A bone repair material using cervus flacca polypeptide and a preparation method thereof

Abstract

This invention relates to the field of bone repair materials technology, and discloses a bone repair material utilizing deer bone peptides and its preparation method. The method includes the preparation of deer bone extract, the preparation of melon seed extract, the formulation of a composite peptide solution, the preparation of a carrier base solution, and the compounding and molding. By using a composite peptide solution containing deer bone peptides and melon seed peptides, the synergistic effect of the two peptides enables the material to possess excellent osteoblast proliferation, differentiation, and anti-inflammatory activity, thereby promoting the repair and regeneration of bone defects and enhancing the bioactivity and therapeutic targeting of the material. By mixing the composite peptide solution with a carrier base solution containing a biocompatible carrier material and adding a cross-linking agent for cross-linking, a stable structure is formed between the peptides and the carrier material, thereby ensuring that the material has good mechanical support properties and a controllable degradation rate, ensuring that the material maintains its morphology in the early stages of implantation and guides new bone growth in an orderly manner over time.

Description

Technical Field

[0001] This invention relates to the field of bone repair materials technology, specifically to a bone repair material utilizing deer antler polypeptide and its preparation method. Background Technology

[0002] Deer bone polypeptide is formulated from extracts of deer bone and melon seeds. Traditional Chinese medicine believes that deer bone is sweet and slightly hot, and has the effects of nourishing deficiency and strengthening bones and muscles, and treating rheumatic pain in the limbs and cold numbness in the muscles and bones. Melon seeds have the effects of dispersing nodules and relieving pain. The combination of the two is used to dispel cold, dispel wind and dampness, and relieve pain, thus having a therapeutic effect on rheumatoid arthritis.

[0003] Currently, in the field of bone defect repair materials, traditional materials often lack specific active ingredient combinations that can effectively simulate the bone tissue microenvironment and synergistically promote osteoblast behavior, which may lead to poor bone integration efficiency or inflammatory response.

[0004] Therefore, a bone repair material using deer antler polypeptide and its preparation method are proposed to solve the above problems. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a bone repair material utilizing deer antler polypeptide and its preparation method, solving the problem mentioned in the background art of lacking a specific combination of active ingredients that can effectively simulate the bone tissue microenvironment and synergistically promote osteoblast behavior, which may lead to poor bone integration efficiency or inflammatory response.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a bone repair material using deer antler polypeptide, comprising the following steps: Step 1: Preparation of deer bone extract. The pretreated deer bone raw material is crushed to obtain deer bone powder. The deer bone powder is mixed with the first extraction solvent for the first extraction. After solid-liquid separation, the first extract and the first bone residue are obtained. The first bone residue is mixed with the second extraction solvent for the second extraction. After solid-liquid separation, the second extract is obtained. The first extract and the second extract are combined, concentrated, precipitated with alcohol, and dried to obtain deer bone extract. Step 2: Preparation of melon seed extract. The pretreated melon seed raw material is crushed to obtain melon seed powder. The melon seed powder is mixed with the third extraction solvent and extracted. After solid-liquid separation, the third extract is obtained. The third extract is concentrated, precipitated with alcohol, and dried to obtain melon seed extract. Step 3: Preparation of the complex polypeptide solution. The deer bone extract prepared in Step 1 and the melon seed extract prepared in Step 2 are dissolved in phosphate buffer in a certain proportion to obtain the complex polypeptide solution. The weight ratio of deer bone polypeptide in the deer bone extract to melon seed polypeptide in the melon seed extract is 2:1 to 6:1. Step 4: Preparation of carrier base solution. The biocompatible carrier material is dissolved in a solvent to obtain the carrier base solution. Step 5: Composite and molding. The composite peptide solution is mixed with the carrier base liquid and composited under stirring to obtain a uniform composite slurry. The composite slurry is injected into a mold and subjected to cross-linking curing or freeze-drying treatment to obtain the bone repair material.

[0007] Preferably, in step one, the preprocessing includes: The deer bone raw material is soaked in a sodium hydroxide solution with a concentration of 0.5-1.5 mol / L at 40-60℃ for 1-3 hours. After soaking, it is taken out, rinsed with deionized water until neutral, and then frozen at -20℃ to -40℃ for 12-24 hours before being vacuum freeze-dried.

[0008] Preferably, in step one, the conditions for the first extraction are: The first extraction solvent is a phosphate buffer solution with a pH of 7.0-7.8. The mass-to-volume ratio of deer bone powder to the first extraction solvent is 1g:10-20mL. The extraction temperature is 45-60℃ and the extraction time is 4-8 hours. The conditions for the second extraction were as follows: the second extraction solvent was a phosphate buffer solution with a pH of 7.0-7.8; the mass-to-volume ratio of the first bone residue to the second extraction solvent was 1 g: 5-10 mL; the extraction temperature was 45-60℃; and the extraction time was 2-4 hours.

[0009] Preferably, in step two, the preprocessing includes: The raw melon seeds are dried at 40-50℃ to constant weight, dehulled, pulverized, and passed through an 80-120 mesh sieve to obtain melon seed powder. The extraction conditions were as follows: the third extraction solvent was water, the mass-to-volume ratio of melon seed powder to the third extraction solvent was 1g:15-25mL, the extraction temperature was 60-80℃, and the extraction time was 2-4 hours.

[0010] Preferably, in step three, the total concentration of deer bone polypeptide and melon seed polypeptide in the composite polypeptide solution is 10-50 mg / mL, the concentration of the phosphate buffer is 0.01-0.05 mol / L, and the pH value is 7.2-7.6.

[0011] Preferably, in step four, the biocompatible carrier material is at least one of gelatin, chitosan, hyaluronic acid, sodium alginate, type I collagen, or polylactic acid-glycolic acid copolymer, and the solvent is at least one of water, acetic acid solution, or trifluoroethanol. The biocompatible carrier material in the carrier base solution has a mass-volume concentration of 5%-20%.

[0012] Preferably, in step five, the composite process specifically includes: The complex peptide solution was slowly added dropwise to the carrier base solution under stirring, and the volume ratio of the complex peptide solution to the carrier base solution was controlled to be 1:1 to 1:4. After the addition was completed, the mixture was stirred continuously at 200-500 r / min for 30-60 minutes at room temperature to obtain a uniform complex slurry.

[0013] Preferably, in step five, the crosslinking curing includes: Add a crosslinking agent to the composite slurry, mix evenly, inject into a mold, and let it stand at 25-37℃ for 1-3 hours for crosslinking. After molding, take it out and wash it with deionized water to obtain a hydrogel-like bone repair material. The crosslinking agent is at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, glutaraldehyde, or genipin, and the amount of crosslinking agent added is 0.5%-5% of the mass of the biocompatible carrier material.

[0014] Preferably, in step five, the freeze-drying process includes: The composite slurry is injected into a mold and pre-frozen at -20℃ to -80℃ for 4-12 hours. Then it is transferred to a freeze dryer and freeze-dried for 24-72 hours under conditions of cold trap temperature below -50℃ and vacuum degree below 10Pa to obtain a porous scaffold-shaped bone repair material.

[0015] A bone repair material utilizing deer antler polypeptide, the bone repair material being made by compounding and molding raw materials comprising the following parts by weight: 10-40 parts of compound polypeptide solution and 60-90 parts of carrier base solution. The composite polypeptide solution contains deer bone extract and melon seed extract, wherein the weight ratio of deer bone polypeptide to melon seed polypeptide is 2:1 to 6:1. The carrier base liquid contains a biocompatible carrier material with a mass-volume concentration of 5%-20%.

[0016] Compared with the prior art, the present invention provides a bone repair material using deer antler polypeptide and its preparation method, which has the following beneficial effects: 1. In this invention, by using a composite polypeptide solution containing deer bone polypeptide and melon seed polypeptide, the synergistic effect of the two polypeptides enables the material to have excellent osteoblast proliferation, differentiation and anti-inflammatory activity, thereby promoting the repair and regeneration of bone defects and improving the bioactivity and therapeutic targeting of the material.

[0017] 2. In this invention, by mixing the composite polypeptide solution with a carrier base liquid containing a biocompatible carrier material and adding a crosslinking agent for crosslinking, a stable structure is formed between the polypeptide and the carrier material, thereby ensuring that the material has good mechanical support properties and a controllable degradation rate, and ensuring that the material maintains its shape in the early stage of implantation and guides new bone growth in an orderly manner over time.

[0018] 3. In this invention, the composite slurry is cross-linked and cured or freeze-dried through the compounding and molding steps, so that the final material forms a solid scaffold or hydrogel with a porous interpenetrating structure, which is conducive to osteoblast ingrowth, adhesion and nutrient exchange, thus optimizing the clinical applicability and osteointegration effect of the material. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1: A method for preparing a bone repair material using deer antler polypeptide, comprising the following steps: Step 1: Preparation of deer bone extract. The pretreated deer bone raw material is crushed to obtain deer bone powder. The deer bone powder is mixed with the first extraction solvent for the first extraction. After solid-liquid separation, the first extract and the first bone residue are obtained. The first bone residue is mixed with the second extraction solvent for the second extraction. After solid-liquid separation, the second extract is obtained. The first extract and the second extract are combined, concentrated, precipitated with alcohol, and dried to obtain deer bone extract. Step 2: Preparation of melon seed extract. The pretreated melon seed raw material is crushed to obtain melon seed powder. The melon seed powder is mixed with the third extraction solvent and extracted. After solid-liquid separation, the third extract is obtained. The third extract is concentrated, precipitated with alcohol, and dried to obtain melon seed extract. Step 3: Preparation of the complex polypeptide solution. The deer bone extract prepared in Step 1 and the melon seed extract prepared in Step 2 are dissolved in phosphate buffer in a certain proportion to obtain the complex polypeptide solution. The weight ratio of deer bone polypeptide in the deer bone extract to melon seed polypeptide in the melon seed extract is 2:1. Step 4: Preparation of carrier base solution. The biocompatible carrier material is dissolved in a solvent to obtain the carrier base solution. Step 5: Composite and molding. The composite peptide solution is mixed with the carrier base liquid and composited under stirring to obtain a uniform composite slurry. The composite slurry is injected into a mold and subjected to cross-linking curing or freeze-drying to obtain the bone repair material.

[0021] In step one, preprocessing includes: The deer bone raw material was soaked in a 0.5 mol / L sodium hydroxide solution at 40°C for 1 hour. After soaking, it was taken out, rinsed with deionized water until neutral, and then frozen at -20°C for 12 hours before being vacuum freeze-dried.

[0022] In step one, the conditions for the first extraction are: The first extraction solvent was a phosphate buffer solution with a pH of 7.0. The mass-to-volume ratio of deer bone powder to the first extraction solvent was 1g:10mL. The extraction temperature was 45℃ and the extraction time was 4 hours. The conditions for the second extraction were as follows: the second extraction solvent was a phosphate buffer solution with a pH of 7.0, the mass-to-volume ratio of the first bone residue to the second extraction solvent was 1 g: 5 mL, the extraction temperature was 45 °C, and the extraction time was 2 hours.

[0023] In step two, preprocessing includes: The raw melon seeds were dried at 40℃ to constant weight, dehulled, pulverized, and passed through an 80-mesh sieve to obtain melon seed powder. The extraction conditions were as follows: the third extraction solvent was water, the mass-to-volume ratio of melon seed powder to the third extraction solvent was 1g:15mL, the extraction temperature was 60℃, and the extraction time was 2 hours.

[0024] In step three, the total concentration of deer bone polypeptide and melon seed polypeptide in the complex polypeptide solution is 10 mg / mL, the concentration of phosphate buffer is 0.01 mol / L, and the pH value is 7.2.

[0025] In step four, the biocompatible carrier material is gelatin, and the solvent is water; The biocompatible carrier material has a mass-volume concentration of 5% in the carrier base solution.

[0026] Step five, specifically, includes: The complex peptide solution was slowly added dropwise to the carrier base solution under stirring, and the volume ratio of the complex peptide solution to the carrier base solution was controlled at 1:1. After the addition was completed, the mixture was stirred continuously at 200 r / min for 30 minutes at room temperature to obtain a uniform complex slurry.

[0027] In step five, cross-linking curing includes: Add a crosslinking agent to the composite slurry, mix well, inject into a mold, let stand at 25°C for 1 hour for crosslinking, remove after molding, wash with deionized water to obtain a hydrogel-like bone repair material. The crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the amount of crosslinking agent added is 0.5% of the mass of the biocompatible carrier material.

[0028] Step five, the freeze-drying process includes: The composite slurry was injected into a mold and pre-frozen at -20°C for 4 hours. Then it was transferred to a freeze dryer and freeze-dried for 24 hours under conditions of cold trap temperature below -50°C and vacuum degree below 10Pa to obtain a porous scaffold-shaped bone repair material.

[0029] A bone repair material utilizing deer antler polypeptide, the bone repair material is made by compounding and molding the following raw materials in parts by weight: 10 parts of compound polypeptide solution and 60 parts of carrier base liquid. The complex polypeptide solution contains deer bone extract and melon seed extract, wherein the weight ratio of deer bone polypeptide to melon seed polypeptide is 2:1. The carrier base solution contains 5% by mass / volume of biocompatible carrier material.

[0030] Example 2: A method for preparing a bone repair material using deer antler polypeptide, comprising the following steps: Step 1: Preparation of deer bone extract. The pretreated deer bone raw material is crushed to obtain deer bone powder. The deer bone powder is mixed with the first extraction solvent for the first extraction. After solid-liquid separation, the first extract and the first bone residue are obtained. The first bone residue is mixed with the second extraction solvent for the second extraction. After solid-liquid separation, the second extract is obtained. The first extract and the second extract are combined, concentrated, precipitated with alcohol, and dried to obtain deer bone extract. Step 2: Preparation of melon seed extract. The pretreated melon seed raw material is crushed to obtain melon seed powder. The melon seed powder is mixed with the third extraction solvent and extracted. After solid-liquid separation, the third extract is obtained. The third extract is concentrated, precipitated with alcohol, and dried to obtain melon seed extract. Step 3: Preparation of the complex polypeptide solution. The deer bone extract prepared in Step 1 and the melon seed extract prepared in Step 2 are dissolved in phosphate buffer in a certain proportion to obtain the complex polypeptide solution. The weight ratio of deer bone polypeptide in the deer bone extract to melon seed polypeptide in the melon seed extract is 2:1 to 6:1. Step 4: Preparation of carrier base solution. The biocompatible carrier material is dissolved in a solvent to obtain the carrier base solution. Step 5: Composite and molding. The composite peptide solution is mixed with the carrier base liquid and composited under stirring to obtain a uniform composite slurry. The composite slurry is injected into a mold and subjected to cross-linking curing or freeze-drying to obtain the bone repair material.

[0031] In step one, preprocessing includes: The deer bone raw material was soaked in a 1.0 mol / L sodium hydroxide solution at 50°C for 2 hours. After soaking, it was taken out, rinsed with deionized water until neutral, and then frozen at -30°C for 18 hours before being vacuum freeze-dried.

[0032] In step one, the conditions for the first extraction are: The first extraction solvent was a phosphate buffer solution with a pH of 7.4. The mass-to-volume ratio of deer bone powder to the first extraction solvent was 1 g: 15 mL. The extraction temperature was 52 °C and the extraction time was 6 hours. The conditions for the second extraction were as follows: the second extraction solvent was a phosphate buffer solution with a pH of 7.4, the mass-to-volume ratio of the first bone residue to the second extraction solvent was 1 g: 7.5 mL, the extraction temperature was 52 °C, and the extraction time was 3 hours.

[0033] In step two, preprocessing includes: The raw melon seeds were dried at 45℃ to constant weight, dehulled, pulverized, and passed through a 100-mesh sieve to obtain melon seed powder. The extraction conditions were as follows: the third extraction solvent was water, the mass-to-volume ratio of melon seed powder to the third extraction solvent was 1g:20mL, the extraction temperature was 70℃, and the extraction time was 3 hours.

[0034] In step three, the total concentration of deer bone polypeptide and melon seed polypeptide in the complex polypeptide solution is 30 mg / mL, the concentration of phosphate buffer is 0.03 mol / L, and the pH value is 7.4.

[0035] In step four, the biocompatible carrier material is gelatin, and the solvent is water; The biocompatible carrier material has a mass-volume concentration of 12% in the carrier base solution.

[0036] Step five, specifically, includes: The complex peptide solution was slowly added dropwise to the carrier base solution under stirring, and the volume ratio of the complex peptide solution to the carrier base solution was controlled at 1:2.5. After the addition was completed, the mixture was stirred continuously at 350 r / min for 45 minutes at room temperature to obtain a uniform complex slurry.

[0037] In step five, cross-linking curing includes: Add a crosslinking agent to the composite slurry, mix well, inject into a mold, let stand at 31°C for 2 hours for crosslinking, remove after molding, wash with deionized water to obtain a hydrogel-like bone repair material. The crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the amount of crosslinking agent added is 2.7% of the mass of the biocompatible carrier material.

[0038] Step five, the freeze-drying process includes: The composite slurry was injected into a mold and pre-frozen at -50°C for 8 hours. Then it was transferred to a freeze dryer and freeze-dried for 48 hours under conditions where the cold trap temperature was below -50°C and the vacuum degree was below 10Pa to obtain a porous scaffold-shaped bone repair material.

[0039] A bone repair material utilizing deer antler polypeptide, the bone repair material is made by compounding and molding the following raw materials in parts by weight: 30 parts of compound polypeptide solution and 75 parts of carrier base liquid. The complex polypeptide solution contains deer bone extract and melon seed extract, wherein the weight ratio of deer bone polypeptide to melon seed polypeptide is 4:1. The carrier base solution contains 12% biocompatible carrier material by mass volume.

[0040] Example 3: A method for preparing a bone repair material using deer antler polypeptide, comprising the following steps: Step 1: Preparation of deer bone extract. The pretreated deer bone raw material is crushed to obtain deer bone powder. The deer bone powder is mixed with the first extraction solvent for the first extraction. After solid-liquid separation, the first extract and the first bone residue are obtained. The first bone residue is mixed with the second extraction solvent for the second extraction. After solid-liquid separation, the second extract is obtained. The first extract and the second extract are combined, concentrated, precipitated with alcohol, and dried to obtain deer bone extract. Step 2: Preparation of melon seed extract. The pretreated melon seed raw material is crushed to obtain melon seed powder. The melon seed powder is mixed with the third extraction solvent and extracted. After solid-liquid separation, the third extract is obtained. The third extract is concentrated, precipitated with alcohol, and dried to obtain melon seed extract. Step 3: Preparation of the complex polypeptide solution. The deer bone extract prepared in Step 1 and the melon seed extract prepared in Step 2 are dissolved in phosphate buffer in a certain proportion to obtain the complex polypeptide solution. The weight ratio of deer bone polypeptide in the deer bone extract to melon seed polypeptide in the melon seed extract is 6:1. Step 4: Preparation of carrier base solution. The biocompatible carrier material is dissolved in a solvent to obtain the carrier base solution. Step 5: Composite and molding. The composite peptide solution is mixed with the carrier base liquid and composited under stirring to obtain a uniform composite slurry. The composite slurry is injected into a mold and subjected to cross-linking curing or freeze-drying to obtain the bone repair material.

[0041] In step one, preprocessing includes: The deer bone raw material was soaked in a 1.5 mol / L sodium hydroxide solution at 60°C for 3 hours. After soaking, it was taken out, rinsed with deionized water until neutral, and then frozen at -40°C for 24 hours before being vacuum freeze-dried.

[0042] In step one, the conditions for the first extraction are: The first extraction solvent was a phosphate buffer solution with a pH of 7.8. The mass-to-volume ratio of deer bone powder to the first extraction solvent was 1 g: 20 mL. The extraction temperature was 60 °C and the extraction time was 8 hours. The conditions for the second extraction were as follows: the second extraction solvent was a phosphate buffer solution with a pH of 7.8, the mass-to-volume ratio of the first bone residue to the second extraction solvent was 1 g: 10 mL, the extraction temperature was 60 °C, and the extraction time was 4 hours.

[0043] In step two, preprocessing includes: The raw melon seeds were dried at 50°C to constant weight, shelled, pulverized, and passed through a 120-mesh sieve to obtain melon seed powder. The extraction conditions were as follows: the third extraction solvent was water, the mass-to-volume ratio of melon seed powder to the third extraction solvent was 1g:25mL, the extraction temperature was 80℃, and the extraction time was 4 hours.

[0044] In step three, the total concentration of deer bone peptide and melon seed peptide in the complex peptide solution is 50 mg / mL, the concentration of phosphate buffer is 0.05 mol / L, and the pH value is 7.6.

[0045] In step four, the biocompatible carrier material is gelatin, and the solvent is water; The biocompatible carrier material has a mass-volume concentration of 20% in the carrier base solution.

[0046] Step five, specifically, includes: The composite peptide solution was slowly added dropwise to the carrier base solution under stirring, and the volume ratio of the composite peptide solution to the carrier base solution was controlled at 1:4. After the addition was completed, the mixture was continuously stirred at 500 r / min for 60 minutes at room temperature to obtain a uniform composite slurry.

[0047] In step five, cross-linking curing includes: Add a crosslinking agent to the composite slurry, mix well, inject into a mold, let stand at 37°C for 3 hours for crosslinking, remove after molding, wash with deionized water to obtain a hydrogel-like bone repair material; The crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the amount of crosslinking agent added is 5% of the mass of the biocompatible carrier material.

[0048] Step five, the freeze-drying process includes: The composite slurry was injected into a mold and pre-frozen at -80°C for 12 hours. Then it was transferred to a freeze dryer and freeze-dried for 72 hours under conditions of cold trap temperature below -50°C and vacuum degree below 10Pa to obtain a porous scaffold-shaped bone repair material.

[0049] A bone repair material utilizing deer antler polypeptide, the bone repair material is made by compounding and molding the following raw materials in parts by weight: 40 parts of compound polypeptide solution and 90 parts of carrier base liquid. The complex polypeptide solution contains deer bone extract and melon seed extract, wherein the weight ratio of deer bone polypeptide to melon seed polypeptide is 6:1. The carrier base solution contains 20% biocompatible carrier material by mass volume.

[0050] Comparative Example 1: The difference between this comparative example and Example 1 is that no deer bone extract and melon seed extract were added when preparing the composite polypeptide solution in this comparative example. Only an equal volume of phosphate buffer and carrier base solution were used for composite preparation.

[0051] Comparative Example 2 differs from Example 1 in that no biocompatible carrier material was added when preparing the carrier base solution; only the solvent and the composite polypeptide solution were mixed.

[0052] Comparative Example 3 differs from Example 1 in that no crosslinking agent was added to the composite slurry during the compounding and molding process.

[0053] Comparative Example 4 differs from Example 1 in that the composite slurry was not cross-linked, cured, or freeze-dried during the compounding and molding process; the mixed slurry was simply left to stand and dry.

[0054] The bone repair materials prepared in Examples 1-3 and Comparative Examples 1-4 were subjected to performance tests. The test items and test methods are as follows: Mechanical property testing was conducted using a universal testing machine to measure the material's compressive modulus. In vitro degradation rate test: The sample was immersed in simulated body fluid, oscillated at a constant temperature of 37°C, and periodically removed, dried and weighed to calculate the percentage of mass loss. Cell proliferation rate was tested using the CCK-8 assay. Osteoblasts were co-cultured with the material extract, absorbance was measured, and the relative cell proliferation rate was calculated. The alkaline phosphatase activity test involved seeding osteoblasts onto the material surface, culturing them for a certain period, lysing the cells, and measuring ALP activity using an alkaline phosphatase assay kit to assess the osteogenic induction capacity of the material.

[0055] The deer antler peptide bone repair material prepared using the processes in Examples 1-3 exhibits significantly superior performance compared to the deer antler peptide bone repair material prepared using the processes in Comparative Examples 1-4. This demonstrates that the present invention, by employing a composite peptide solution containing deer bone peptides and melon seed peptides, utilizes the synergistic effect of the two peptides to enable the material to possess excellent osteoblast proliferation, differentiation, and anti-inflammatory activity, thereby promoting the repair and regeneration of bone defects and enhancing the material's bioactivity and therapeutic targeting. By mixing the composite peptide solution with a carrier base liquid containing biocompatible carrier materials and adding a cross-linking agent for cross-linking, a stable structure is formed between the peptides and the carrier materials, ensuring that the material has good mechanical support properties and a controllable degradation rate, ensuring that the material maintains its morphology in the early stages of implantation and guides new bone growth in an orderly manner over time. Through the composite and molding steps, the composite slurry is cross-linked and cured or freeze-dried, resulting in a final material forming a solid scaffold or hydrogel morphology with a porous interpenetrating structure, which is conducive to osteoblast ingrowth, adhesion, and nutrient exchange, optimizing the material's clinical applicability and bone integration effect.

[0056] The deer antler polypeptide bone repair material prepared by the molding process of this invention has good mechanical support properties, controllable degradation rate and excellent osteogenic bioactivity.

[0057] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0058] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a bone repair material using deer antler polypeptide, characterized in that: Includes the following steps: Step 1: Preparation of deer bone extract. The pretreated deer bone raw material is crushed to obtain deer bone powder. The deer bone powder is mixed with the first extraction solvent for the first extraction. After solid-liquid separation, the first extract and the first bone residue are obtained. The first bone residue is mixed with the second extraction solvent for the second extraction. After solid-liquid separation, the second extract is obtained. The first extract and the second extract are combined, concentrated, precipitated with alcohol, and dried to obtain deer bone extract. Step 2: Preparation of melon seed extract. The pretreated melon seed raw material is crushed to obtain melon seed powder. The melon seed powder is mixed with the third extraction solvent and extracted. After solid-liquid separation, the third extract is obtained. The third extract is concentrated, precipitated with alcohol, and dried to obtain melon seed extract. Step 3: Preparation of the complex polypeptide solution. The deer bone extract prepared in Step 1 and the melon seed extract prepared in Step 2 are dissolved in phosphate buffer in a certain proportion to obtain the complex polypeptide solution. The weight ratio of deer bone polypeptide in the deer bone extract to melon seed polypeptide in the melon seed extract is 2:1 to 6:

1. Step 4: Preparation of carrier base solution. The biocompatible carrier material is dissolved in a solvent to obtain the carrier base solution. Step 5: Composite and molding. The composite peptide solution is mixed with the carrier base liquid and composited under stirring to obtain a uniform composite slurry. The composite slurry is injected into a mold and subjected to cross-linking curing or freeze-drying treatment to obtain the bone repair material.

2. The method for preparing a bone repair material using deer antler polypeptide according to claim 1, characterized in that: In step one, the preprocessing includes: The deer bone raw material is soaked in a sodium hydroxide solution with a concentration of 0.5-1.5 mol / L at 40-60℃ for 1-3 hours. After soaking, it is taken out, rinsed with deionized water until neutral, and then frozen at -20℃ to -40℃ for 12-24 hours before being vacuum freeze-dried.

3. The method for preparing a bone repair material using deer antler polypeptide according to claim 1, characterized in that: In step one, the conditions for the first extraction are: The first extraction solvent is a phosphate buffer solution with a pH of 7.0-7.

8. The mass-to-volume ratio of deer bone powder to the first extraction solvent is 1g:10-20mL. The extraction temperature is 45-60℃ and the extraction time is 4-8 hours. The conditions for the second extraction were as follows: the second extraction solvent was a phosphate buffer solution with a pH of 7.0-7.8; the mass-to-volume ratio of the first bone residue to the second extraction solvent was 1 g: 5-10 mL; the extraction temperature was 45-60℃; and the extraction time was 2-4 hours.

4. The method for preparing a bone repair material using deer antler polypeptide according to claim 1, characterized in that: In step two, the preprocessing includes: The raw melon seeds are dried at 40-50℃ to constant weight, dehulled, pulverized, and passed through an 80-120 mesh sieve to obtain melon seed powder. The extraction conditions were as follows: the third extraction solvent was water, the mass-to-volume ratio of melon seed powder to the third extraction solvent was 1g:15-25mL, the extraction temperature was 60-80℃, and the extraction time was 2-4 hours.

5. The method for preparing a bone repair material using deer antler polypeptide according to claim 1, characterized in that: In step three, the total concentration of deer bone polypeptide and melon seed polypeptide in the composite polypeptide solution is 10-50 mg / mL, the concentration of the phosphate buffer is 0.01-0.05 mol / L, and the pH value is 7.2-7.

6.

6. The method for preparing a bone repair material using deer antler polypeptide according to claim 1, characterized in that: In step four, the biocompatible carrier material is at least one of gelatin, chitosan, hyaluronic acid, sodium alginate, type I collagen, or polylactic acid-glycolic acid copolymer. The solvent is at least one of water, acetic acid solution or trifluoroethanol; The biocompatible carrier material in the carrier base solution has a mass-volume concentration of 5%-20%.

7. The method for preparing a bone repair material using deer antler polypeptide according to claim 1, characterized in that: Step five specifically includes: The complex peptide solution was slowly added dropwise to the carrier base solution under stirring, and the volume ratio of the complex peptide solution to the carrier base solution was controlled to be 1:1 to 1:

4. After the addition was completed, the mixture was stirred continuously at 200-500 r / min for 30-60 minutes at room temperature to obtain a uniform complex slurry.

8. The method for preparing a bone repair material using deer antler polypeptide according to claim 1, characterized in that: In step five, crosslinking and curing includes: Add a crosslinking agent to the composite slurry, mix evenly, inject into a mold, and let it stand at 25-37℃ for 1-3 hours for crosslinking. After molding, take it out and wash it with deionized water to obtain a hydrogel-like bone repair material. The crosslinking agent is at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, glutaraldehyde, or genipin, and the amount of crosslinking agent added is 0.5%-5% of the mass of the biocompatible carrier material.

9. The method for preparing a bone repair material using deer antler polypeptide according to claim 1, characterized in that: In step five, the freeze-drying process includes: The composite slurry is injected into a mold and pre-frozen at -20℃ to -80℃ for 4-12 hours. Then it is transferred to a freeze dryer and freeze-dried for 24-72 hours under conditions of cold trap temperature below -50℃ and vacuum degree below 10Pa to obtain a porous scaffold-shaped bone repair material.

10. A bone repair material utilizing deer antler polypeptide, prepared by any one of the preparation methods of bone repair material utilizing deer antler polypeptide as described in claims 1-9, characterized in that: The bone repair material is made from raw materials containing the following parts by weight through compounding and molding: 10-40 parts of compound polypeptide solution and 60-90 parts of carrier base solution. The composite polypeptide solution contains deer bone extract and melon seed extract, wherein the weight ratio of deer bone polypeptide to melon seed polypeptide is 2:1 to 6:

1. The carrier base liquid contains a biocompatible carrier material with a mass-volume concentration of 5%-20%.