CN118121760A

CN118121760A - Preparation method of composite bone powder with bioactivity and adhesiveness

Info

Publication number: CN118121760A
Application number: CN202410545852.2A
Authority: CN
Inventors: 吴冬冬; 陈海金; 高占山; 康田梦; 骆秋豪; 叶浩楠
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2024-05-06
Filing date: 2024-05-06
Publication date: 2024-06-04
Anticipated expiration: 2044-05-06
Also published as: CN118121760B

Abstract

The invention discloses a preparation method of composite bone powder with bioactivity and adhesiveness, which relates to the technical field of bone powder preparation and comprises the following steps: sintering hydroxyapatite powder at high temperature, adding the powder into maleimide-polyethylene glycol-maleimide aqueous solution, uniformly mixing the powder with the mixed aqueous solution of OGP polypeptide and Switcch polypeptide, adding zinc sulfate heptahydrate aqueous solution, forming gel, drying and crushing to obtain the composite bone powder with bioactivity and adhesiveness. The composite bone powder with bioactivity and adhesiveness prepared by the method provided by the invention has the advantages of high osteogenesis activity, proper degradation rate, super strong wet adhesiveness and the like, has strong self-healing capacity and shape adaptability, can be adhered to irregular bone defect positions, and effectively solves the problems of weak adhesiveness and low mechanical stability of the existing bone glue to bone tissues.

Description

Preparation method of composite bone powder with bioactivity and adhesiveness

Technical Field

The invention relates to the technical field of bone meal preparation, in particular to a preparation method of composite bone meal with bioactivity and adhesiveness.

Background

Critical size bone defects are defined as defects that fail to achieve spontaneous bone fusion without further surgical intervention. The gold standard for clinical reconstruction of these defects is autologous bone grafting, but its use is limited due to the low availability of bone grafting and the incidence of donor sites. Allografts, while solving the source problem compared to autografts, still have certain limitations such as the risk of immune rejection and infection transmission. In addition, the low induced bone activity and high cost of allograft bone also limit its clinical use. In the case of xenografts, the problems of failure to integrate with host tissue, obvious graft rejection, and the like remain unsolved. In order to solve these problems, biological materials such as metals or alloys, bioceramics and polymers are increasingly receiving attention as substitute materials for bone grafting. However, these materials also have limitations, and metallic materials are not degradable and cause chronic inflammation. Bioceramics have the disadvantages of brittleness, poor processability, poor mechanical properties, and the like. The polymer material, especially the synthetic material, releases toxic components during degradation. In the case of severe highly comminuted fractures or large bleeding, it becomes extremely difficult and time consuming to surgically splice small fragments with concomitant sustained bleeding, which can cause serious complications and even be life threatening. Immediate hemostasis and firm fixation of complex bone fragments are critical. In this case, bone cement is a promising fixation method with bone fragment defects. Bone cement including Cyanoacrylate (CA), polymethyl methacrylate (PMMA) and Calcium Phosphate Cement (CPC) is a clinically common bone repair material. However, such adhesives still have some limitations. For example, CA is poorly biodegradable and biocompatible. While PMMA lacks inherent adhesive properties, causes significant thermal necrosis, and leaks out toxic monomers. In addition, CPC has poor mechanical properties, is easily collapsed in a humid environment, and limits its clinical applications.

Accordingly, various types of hydrogel bone cements such as fibrin glue, polysaccharide, silk mucin, etc. have been studied in view of the above problems. Although these hydrogel bone gels exhibit satisfactory biosafety and biodegradability, they still lack sufficient adhesion to bone tissue, particularly in wet environments with continuous bleeding, especially for bones (e.g., femur) that require stable fixation. In a wet bone environment, the requirements for strong adhesion strength and mechanical stability of the material are high. The ideal bone cement should be bioabsorbable and easily degraded to be biocompatible, without the need for additional surgical removal of the fixture. In addition, to accelerate structural repair and healing of severe fractures, the novel bone cement should also have osteogenic properties.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a preparation method of the composite bone powder with bioactivity and adhesiveness, which has the advantages of high osteogenesis activity, proper degradation rate, super strong wet adhesiveness and the like, has strong self-healing capacity and shape adaptability, can be adhered to irregular bone defect positions, and effectively solves the problems of weak adhesiveness and low mechanical stability of the existing bone cement to bone tissues.

In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows: provides a preparation method of composite bone powder with bioactivity and adhesiveness, which comprises the following steps:

S1, dissolving OGP polypeptide in deionized water to prepare an OGP polypeptide aqueous solution, dissolving Switcch polypeptide in deionized water to prepare a Switcch polypeptide aqueous solution, uniformly mixing the OGP polypeptide aqueous solution and the Switcch polypeptide aqueous solution, and regulating the pH value to obtain a mixed solution I;

S2, dissolving maleimide-polyethylene glycol-maleimide in deionized water to prepare a maleimide-polyethylene glycol-maleimide aqueous solution, then adding high-temperature sintered hydroxyapatite powder, and performing ultrasonic treatment until the hydroxyapatite powder is uniformly distributed to obtain a mixed solution II;

S3, uniformly mixing the mixed solution I obtained in the step S1 and the mixed solution II obtained in the step S2, and placing the mixed solution I and the mixed solution II on a shaking table for reaction to obtain a mixed solution III;

S4, adding a zinc sulfate heptahydrate aqueous solution into the mixed solution III obtained in the step S3, and placing the mixed solution on a shaking table for reaction to obtain the composite bone glue;

S5, drying and crushing the composite bone glue obtained in the step S4 to obtain the composite bone powder with bioactivity and adhesiveness.

Further, in step S1, the amino acid sequence of the OGP polypeptide is shown as SEQ ID No.1, and the amino acid sequence of the Switcch polypeptide is shown as SEQ ID No. 2.

Further, the OGP polypeptides and Switcch polypeptides were attached to the resin using solid phase synthesis.

Further, a method for preparing OGP polypeptides and Switcch polypeptides comprises the steps of:

(1) Washing the resin three times by using N, N-dimethylformamide solution, washing the residual N, N-dimethylformamide solution in the resin by using dichloromethane, and drying the resin by using a suction filtration instrument;

(2) Taking 250 mu L of 1, 2-ethanedithiol solution with the concentration of 98vt percent, triisopropylchlorosilane solution with the concentration of 98vt percent and deionized water respectively, fixing the volume to 10mL by trifluoroacetic acid to obtain a lysate, adding the lysate into resin, and then cracking the lysate on a shaking table at the speed of 35r/min for 2.5h to strip the polypeptide from the resin and enter the solution;

(3) Separating the solution and the resin by using a solid phase synthesis tube, collecting polypeptide liquid, and blowing away trifluoroacetic acid in the solution by using nitrogen;

(4) Centrifuging the polypeptide solution in a high-speed centrifuge with anhydrous diethyl ether at low temperature of 4deg.C and at 8000r/min for three times to obtain crude polypeptide product;

(5) Drying the crude product with nitrogen, and then dissolving the crude polypeptide product with an acetonitrile solution with the concentration of 20vt percent to prepare a mixed phase solution of the crude polypeptide product with the concentration of 10 mg/mL;

(7) Purification of the crude product by HPLC: liquid phase method of OGP polypeptide using C18 column: the phase A is a mixed solution of deionized water and trifluoroacetic acid, the phase B is a mixed solution of acetonitrile and trifluoroacetic acid, wherein the concentration of the trifluoroacetic acid is 1vt per mill, the flow rate is 20mL/min, the initial gradient is 90% of the phase A, 10% of the phase B is after 30min, and the phase A is 10% of the phase B is 90%; liquid phase method of Switcch polypeptides: the phase A is a mixed solution of deionized water and trifluoroacetic acid, the phase B is a mixed solution of acetonitrile and trifluoroacetic acid, wherein the concentration of the trifluoroacetic acid is 1vt per mill, the flow rate is 20ml/min, the initial gradient is 95% of the phase A, 5% of the phase B is 5% after 30min, and the phase B is 95%; purifying to obtain polypeptide pure product solution;

(8) Pressurizing and rotary steaming the polypeptide pure product liquid, wherein the rotary steaming temperature is 52 ℃ and the rotating speed is 50r/min; z until the liquid is concentrated to 25-30mL;

(9) Quick freezing with liquid nitrogen to obtain solid, and lyophilizing in a lyophilizer for 2-3d to obtain pure OGP polypeptide and Switcch polypeptide.

Further, the volume ratio of the OGP polypeptide aqueous solution to the Switcch polypeptide aqueous solution is 0.5-1.5:0.5-1.5.

Further, the volume ratio of the OGP polypeptide aqueous solution to Switcch polypeptide aqueous solution was 1:1.

Further, the concentration of the OGP polypeptide aqueous solution is 33.5-34.5g/L.

Further, the concentration of the OGP polypeptide aqueous solution is 34g/L.

Further, the concentration of Switcch polypeptide aqueous solution is 76.5-77.5g/L.

Further, the concentration of Switcch polypeptide aqueous solution was 77g/L.

Further, the pH is adjusted to 7.5-8.5.

Further, the pH was adjusted to 8.

Further, the pH was adjusted with an aqueous NAOH solution having a concentration of 1 nmol/L.

Further, in step S2, the concentration of the maleimide-polyethylene glycol-maleimide aqueous solution is 209.5-210.5g/L.

Further, the concentration of the maleimide-polyethylene glycol-maleimide aqueous solution was 210g/L.

Further, the hydroxyapatite powder is heated from room temperature to 1000-1200 ℃ at a speed of 4-6 ℃/min, and is sintered for 1.5-2.5h.

Further, the hydroxyapatite powder is heated from room temperature to 1100 ℃ at a rate of 5 ℃/min and sintered for 1.5-2.5 hours.

Further, the adding amount of the hydroxyapatite powder is 45-55% of the sum of the masses of the OGP polypeptide, switcch polypeptide and maleimide-polyethylene glycol-maleimide.

Further, the added amount of the hydroxyapatite powder is 50% of the sum of the mass of the OGP polypeptide, switcch polypeptide and maleimide-polyethylene glycol-maleimide.

In step S3, the volume ratio of the first mixed solution to the second mixed solution is 0.5-1.5:0.5-1.5, and the reaction is carried out for 1-2 hours.

Further, the volume ratio of the first mixed solution to the second mixed solution is 1:1.

In the step S4, the volume ratio of the mixed solution III to the zinc sulfate heptahydrate aqueous solution is 19.5-20.5:0.5-1.5.

Further, the volume ratio of the mixed solution III to the zinc sulfate heptahydrate aqueous solution is 20:1.

Further, the concentration of the aqueous solution of zinc sulfate heptahydrate is 50-55g/L.

Further, the reaction is carried out for 11-13h.

Further, the reaction was carried out for 12 hours.

Further, in step S5, the mixture is sufficiently dried at room temperature.

The composite bone powder with bioactivity and adhesiveness is prepared by the preparation method of the composite bone powder with bioactivity and adhesiveness.

In summary, the invention has the following beneficial effects:

1. The osteogenic growth peptide (osteogenic growth peptide, OGP) used in the present invention can promote proliferation, differentiation, alkaline phosphatase activity and matrix mineralization of osteoblast lineage cells. OGP regulates the expression of Transforming Growth Factors (TGFs), insulin-like growth factors (IGFs) and Basic Fibroblast Growth Factors (BFGF), thereby increasing bone formation and trabecular bone density in vivo. Switcch contain adjacent histidines on the peptide chain, and the imidazole group of the histidines forms a covalent bond with the bioactive metal Zn ²⁺. Zn ²⁺ can regulate and control the polarization of macrophages in vivo to regulate the local immune microenvironment, and can obviously enhance the mechanical fixation of bone grafting and the growth rate of new bones. The adhesive based on Switcch polypeptides does not affect the adhesiveness and self-healing ability of Switcch adhesive after the OGP polypeptide is added. OGP polypeptide is modified, the OGP peptide is connected with cysteine at the N end of a peptide chain and between OGP (10-14) and leucine, and sulfydryl on the cysteine and maleimide-polyethylene glycol-maleimide (Mal-PEG-Mal) undergo click reaction. The hydroxyapatite is a main inorganic component of human and animal bones, can realize chemical bond combination with organism tissues on interfaces, has certain solubility in vivo, can release ions harmless to organisms, can participate in metabolism in vivo, has stimulation or induction effect on hyperosteogeny, can promote repair of defective tissues, and shows bioactivity.

2. In the invention, the active polypeptides OGP and Switcch react with Mal-PEG-Mal, OGP (10-14) is grafted on the hydrogel structure instead of simply wrapping the hydrogel, and the active polypeptides are not diffused after the hydrogel is degraded in vivo and still connected on the hydrogel framework, so that the contact time of the hydrogel and the defect is prolonged, and a better osteogenic effect is achieved. The hydroxyapatite is firmly adhered in the hydrogel system, so that the diffusion of the hydroxyapatite is greatly reduced.

3. The composite bone powder OGP.Sw-PEG@HAp _0.5 with bioactivity and adhesiveness prepared by the method provided by the invention has strong adhesiveness after being converted into bone cement in a wet bone environment, can be firmly adhered to a bone defect part, has strong self-healing capacity and shape adaptability, and has three elements of osteogenesis: OGP active polypeptide, switcch polypeptide and hydroxyapatite all have the osteogenesis effect, and the osteogenesis effect of the compound bone powder is promoted by the synergistic effect of the OGP active polypeptide, switcch polypeptide and hydroxyapatite, and the osteogenesis effect of the compound bone powder is not the simple addition of the three, and the contact time of the hydrogel and a bone defect part is prolonged due to the design of the hydrogel, so that the osteogenesis effect is more obvious.

4. The method provided by the invention adopts a complete OGP peptide chain, retains inactive partial amino acid and modifies the inactive partial amino acid, prepares modified OGP, switcch peptide, zn ²⁺ and PEG into hydrogel through ion coordination and click reaction, so as to realize bone repair function, determine a novel method for stimulating osteoblast function and forming new bone, and provide a novel idea for regenerating irregular bone defects.

Drawings

FIG. 1 is a graph showing the effect of bioactive and adhesive composite bone powder filling the femoral condyle defect of a pig;

FIG. 2 is a graph showing the effect of bioactive and adhesive composite bone meal filling between irregularly defective pig bones;

FIG. 3 is a graph showing the effect of bioactive and adhesive composite bone meal filling in the femur of an irregularly defective rat;

FIG. 4 is a graph showing the effect of bioactive and adhesive composite bone meal filling between smooth glass surfaces;

FIG. 5 is a graph showing comparison of absorbance at 450nm of cells treated with different bone meal;

FIG. 6 is a graph showing the effect of different bone meal on cell activity;

FIG. 7 is a fluorescence image under a confocal laser microscope of cells treated with different bone meal;

FIG. 8 is a Micro CT image of a rat femoral condyle defect model filled with different bone powders at different time points;

Fig. 9 is a graph comparing bone volume fractions at various time points for rat femoral defect models filled with different bone powders.

Detailed Description

The present invention will be further described with reference to examples and drawings, but the present invention is not limited thereto.

Example 1

A preparation method of composite bone powder with bioactivity and adhesiveness comprises the following steps:

S1, dissolving OGP polypeptide in deionized water to prepare an OGP polypeptide aqueous solution with the concentration of 34g/L, dissolving Switcch polypeptide in deionized water to prepare a Switcch polypeptide aqueous solution with the concentration of 77g/L, uniformly mixing 100 mu L of OGP polypeptide aqueous solution and 100 mu L Switcch polypeptide aqueous solution, and adding 30 mu L of NAOH aqueous solution with the concentration of 1nmol/L to adjust the pH value to 8 to obtain a mixed solution I;

S2, dissolving maleimide-polyethylene glycol-maleimide into deionized water to prepare a maleimide-polyethylene glycol-maleimide aqueous solution with the concentration of 210g/L, then heating 45mg of hydroxyapatite powder from room temperature to 1100 ℃ at the speed of 5 ℃/min, sintering at a high temperature for 2 hours, adding 230 mu L of maleimide-polyethylene glycol-maleimide aqueous solution, and placing into an ultrasonic instrument until the hydroxyapatite powder is uniformly distributed to obtain a mixed solution II;

S3, uniformly mixing 230 mu L of the first mixed solution obtained in the step S1 and 230 mu L of the second mixed solution obtained in the step S2, and placing the mixed solution on a shaking table for reaction for 1.5 hours to obtain a third mixed solution;

S4, adding 21.5 mu L of a zinc sulfate heptahydrate aqueous solution with the concentration of 51.1g/L into 430 mu L of the mixed solution III obtained in the step S3, and placing the mixed solution on a shaking table for reacting for 12 hours to obtain the composite bone glue;

and S5, fully drying and crushing the composite bone glue obtained in the step S4 at room temperature to obtain the composite bone powder with bioactivity and adhesiveness.

Example 2

s1, dissolving OGP peptide in deionized water to prepare an OGP polypeptide aqueous solution with the concentration of 33.5g/L, dissolving Switcch polypeptide in deionized water to prepare a Switcch polypeptide aqueous solution with the concentration of 76.5g/L, uniformly mixing 101 mu L of OGP polypeptide aqueous solution and 101 mu L Switcch polypeptide aqueous solution, and adding 22 mu L of NAOH aqueous solution with the concentration of 1nmol/L to adjust the pH value to 7.5 to obtain a mixed solution I;

s2, dissolving maleimide-polyethylene glycol-maleimide into deionized water to prepare a maleimide-polyethylene glycol-maleimide aqueous solution with the concentration of 209.5g/L, then heating 45mg of hydroxyapatite powder from room temperature to 1000 ℃ at the speed of 4 ℃/min, sintering at a high temperature for 2.5 hours, adding 200 mu L of maleimide-polyethylene glycol-maleimide aqueous solution, and placing into an ultrasonic instrument until the hydroxyapatite powder is uniformly distributed to obtain a mixed solution II;

S3, uniformly mixing 229 mu L of the first mixed solution obtained in the step S1 and 200 mu L of the second mixed solution obtained in the step S2, and placing the mixed solution on a shaking table for reaction for 1h to obtain a third mixed solution;

s4, adding 20 mu L of zinc sulfate heptahydrate water solution with the concentration of 55g/L into 429 mu L of the mixed solution III obtained in the step S3, and placing the mixed solution on a shaking table for reacting for 11 hours to obtain the composite bone glue;

s5, fully drying and crushing the composite bone glue obtained in the step S4 at room temperature to obtain the composite bone powder with bioactivity and adhesiveness.

Example 3

s1, dissolving OGP polypeptide in deionized water to prepare an OGP polypeptide aqueous solution with the concentration of 34.5g/L, dissolving Switcch polypeptide in deionized water to prepare a Switcch polypeptide aqueous solution with the concentration of 77.5g/L, uniformly mixing 99 mu L of OGP polypeptide aqueous solution and 99 mu L Switcch polypeptide aqueous solution, and adding 38 mu L of NAOH aqueous solution with the concentration of 1nmol/L to adjust the pH value to 8.5 to obtain a mixed solution I;

s2, dissolving maleimide-polyethylene glycol-maleimide into deionized water to prepare a maleimide-polyethylene glycol-maleimide aqueous solution with the concentration of 210.5g/L, then heating 45mg of hydroxyapatite powder from room temperature to 1200 ℃ at the speed of 6 ℃/min, sintering at a high temperature for 1.5 hours, adding 199.5 mu L of the maleimide-polyethylene glycol-maleimide aqueous solution into an ultrasonic instrument until the hydroxyapatite powder is uniformly distributed, and obtaining a mixed solution II;

s3, uniformly mixing 236 mu L of the first mixed solution obtained in the step S1 and 199.5 mu L of the second mixed solution obtained in the step S2, and placing the mixed solution on a shaking table for reacting for 2.5 hours to obtain a third mixed solution;

S4, adding 22 mu L of zinc sulfate heptahydrate water solution with the concentration of 50g/L into 435.5 mu L of the mixed solution III obtained in the step S3, and placing the mixed solution III on a shaking table for reaction for 13 hours to obtain the composite bone glue;

s5, drying the composite bone glue obtained in the step S4 completely at room temperature, and crushing to obtain the composite bone glue with bioactivity and adhesiveness.

Comparative example 1

Comparative example 1 differs from example 1 in that comparative example 1 does not contain an aqueous OGP polypeptide solution and hydroxyapatite powder. (Sw-PEG)

Comparative example 2

Comparative example 2 differs from example 1 in that comparative example 2 does not contain an aqueous solution of the OGP polypeptide. (Sw-PEG@HAp _0.5)

Comparative example 3

Comparative example 3 differs from example 1 in that comparative example 3 does not contain an aqueous OGP polypeptide solution, an aqueous Switcch polypeptide solution, and an aqueous maleimide-polyethylene glycol-maleimide solution. (HAp)

Test example 1

20Mg of the bioactive and adhesive composite bone powder prepared in example 1 was filled in the space between the femoral condyle defect of a pig, the bone of an irregularly defective pig, the femur of an irregularly defective rat and the smooth glass surface, and then 75. Mu.L of deionized water was added to stir for 10 seconds, and the adhesion was tested, and the results are shown in FIGS. 1 to 4, respectively.

As can be seen from fig. 1 to 4, the composite bone powder with bioactivity and adhesiveness can be adhered to various materials after adding water, and the bone powder can be made into hydrogel which can be self-adaptive to various materials and has strong adhesiveness.

Test example 2

To verify the bioactivity, the composite bone powder prepared in example 1, comparative example 1 and comparative example 2 was prepared into hydrogel by adding water.

Material preparation: preparing a material leaching solution according to the international characterization of 0.2g/mL, leaching different hydrogels with a volume of 200 mu L and a concentration of 26wt% into 1mL of alpha-MEM complete medium respectively; cell preparation: using ninety-six well plates, each well plate was plated with 8 ten thousand BMSC cells from rats and attached for 24h; the medium was aspirated and the aqueous gel extract was added and incubated for 1, 3 and 5 days, respectively. The extract was aspirated, 10% CCK8 was added, 100. Mu.L per well was incubated for 2 hours in the absence of light, and absorbance at 450nm was measured with an enzyme-labeled instrument, and the results are shown in FIG. 5, and the cell activities are shown in FIG. 6, and in FIG. 6, the experimental results of the blank control group, comparative example 1, comparative example 2 and example 1 were shown in the order from left to right in each group.

As can be seen from FIG. 5, the bone powder prepared in example 1, comparative example 1 and comparative example 2 were nontoxic.

As can be seen from FIG. 6, the bone powder prepared in example 1, comparative example 1 and comparative example 2 are nontoxic and have the effect of promoting cell proliferation, wherein the composite bone powder material prepared in example 1 is particularly remarkable in performance and better promotes proliferation of osteoblast cells.

Test example 3

To verify the biosafety, the composite bone powder prepared in example 1, comparative example 2 and comparative example 3 were prepared into hydrogel by adding water, respectively.

Material preparation: preparing a material leaching solution according to the international characterization of 0.2g/mL, leaching different hydrogels with the volume of 200 mu L and the mass fraction of 26% into 1mL of alpha-MEM complete medium respectively; cell preparation: using a glass bottom dish, spreading BMSC cells of 1 ten thousand rats on each pore plate, and adhering for 24 hours; absorbing and discarding the culture medium, adding leaching solution of the aqueous gel, and respectively culturing for 1, 3, 5 and 7 days; the leaching solution was sucked and removed, FADI dye solution with a concentration of one thousandth was added, 1mL of each dish was dyed for 3min in a dark place, and the result was observed under a laser confocal microscope, as shown in FIG. 7, wherein each column was the experimental results of a blank control group, a comparative example 1, a comparative example 2 and an example 1 in order from left to right.

As can be seen from FIG. 7, the cell viability of the added bone meal prepared in example 1, comparative example 1 and comparative example 2 was high, further demonstrating its non-toxicity.

Test example 4

15 Biological adult SD male rats with weights between 210 and 220g are selected and divided into 5 groups, the inner sides of the femoral condyles of the left and right hind legs are molded, the models are cylinders with diameters of 3mm and heights of 6mm, no material is filled in the model 1, and the bone powder prepared in the example 1, the comparative example 2 and the comparative example 3 is filled in the model 2 to 5 groups. After the bone powder is filled in, the bone powder is soaked by intracondylar blood to form hydrogel, and the hydrogel has strong adhesiveness and is firmly adhered to the defect. 3 mice in each group were cultured for 4 weeks, 8 weeks and 12 weeks, respectively, the femur of the rat was scanned by Micro CT and a defect model was reconstructed, as shown in FIG. 8, the defect was observed, and the amounts of the new bone were compared, as shown in FIG. 9, and in FIG. 9, each group was a blank control group, comparative example 1, comparative example 2, comparative example 3 and experimental result of example 1 in this order from left to right.

As can be seen from fig. 8 to 9, the amount of new bone in the femur of the rat filled with the composite bone powder with bioactivity and adhesiveness obtained in example 1 was significantly superior to that of the other groups, indicating that the composite bone powder with bioactivity and adhesiveness obtained in example 1 had strong osteogenesis properties.

In conclusion, the composite bone powder with bioactivity and adhesiveness prepared by the method provided by the invention has super-strong wet adhesiveness, bioactivity and strong osteogenesis, and can promote proliferation of osteoblast cells.

Although specific embodiments of the invention have been described in detail with reference to the accompanying drawings, it should not be construed as limiting the scope of protection of the present patent. Various modifications and variations which may be made by those skilled in the art without the creative effort are within the scope of the patent described in the claims.

Claims

1. The preparation method of the composite bone powder with bioactivity and adhesiveness is characterized by comprising the following steps:

2. The method for preparing bioactive and adhesive composite bone powder according to claim 1, wherein in step S1, the amino acid sequence of the OGP polypeptide is shown in SEQ ID No.1, and the amino acid sequence of the Switcch polypeptide is shown in SEQ ID No. 2.

3. The method for preparing bioactive and adhesive composite bone powder according to claim 1, wherein in step S1, the volume ratio of the OGP polypeptide aqueous solution to the Switcch polypeptide aqueous solution is 0.5-1.5:0.5-1.5, and the concentration of the OGP polypeptide aqueous solution is 33.5-34.5g/L; the concentration of Switcch polypeptide aqueous solution is 76.5-77.5g/L.

4. The method for preparing bioactive and adhesive composite bone powder according to claim 1, wherein in step S1, the pH is adjusted to 7.5-8.5.

5. The method for preparing bioactive and adhesive composite bone powder of claim 1, wherein in step S2, the concentration of the maleimide-polyethylene glycol-maleimide aqueous solution is 209.5-210.5g/L.

6. The method for preparing bioactive and adhesive composite bone powder as claimed in claim 1, wherein in step S2, the hydroxyapatite powder is heated from room temperature to 1000-1200 ℃ at a rate of 4-6 ℃/min and sintered for 1.5-2.5 hours; the adding amount of the hydroxyapatite powder is 45-55% of the total mass of the OGP polypeptide, switcch polypeptide and maleimide-polyethylene glycol-maleimide.

7. The method for preparing bioactive and adhesive composite bone powder according to claim 1, wherein in step S3, the volume ratio of the first mixed solution to the second mixed solution is 0.5-1.5:0.5-1.5, and the reaction is performed for 1-2 hours.

8. The method for preparing bioactive and adhesive composite bone powder as claimed in claim 1, wherein in the step S4, the volume ratio of the mixed solution three to the zinc sulfate heptahydrate aqueous solution is 19.5-20.5:0.5-1.5, the concentration of the zinc sulfate heptahydrate aqueous solution is 50-55g/L, and the reaction is 11-13h.

9. The method of preparing bioactive and adhesive composite bone powder of claim 1, wherein in step S5, the bone powder is dried substantially at room temperature.

10. The bioactive and adhesive composite bone meal prepared by the method for preparing bioactive and adhesive composite bone meal according to any one of claims 1-9.