the amino acid sequence of the self-assembling KLD-12 peptide comprises the sequence shown in SEQ ID NO: 4. In some embodiments, the amino acid sequence of the self-assembling KLD-12 peptide is shown in SEQ ID NO: 4.

[0066] In some embodiments, n is 3, 4, 5, 6, 7, or an integer between any two values. In some embodiments, n is an integer between 3 and 5.

[0067] Different regions of the biomimetic active peptide, namely n 3,4-L-dihydroxyphenylalanine, E7 peptide and self-assembling polypeptide are operably connected.

[0068] In some embodiments, different regions are connected by a linker, such as a flexible linker, to reduce mutual interference between the regions.

[0069] In the third aspect of the present application, a nucleic acid molecule is provided, which encodes the biomimetic active peptide precursor as described in the first aspect.

[0070] In the present application, unless otherwise specified, "nucleic acid" has the commonly known meaning in the technical field, also known as "polynucleotide", which is a molecule formed by multiple nucleotide monomers.

[0071] The nucleic acid molecule described in the present application may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0072] In a fourth aspect of the present application, a method for preparing modified bone powder is provided, comprising the following steps:

[0073] The solution of the biomimetic active peptide and the biological bone deproteinized particles as described in the second aspect are uniformly dispersed in the solution and freeze-dried.

[0074] In the present application, biomimetic active peptides are creatively used to modify biological bone deproteinized particles. DOPA in the biomimetic active peptides can chelate with the metal between bone particles, enhance its specific binding with bone particles, promote the enrichment of calcium ions on the surface of the material, make the concentration of calcium ions reach an oversaturated state, provide nucleation sites for apatite, and promote the regeneration and repair performance of bone defect sites.

[0075] Unless otherwise specified, the term "biological bone deproteinized particles" in this application refers to biological bone particles after deproteinization.

[0076] The deproteinization method can be conventional in the art. For example, in some embodiments, a deproteinization method using hydrogen peroxide and ethylenediamine solution is used.

[0077] In some embodiments, the biological bone deproteinized particles are prepared by a method comprising the following steps:

[0078] 1) Take animal bone particles, and use isopropanol and purified water to wash them 3 to 6 times by repeated alternating oscillation to obtain defatted bone particles, wherein the mass ratio of the animal bone particles to isopropanol is 1:(2 to 4);

[0079] 2) adding the defatted bone particles to hydrogen peroxide, allowing the particles to stand for 12 to 24 hours, discarding the hydrogen peroxide, placing the particles in purified water for 3 to 6 times of shaking washing, drying the particles at 75 to 100° C. for 5 to 7 hours, and passing the particles through a 10 to 20 mesh sieve to obtain sieved bone particles;

[0080] 3) adding the sieved bone particles to a 70%-100% ethylenediamine solution and heating to a slight boil, then maintaining at 100-125° C. for 7-9 hours, discarding the waste liquid, adding the particles to purified water and shaking and washing at least once, and repeating step 3) twice;

[0081] 4) The bone particles obtained in step 3) are washed 8 to 20 times with purified water and at least once with water for injection. The washing process takes 3 to 6 minutes per time. During the washing process, the container is turned over and the waste liquid is discarded to obtain deproteinized bone particles.

[0082] The particle size of the biological bone deproteinized particles can be conventional in the art, for example, the biological bone deproteinized particles can be obtained by passing through a sieve with a pore size of 14 meshes.

[0083] In some embodiments, the preparation method comprises the following steps:

[0084] The biomimetic active peptide described in the second aspect is mixed with the biological bone deproteinized particles in the form of a solution.

[0085] If there is no special explanation, the term "mixing in the form of a solution" means mixing the biomimetic active peptide and the biological bone deproteinized particles in a solution.

[0086] In some embodiments, the preparation method comprises: mixing a solution of the biomimetic active peptide as described in the second aspect with a mass concentration of 1% to 10% with biological bone deproteinized particles, and freeze-drying the mixture.

[0087] The freeze-drying method in the present application can be conventional in the art. In some embodiments, freeze-drying is performed using a vacuum freeze dryer. In some embodiments, the model of the vacuum freeze dryer is LGJ-50F.

[0088] In some embodiments, the freeze-drying process includes: pre-freezing at -50°C in a vacuum freeze dryer (model: LGJ-50F) (0.7㎡ silicone oil heating), pre-vacuuming at -40°C, primary drying at -20°C, and sequentially drying at 0°C, 20°C, and 25°C.

[0089] In some embodiments, the weight ratio of the solution of the biomimetic active peptide to the biological bone deproteinized particles is 100: (1 to 10). In some embodiments, the weight ratio of the solution of the biomimetic active peptide to the biological bone deproteinized particles is 100: 1. In some embodiments, the weight ratio of the solution of the biomimetic active peptide to the biological bone deproteinized particles is 100: 2. In some embodiments, the weight ratio of the solution of the biomimetic active peptide to the biological bone deproteinized particles is 100: 3. In some embodiments, the weight ratio of the solution of the biomimetic active peptide to the biological bone deproteinized particles is 100: 4. In some embodiments, the weight ratio of the solution of the biomimetic active peptide to the biological bone deproteinized particles is 100: 5. In some embodiments, the weight ratio of the solution of the biomimetic active peptide to the biological bone deproteinized particles is 100: 6. In some embodiments, the weight ratio of the solution of the biomimetic active peptide to the biological bone deproteinized particles is 100: 7. In some embodiments, the weight ratio of the solution of the biomimetic active peptide to the biological bone deproteinized particles is 100: 8. In some embodiments, the weight ratio of the solution of the biomimetic active peptide to the biological bone deproteinized particles is 100: 9. In some embodiments, the weight ratio of the solution of the biomimetic active peptide to the biological bone deproteinized particles is 100: 10.

[0090] In some of the methods, in the step of mixing the solution of the biomimetic active peptide as described in the second aspect with a mass concentration of 1% to 10% with the biological bone deproteinized particles, the mixing is performed by shaking.

[0091] In some embodiments, the oscillation is selected from one or more of ultrasonic oscillation and vortex oscillation. In some embodiments, the oscillation time is 10 min to 30 min.

[0092] In the present application, the biological bone in the biological bone deproteinized particles may be derived from mammals, such as one or more of pigs, cattle, sheep and pigs.

[0093] In the fifth aspect of the present application, there is provided modified bone powder prepared by the preparation method described in the fourth aspect.

[0094] In a sixth aspect of the present application, a method for preparing a bone regeneration and repair material is provided, comprising the following steps:

[0095] Mixing the modified bone powder and hemostatic cement as described in the fifth aspect; and

[0096] Irradiation sterilization to prepare bone regeneration and repair materials.

[0097] The term "hemostatic clay" in this application refers to a substance with a physical form between gel and clay that has a hemostatic function. In some embodiments, the hemostatic clay contains or does not contain a component that can stop bleeding alone. Without wishing to be limited by any theory, it is believed that the hemostatic clay in this application exerts its hemostatic effect at least through the following mechanism: it can effectively adhere to the bone injury site to act as a physical barrier, and at the same time can effectively absorb excess water in the blood to inhibit the fluidity of the blood, thereby stimulating the physiological hemostatic mechanism, and playing a physical hemostatic and physiological hemostatic effect.

[0098] In some embodiments, the hemostatic putty contains at least a high molecular weight polysaccharide and a solvent.

[0099] In the present application, the polymer polysaccharide is selected from the group consisting of sodium hyaluronate, oxidized regenerated cellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose, carboxymethyl chitosan and sodium alginate. The hemostatic glue may contain only one polymer polysaccharide from the above group, such as sodium hyaluronate, oxidized regenerated cellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose, carboxymethyl chitosan or sodium alginate, or may contain two or more polymer polysaccharides from the above group.

[0100] In the present application, the solvent is selected from one or more of the group consisting of water and vitamin E acetate.

[0101] Wherein, the weight ratio of the high molecular weight polysaccharide to the solvent may be (5-20): (20-80). In some embodiments, the mass ratio of the high molecular weight polysaccharide to the solvent is 40:20.

[0102] The preparation of the hemostatic putty includes uniformly mixing the high molecular weight polysaccharide and the solvent.

[0103] In some embodiments, the stirring time is 2 h to 8 h, for example, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h, 6.5 h, 7 h, 7.5 h, 8 h, or a range or value between any two values.

[0104] Depending on the needs of the preparation process, for example, to facilitate better mixing of the high molecular weight polysaccharide and the solvent, heating can be optionally performed during the mixing process. Exemplarily, in one embodiment, the preparation of the hemostatic glue comprises: heating 40 parts of vitamin E acetate at 70° C., adding 20 parts of sodium carboxymethyl cellulose to the heated vitamin E acetate and mixing evenly, and then cooling at room temperature to obtain the hemostatic glue.

[0105] In some embodiments, the hemostatic glue further comprises stearate and poloxamer. The poloxamer is one or more of the group consisting of poloxamer 188, poloxamer 338, poloxamer 407, poloxamer 105, poloxamer 124, poloxamer 184, poloxamer 237, poloxamer 335, poloxamer 403 and poloxamer 401.

[0106] In some embodiments, the hemostatic glue comprises stearate, poloxamer, a high molecular weight polysaccharide and a solvent, and the weight ratio of the stearate, the poloxamer, the high molecular weight polysaccharide and the solvent is (5-20): (5-20): (5-20): (20-80). Without wishing to be limited by any theory, it is found that when the relative content (weight ratio) of each component of the hemostatic glue is not within the above numerical range, when the relative content (weight ratio) of each component of the hemostatic glue is not within the above range, the prepared hemostatic glue may be loose (unformed) or have poor adhesion.

[0107] In some embodiments, the weight ratio of the modified bone powder, the stearate, the poloxamer, the high molecular weight polysaccharide and the solvent is (5-20): (5-20): (5-20): (5-20): (20-80).

[0108] In some embodiments, the weight ratio of the modified bone powder, the stearate, the poloxamer, the high molecular weight polysaccharide and the solvent is (10-20): (10-15): (10-15): (10-15): (20-40).

[0109] In some embodiments, the weight ratio of the modified bone powder, the stearate, the poloxamer, the high molecular weight polysaccharide and the solvent is 20:15:10:15:40.

[0110] In some embodiments, the weight ratio of the modified bone powder, the stearate, the poloxamer, the high molecular weight polysaccharide and the solvent is 20:10:15:15:40.

[0111] In some embodiments, the weight ratio of the modified bone powder, the stearate, the poloxamer, the high molecular weight polysaccharide and the solvent is 10:10:10:10:20.

[0112] In some embodiments, the weight ratio of the modified bone powder, the stearate, the poloxamer, the high molecular weight polysaccharide and the solvent is 12:15:15:15:25.

[0113] In the step of mixing the modified bone powder and the hemostatic cement, the mixing method can be conventional in the art, such as stirring. In some embodiments, an electric stirrer is used for stirring. In some embodiments, the stirring speed is 20 rpm to 100 rpm, such as 20 rpm, 25 rpm, 30 rpm, 35 rpm, 40 rpm, 45 rpm, 50 rpm, 55 rpm, 60 rpm, 65 rpm, 70 rpm, 75 rpm, 80 rpm, or a range or value between any two values.

[0114] In some embodiments, the intensity of the irradiation is 10 K to 40 K. In some embodiments, the intensity of the irradiation is 10 K, 15 K, 20 K, 25 K, 30 K, 35 K, 40 K, or a range or value between any two values.

[0115] In the seventh aspect of the present application, a bone regeneration repair material prepared using the preparation method described in the sixth aspect is provided.

[0116] The new multifunctional bone regeneration and repair material with self-assembling polypeptide in a clay state, which is prepared by the combined action of biomimetic active peptides, biological bone deproteinized particles and hemostatic clay, has plasticity, high adhesion, hemostasis, degradability and high osteogenic activity. It can be directly used in the clinical treatment of complex bone injury wounds to improve the osteogenic repair ability of the injured area.

[0117] In the eighth aspect of the present application, there is provided use of the biomimetic active peptide precursor as described in the first aspect, the biomimetic active peptide as described in the second aspect, or the modified bone powder as described in the fifth aspect in the preparation of a product for treating bone injuries.

[0118] Exemplary bone injuries include, but are not limited to, bone surface bleeding, bone defects, and the like.

[0119] In some embodiments, the product comprises a bone regeneration repair material. In some embodiments, the product is a bone regeneration repair material.

[0120] In the ninth aspect of the present application, a method for treating bone damage using the bone regeneration and repair material as described in the seventh aspect is provided.

[0121] In some embodiments, the method includes administering the bone regeneration repair material to an affected area of ​​a subject.

[0122] In the tenth aspect of the present application, a gene expression cassette is provided, which contains a promoter, the nucleic acid molecule as described in the third aspect, and a terminator.

[0123] In the eleventh aspect of the present application, a recombinant vector is provided, which comprises a backbone vector and one or more of the following items:

[0124] (1) The nucleic acid molecule according to the third aspect; and

[0125] (2) The gene expression cassette as described in the tenth aspect.

[0126] In the twelfth aspect of the present application, a recombinant cell is provided, which comprises one or more of the nucleic acid molecule as described in the third aspect, the gene expression cassette as described in the tenth aspect, and the recombinant vector as described in the eleventh aspect.

[0127] Some embodiments are provided below.

[0128] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0129] Example 1

[0130] Step 1: Preparation of biomimetic active peptides: (DOPA)4-EPLQLKM-RADA16 was artificially synthesized using an automatic peptide synthesizer according to the following amino acid sequence (DOPA-DOPA-DOPA-DOPA)-Glu-Pro-Leu-Gln-Leu-Lys-Met-Arg-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Met, wherein DOPA is 3,4-dihydroxyphenylalanine.

[0131] Step 2: (DOPA)4-EPLQLKM-RADA16 is prepared into an aqueous solution with a concentration of 5wt%, and mixed according to the weight ratio of biomimetic active peptide solution: biological bone deproteinized particles = 100:10, ultrasonically shaken for 30 minutes, placed in a vacuum freeze dryer (model: LGJ-50F) (0.7㎡ silicone oil heating), pre-frozen at -50°C, pre-vacuumed at -40°C, dried once at -20°C, and dried at 0°C, 20°C, and 25°C in sequence. After freeze-drying, the biomimetic active peptide modified bone powder is obtained (the freeze-drying method used in the following embodiments and comparative examples is the same as that of this embodiment).

[0132] Step 3: Mix the biomimetic active peptide modified bone powder prepared above with calcium stearate, poloxamer 407, sodium carboxymethyl cellulose and water in a weight ratio of biomimetic active peptide modified bone powder: calcium stearate: poloxamer 407: sodium carboxymethyl cellulose: water = 20:15:10:15:40 at room temperature, stir with an electric stirrer at a speed of 100 rpm for 4 hours, shape and package after stirring evenly, and sterilize by 25K irradiation.

[0133] The bone regeneration repair material was prepared, and the results showed that it had good plasticity ( Figure 1 ).

[0134] The preparation method of biological bone deproteinized particles comprises the following steps:

[0135] 1) animal bone particles are taken and washed with isopropanol and purified water by repeated alternating oscillation for 3 to 6 times to obtain defatted bone particles, wherein the mass ratio of the animal bone particles to isopropanol is 1:2.5;

[0136] 2) adding the defatted bone particles to hydrogen peroxide, allowing the particles to stand for 12 to 24 hours, discarding the hydrogen peroxide, placing the particles in purified water for 3 to 6 times of shaking washing, drying the particles at 75 to 100° C. for 5 to 7 hours, and passing the particles through a 14-mesh sieve to obtain sieved bone particles;

[0137] 3) adding the sieved bone particles into a 70%-100% ethylenediamine solution and heating to a slight boil, then maintaining the solution at 100-125° C. for 7-9 hours, discarding the waste liquid, adding the particles into purified water and shaking and washing them for 3 times, and repeating step 3) twice;

[0138] 4) The bone particles obtained in step 3) are washed 15 times with purified water and washed twice with injection water, with the washing process taking 3 to 6 minutes per time. During the washing process, the container is turned over and the waste liquid is discarded to obtain biological bone deproteinized bone particles.

[0139] The biological bone deproteinized particles in the following examples and comparative examples were all prepared using this method.

[0140] Example 2

[0141] Step 1: Preparation of biomimetic active peptides: Use an automatic peptide synthesizer to artificially synthesize (DOPA)6-EPLQLKM-RADA16 according to the following amino acid sequence (DOPA-DOPA-DOPA-DOPA-DOPA-DOPA)-Glu-Pro-Leu-Gln-Leu-Lys-Met-Arg-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Met.

[0142] Step 2: Prepare (DOPA)6-EPLQLKM-RADA16 into an aqueous solution with a concentration of 1wt%, mix it in a weight ratio of 100:10 for the biomimetic active peptide solution and biological bone deproteinized particles, use ultrasonic vibration for 20 minutes, and freeze-dry it in a freeze dryer to obtain the biomimetic active peptide modified bone powder.

[0143] Step 3: Mix the biomimetic active peptide modified bone powder prepared above with calcium stearate, poloxamer 188, sodium carboxymethyl cellulose and water in a weight ratio of biomimetic active peptide modified bone powder: calcium stearate: poloxamer 188: sodium carboxymethyl cellulose: vitamin E acetate = 20:10:15:15:40 at room temperature, stir with an electric stirrer at a speed of 50 rpm for 5 hours, shape and package after stirring evenly, and sterilize by 25K irradiation to prepare a bone repair material.

[0144] Example 3

[0145] Step 1: Preparation of biomimetic active peptides: Use an automatic peptide synthesizer to artificially synthesize (DOPA)6-EPLQLKM-IEIK-13 according to the following amino acid sequence (DOPA-DOPA-DOPA-DOPA-DOPA-DOPA)-Glu-Pro-Leu-Gln-Leu-Lys-Met-Arg-Ala-Asp-Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile-Glu-Ile-Lys-Ile-Met.

[0146] Step 2: Prepare (DOPA)6-EPLQLKM-IEIK-13 into a 10wt% aqueous solution, mix it in a weight ratio of 100:1 for the biomimetic active peptide solution and biological bone deproteinized particles, use ultrasonic vibration for 10 minutes, and freeze-dry it in a freeze dryer to obtain the biomimetic active peptide modified bone powder.

[0147] Step 3: Mix the biomimetic active peptide modified bone powder prepared above with calcium stearate, poloxamer 105, sodium alginate and water in a weight ratio of biomimetic active peptide modified bone powder: calcium stearate: poloxamer 105: sodium alginate: water = 10:10:10:10:20 at room temperature, stir with an electric stirrer at a speed of 100 rpm and a stirring time of 8 hours, shape and package after stirring evenly, and sterilize by 25K irradiation to prepare a bone repair material.

[0148] Example 4

[0149] Step 1: Preparation of biomimetic active peptides: Use an automatic peptide synthesizer to artificially synthesize (DOPA)6-EPLQLKM-KLD-12 according to the following amino acid sequence (DOPA-DOPA-DOPA-DOPA-DOPA-DOPA)-Glu-Pro-Leu-Gln-Leu-Lys-Met-Arg-Ala-Asp-Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu-Met.

[0150] Step 2: Prepare (DOPA)6-EPLQLKM-KLD-12 into an aqueous solution with a concentration of 3wt%, mix it in a weight ratio of 100:8 for the biomimetic active peptide solution and biological bone deproteinized particles, use ultrasonic vibration for 15 minutes, and freeze-dry it in a freeze dryer to obtain the biomimetic active peptide modified bone powder.

[0151] Step 3: Mix the biomimetic active peptide modified bone powder prepared above with calcium stearate, poloxamer 124, sodium hyaluronate and vitamin E acetate in a weight ratio of biomimetic active peptide modified bone powder: calcium stearate: poloxamer 124: sodium alginate: vitamin E acetate = 12:15:15:15:25 at room temperature, stir with an electric stirrer at a speed of 60 rpm and a stirring time of 4 hours. After stirring evenly, shape and package, and sterilize by 25K irradiation to prepare a bone repair material.

[0152] Comparative Example 1

[0153] Similar to Example 1, the difference is that the number of DOPA is adjusted to 2.

[0154] Step 1: Preparation of biomimetic active peptides: Use an automatic peptide synthesizer to artificially synthesize (DOPA)2-EPLQLKM-RADA16 according to the following amino acid sequence (DOPA-DOPA)-Glu-Pro-Leu-Gln-Leu-Lys-Met-Arg-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Met.

[0155] Step 2: (DOPA)2-EPLQLKM-RADA16 was prepared into an aqueous solution with a concentration of 1 wt%, and mixed according to a weight ratio of biomimetic active peptide solution: biological bone deproteinized particles = 100:2, ultrasonically shaken for 10 minutes, and freeze-dried in a freeze dryer to obtain biomimetic active peptide modified bone powder;

[0156] Step 3: Mix the biomimetic active peptide modified bone powder prepared above with calcium stearate, poloxamer 188, sodium carboxymethyl cellulose and water in a weight ratio of biomimetic active peptide modified bone powder: calcium stearate: poloxamer 188: sodium carboxymethyl cellulose: water = 15:15:5:15:40 at room temperature, stir with an electric stirrer at a speed of 100 rpm and a stirring time of 2 hours, shape and package after stirring evenly, and sterilize by 25K irradiation to prepare a bone repair material.

[0157] Comparative Example 2

[0158] Similar to Example 1, the difference is that the number of DOPAs is adjusted to 8.

[0159] Step 1: Preparation of biomimetic active peptides: Use an automatic peptide synthesizer to artificially synthesize (DOPA)8-EPLQLKM-RADA16 according to the following amino acid sequence (DOPA-DOPA-DOPA-DOPA-DOPA-DOPA-DOPA-Glu-Pro-Leu-Gln-Leu-Lys-Met-Arg-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Met.

[0160] Step 2: (DOPA)8-EPLQLKM-RADA16 was prepared into an aqueous solution with a concentration of 0.5wt%, and mixed according to the weight ratio of biomimetic active peptide solution: biological bone deproteinized particles = 100:5, and ultrasonically vibrated for 20 minutes, and freeze-dried in a freeze dryer to obtain biomimetic active peptide modified bone powder;

[0161] Step 3: Mix the prepared biomimetic active peptide modified bone powder with calcium stearate, poloxamer 407, carboxymethyl chitosan and water at a weight ratio of 15:20:10:15:40 at room temperature, stir with an electric stirrer at a speed of 80 rpm for 4 hours, shape and package after stirring evenly, and sterilize by 25K irradiation to obtain a bone repair material.

[0162] Comparative Example 3

[0163] Similar to Example 1, except that no (DOPA)4-EPLQLKM-RADA16 was added, and only bone meal and cement were contained.

[0164] The bone powder was mixed with calcium stearate, poloxamer 407, sodium carboxymethyl cellulose and water at a weight ratio of 20:15:10:15:40 of biomimetic active peptide-modified bone powder: calcium stearate: poloxamer 407: sodium carboxymethyl cellulose: water at room temperature, and stirred with an electric stirrer at a speed of 100 rpm for 4 hours. After stirring evenly, the mixture was molded, packaged and sterilized by 25K irradiation to prepare a bone repair material.

[0165] Comparative Example 4

[0166] Similar to Example 1, the difference is that DOPA, E7 peptide and RADA16 peptide are not connected together, but mixed and added.

[0167] Step 1: Preparation of DOPA: Use an automatic peptide synthesizer to artificially synthesize (DOPA)4, EPLQLKM and RADA16 peptides according to the following amino acid sequences (DOPA-DOPA-DOPA-DOPA)–Met, Glu-Pro-Leu-Gln-Leu-Lys-Met-Arg-Ala-Asp-Met and Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala–Met, respectively.

[0168] Step 2: (DOPA)4, EPLQLKM and RADA16 peptides were prepared into a 5wt% aqueous solution according to a weight ratio of (DOPA)4:EPLQLKM:RADA16=1:1:1, and then the peptide solution: biological bone deproteinized particles were mixed according to a weight ratio of 100:10, ultrasonically shaken for 30 minutes, and freeze-dried in a freeze dryer to obtain biomimetic active peptide modified bone powder;

[0169] Step 3: Mix the biomimetic active peptide modified bone powder prepared above with calcium stearate, poloxamer 407, sodium carboxymethyl cellulose and water in a weight ratio of biomimetic active peptide modified bone powder: calcium stearate: poloxamer 407: sodium carboxymethyl cellulose: water = 20:15:10:15:40 at room temperature, stir with an electric stirrer at a speed of 100 rpm and a stirring time of 4 hours, shape and package after stirring evenly, and sterilize by 25K irradiation to prepare a bone repair material.

[0170] Comparative performance:

[0171] 1. Adhesion strength:

[0172] The specific operation process is as follows: two cortical bone rings with a width of 10 mm are prepared from bovine femurs, and an equal amount of test material is evenly applied to the surface of one of the cortical bone rings (S). After the other cortical bone ring is in contact with the sample, a pressure of 78 N is applied at a speed of 1 mm / min for 1 min. Then the two sides of the bone ring are connected to the tensile fixture of the multifunctional mechanical testing machine, and the two bone rings are pulled apart at a speed of 1 mm / min until the bond breaks, and the maximum tensile force F at the break is recorded, and the bond strength (MPa) = F / S is calculated. The experimental results are shown in Table 1.

[0173] Table 1 Adhesion strength results of examples and comparative examples

[0174]

[0175] 2. The results of the effectiveness evaluation of rabbit skull defects carried out in Example 1 and Example 3 are as follows:

[0176] 12 New Zealand rabbits were used as experimental animals and were divided into 4 groups on average. The samples of Example 1 and Example 3 were used as experimental group 1 and experimental group 2 respectively, and bioactive glass and self-setting calcium phosphate were used as test control group 1 and control group 2 respectively, and the rest were used as blank groups. Defect model was made at New Zealand rabbit skull, and skin, soft tissue and periosteum were cut successively along the midline of the skull using sterile surgical instruments, and skin and periosteum flap were turned back to the rear of the parietal bone, and bone defects with a diameter of 6mm and a depth of about 1mm were drilled at 1cm from the orbit and 1cm from the ear on the sagittal line, until blood gushed out of the bone defect area, and double-layer cortex was drilled, and the dura mater was not injured. The samples in Example 1 and Example 2, the bioactive glass of the control group, and the self-setting calcium phosphate of the control group were implanted into the skull defect of the animal respectively. After the operation, the periosteum was used to cover the material, and the skin and periosteum tissue flap were reset. After 2 weeks, the defect was observed to have a repair effect. After the operation, the periosteum was used to cover the material, and the skin and periosteum tissue flap were reset. After 2 weeks, the defect was observed to have a repair effect.

[0177] Table 2 Results of bone volume fraction at defect site after 2 weeks of sampling

[0178]

[0179] According to the bonding strength results of the above embodiments and comparative examples (see Table 1), the bonding strength of the bone repair materials prepared in embodiments 1 to 4 is higher than that of the control group; at the same time, according to the effectiveness results of rabbit skull defects, the bone defect repair effects of the bone repair materials prepared in embodiments 1 and 3 were compared with the conventional bone repair materials bioactive glass and self-solidifying calcium phosphate on the market. It can be seen that the bone volume fractions of experimental groups 1 and 2 were significantly higher than those of the control group 2 weeks after the operation, and the MicroCT results showed that experimental groups 1 and 2 had obvious new bone formation compared with the control group ( Figure 2~Figure 7).

[0180] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

[0181] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A biomimetic active peptide precursor, characterized in that: It comprises m tyrosines, an E7 peptide and a self-assembling polypeptide connected in sequence from the N segment to the C terminus; Wherein, the amino acid sequence of the E7 peptide comprises the sequence shown in SEQ ID NO: 1; m is an integer between 2 and 8; Optionally, the amino acid sequence of the biomimetic active peptide precursor comprises a sequence as shown in any one of SEQ ID NOs: 5 to 8.

2. A biomimetic active peptide, characterized in that: It comprises n 3,4-L-dihydroxyphenylalanine, E7 peptide and self-assembling polypeptide connected in sequence from N segment to C terminal; Wherein, the amino acid sequence of the E7 peptide comprises the sequence shown in SEQ ID NO: 1; n is an integer between 2 and 8; Optionally, the biomimetic active peptide is obtained by oxidizing the biomimetic active peptide precursor according to claim 1.

3. The biomimetic active peptide precursor according to claim 1 or the biomimetic active peptide according to claim 2, characterized in that: The self-assembling polypeptide is selected from the group consisting of a self-assembling KLD-12 peptide, a self-assembling RADA16 peptide, a self-assembling IEIK-13 peptide, a self-assembling KLD-12 peptide derivative, a self-assembling RADA16 peptide derivative, and a self-assembling IEIK-13 peptide derivative; Optionally, the self-assembling polypeptide is a self-assembling RADA16 peptide; the amino acid sequence of the self-assembling RADA16 peptide optionally comprises the sequence shown in SEQ ID NO: 2; Optionally, the self-assembling polypeptide is a self-assembling IEIK-13 peptide; the amino acid sequence of the self-assembling IEIK-13 peptide optionally comprises the sequence shown in SEQ ID NO: 3; Optionally, the self-assembling polypeptide is a self-assembling KLD-12 peptide; the amino acid sequence of the self-assembling KLD-12 peptide optionally comprises a sequence as shown in SEQ ID NO:

4.

4. A nucleic acid molecule, characterized in that It encodes the biomimetic active peptide precursor as described in claim 1 or 3.

5. A method for preparing modified bone meal, characterized in that: It includes the following steps: The solution of the biomimetic active peptide as claimed in claim 2 or 3 and the biological bone deproteinized particles are uniformly dispersed in the solution and freeze-dried; Optionally, the preparation method comprises the steps of: mixing a solution of the biomimetic active peptide as claimed in claim 2 or 3 with a mass concentration of 1% to 10% with biological bone deproteinized particles, and freeze-drying the mixture; Further optionally, the preparation method satisfies one or more of the following conditions: The weight ratio of the biomimetic active peptide solution to the biological bone deproteinized particles is 100:(1-10); Mixing by shaking, wherein the shaking can be selected from one or more of ultrasonic shaking and vortex shaking, and the shaking time can be 10 min to 30 min; and The biological bone in the biological bone deproteinized particles is derived from one or more of bovine bones and pig bones.

6. The modified bone powder prepared by the preparation method according to claim 5.

7. A method for preparing a bone regeneration and repair material, characterized in that: It includes the following steps: Mixing the modified bone powder as claimed in claim 6 and hemostatic cement; and Irradiation sterilization to prepare bone regeneration and repair materials; Wherein, the weight ratio of the modified bone powder to the hemostatic cement is (5-20): (35-140); Optionally, the mixing method is stirring; wherein: the stirring speed can be optionally 20 rpm~100 rpm, and the stirring time can be optionally 2h~8h.

8. The preparation method according to claim 7, characterized in that: The hemostatic putty comprises stearate, poloxamer, high molecular weight polysaccharide and solvent; Optionally, the hemostatic putty satisfies one or more of the following characteristics: (I) the polymer polysaccharide is one or more selected from the group consisting of sodium hyaluronate, oxidized regenerated cellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose, carboxymethyl chitosan and sodium alginate; (II) the poloxamer is selected from one or more of the group consisting of poloxamer 188, poloxamer 338, poloxamer 407, poloxamer 105, poloxamer 124, poloxamer 184, poloxamer 237, poloxamer 335, poloxamer 403 and poloxamer 401; (III) the solvent comprises one or more of water and vitamin E acetate; (iv) The weight ratio of the modified bone powder, the stearate, the poloxamer, the high molecular weight polysaccharide and the solvent is (5-20):(5-20):(5-20):(5-20):(20-80).

9. A bone regeneration and repair material prepared by the preparation method according to claim 7 or 8.

10. Use of the biomimetic active peptide precursor according to claim 1 or 3, the biomimetic active peptide according to claim 2 or 3, or the modified bone powder according to claim 6 in the preparation of a product for treating bone damage; Optionally, the product includes bone regeneration and repair materials.

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