CN110898258A

CN110898258A - Antibacterial collagen-based bone repair material

Info

Publication number: CN110898258A
Application number: CN201911261309.5A
Authority: CN
Inventors: 张正男; 段书霞; 韩涵; 付迎坤; 孙海鹏; 石沛龙; 崔彬彬; 邵蕊娜; 韩修恒; 田崇; 周静; 郝明; 严子跃; 佘开江; 姬鹏远; 王喜卫; 段晓堂
Original assignee: Henan Yadu Industrial Co Ltd
Current assignee: Beijing Shunkang Medical Management Consulting Co ltd
Priority date: 2019-12-10
Filing date: 2019-12-10
Publication date: 2020-03-24
Anticipated expiration: 2039-12-10
Also published as: CN110898258B

Abstract

The invention discloses an antibacterial collagen-based bone repair material which comprises a bone repair material main body, a graphene oxide, polydopamine, a polypyrrole self-assembly layer doped with sulfosalicylic acid and bone morphogenetic protein-2 fixedly carried in the self-assembly layer, wherein the bone repair material main body is composed of zinc oxide/hydroxyapatite whiskers, type I collagen, polyvinyl alcohol and a cross-linking agent. The surface and the interior of the material have good antibacterial effect, inflammation and infection in the bone grafting process can be avoided to the maximum extent, the used raw materials are nontoxic and harmless, the material has good biocompatibility, the degradation speed of the material is relatively consistent with the bone defect repair speed, the polypyrrole-polydopamine coating is self-assembled on the surface of the bone repair material, the bone morphogenetic protein-2 can be firmly loaded and stably released, the utilization rate of the bone morphogenetic protein-2 is improved, and complications caused by overhigh concentration of the bone morphogenetic protein-2 are greatly avoided.

Description

Antibacterial collagen-based bone repair material

Technical Field

The invention relates to the technical field of biomedical materials, in particular to an antibacterial collagen-based bone repair material.

Background

An ideal bone repair material should have good biocompatibility, degradability, osteoconductivity, and osteoinductivity. Natural bone is composed mainly of apatite and collagen. Therefore, bone repair materials composed of hydroxyapatite and collagen have been widely studied.

The bone repair materials generally have no antibacterial property, and after bone transplantation, serious bacterial and fungal infection is often caused, so that the number of patients who die each year because of organ transplantation-induced infection is huge. The mode of injecting and orally taking the medicine to prevent the infection is poor in effect, and the medicine can quickly enter a circulatory system after entering a human body, so that the medicine concentration at the bone transplantation part is low. The medicine can enter other organs along with the blood, and causes toxic and side effects. At the cellular level, the cause of infection is: bacteria adhere to the bone graft and subsequently form a biofilm, which in turn induces infection. The existing antibacterial bone repair material mostly achieves the antibacterial effect by loading antibiotics, is difficult to realize the controlled release of the antibiotics, can cause harm to human bodies when the concentration of the antibiotics is too high, achieves the antibacterial effect by modifying the antibacterial agent on the surface, generally has no antibacterial performance inside, easily causes inflammation and infection in the recovery period, and has important significance in finding components which can sterilize and inhibit bacteria and have bone conduction activity and preparing bone repair materials with antibacterial performance on the surface and inside.

Among the known growth factors, bone morphogenetic protein-2 (BMP-2) has the strongest osteogenesis effect. The existing research proves that BMP-2 has an important regulation effect on the differentiation and functions of osteoblasts and chondrocytes, can induce the undifferentiated mesenchymal stem cells to proliferate and differentiate in the direction of osteoblasts by crossing species, promotes osteogenesis and enables fibroblasts to have osteogenic phenotype. However, BMP-2 diffuses rapidly in body fluids, has a short half-life, is easily enzymatically hydrolyzed, decreases therapeutic concentration rapidly, and does not continuously stimulate target cells to fully exert its inducing activity. Promoting osteogenesis by increasing the dosage of BMP-2 results in a series of complications due to too high a dosage, may cause tissue swelling, inflammation, promotes undesirable ectopic osteogenesis, and may form cyst-like lesions in callus. Therefore, the bone repair material obtained by simply compounding the BMP-2 and the matrix material has uncertain porosity, uncontrollable concentration and release time of the BMP-2 and unsatisfactory repair effect. Therefore, the current research focus is to combine BMP-2 with a carrier to establish a stable BMP-2 release system, reduce the release of BMP-2 to maintain the local concentration of the implantation site, reduce the incidence of complications and enable BMP-2 to play a greater role.

Therefore, the development of bone repair materials with antibacterial effect, controllable release of bone morphogenetic protein-2, reduced complication rate and ideal repair effect is urgently needed.

Disclosure of Invention

The invention provides an antibacterial collagen-based bone repair material which has an antibacterial effect, can regularly release bone morphogenetic protein-2, reduces the incidence rate of complications and has an ideal repair effect.

The purpose of the invention is realized by the following technical scheme:

an antibacterial collagen-based bone repair material comprises a bone repair material main body consisting of zinc oxide/hydroxyapatite whiskers, type I collagen, polyvinyl alcohol and a cross-linking agent, a graphene oxide, polydopamine, a polypyrrole self-assembly layer doped with sulfosalicylic acid and bone morphogenetic protein-2 immobilized on the self-assembly layer.

Preparing zinc oxide/hydroxyapatite crystal whisker: dispersing zinc nitrate and polyethylene glycol 6000 in anhydrous ethanol, and heating in water bath to dissolve to obtain clear solution; weighing zinc nitrate and polyethylene glycol according to the mass ratio of 1:2, adding the zinc nitrate and the polyethylene glycol together with hydroxyapatite whisker (HAPw) dried for more than 24 hours at constant temperature into absolute ethyl alcohol, ultrasonically dispersing, slowly dropping the clarified liquid into the HAPw dispersion liquid under magnetic stirring, adjusting the pH, carrying out water bath reaction, ultrasonically dispersing after the reaction is finished, evaporating the solvent to dryness, drying, placing in a resistance furnace, heating to 800 ℃ by a program, calcining for 1 hour, and naturally cooling to room temperature to obtain the zinc oxide/hydroxyapatite whisker (ZnO/HAPw).

The preparation method of the antibacterial collagen-based bone repair material comprises the following steps:

step one, preparing a bone repair material: dissolving type I collagen in glacial acetic acid solution, adding zinc oxide/hydroxyapatite whisker (ZnO/HAPw) after the type I collagen is completely dissolved, uniformly stirring, adding polyvinyl alcohol solution, continuously stirring to obtain mixed solution, filling the mixed solution into a mold, freezing, vacuum drying, taking out, placing the mixed solution into a cross-linking agent solution for cross-linking, taking out the material, soaking, forming holes, and freezing and drying again to obtain the collagen;

step two, amination modification of the bone repair material: performing amination modification on the bone repair material prepared in the step two by soaking in a polyethyleneimine solution, taking out and cleaning, drying, then soaking in a hydrochloric acid solution, taking out and cleaning, and drying for later use;

step three, preparing a self-assembled layer: immersing the bone repair material modified by the triamination in the step into a graphene oxide dispersion liquid, enabling the surface of the bone repair material to be self-assembled with a layer of graphene oxide, taking out the graphene oxide, washing with water, then placing the bone repair material into a three-electrode system of calcium nitrate solution electrolyte added with doping agents of sulfosalicylic acid, pyrrole monomers and dopamine monomers, carrying out polypyrrole-polydopamine layer-by-layer self-assembly under the alternating action of two pulse voltages, and controlling the layer number of the polypyrrole-polydopamine by setting the circulating times of the two pulses;

step four, immobilizing bone morphogenetic protein-2: and (3) cleaning the bone repair material subjected to the four steps of assembly with deionized water, drying in vacuum, immersing in a bone morphogenetic protein-2 solution, taking out a sample immobilized with the bone morphogenetic protein-2, and drying in an oven at 37 ℃ to obtain the antibacterial collagen-based bone repair material.

Further, the mass fraction of acetic acid in the glacial acetic acid solution in the first step is 1%.

Further, the mass ratio of the polyvinyl alcohol, the ZnO/HAPw and the type I collagen in the mixed solution in the step one is 15:1: 2.

Further, in the first step, the polyvinyl alcohol solution is PVA-2400 solution with the mass fraction of 7%.

Further, in the first step, the crosslinking agent solution is a 95% ethanol solution of EDC-NHS, and the crosslinking time is 24 h.

Further, the step one of impregnating and pore-forming specifically comprises the following steps: first, the material was taken out and immersed in Na₂HPO₄The solution is put for 2 to 3 hours, and then the material is taken out to be soakedAnd (4) putting the NaCl solution into the NaCl solution for 18-30 hours, periodically replacing the NaCl solution, and then washing the NaCl solution by shaking with a large amount of distilled water until NaCl is completely separated out.

Further, in the third electrolyte, the concentration of calcium nitrate is 10mM, the concentration of pyrrole is 10mM, the concentration of dopamine is 5mM, and the concentration of sulfosalicylic acid is 20 mM.

Further, the voltage of the first pulse in the third step is 1V, and the time is 50 s; the second pulse voltage was-0.8V for 80 s.

According to the invention, zinc nitrate is used as a precursor, polyethylene glycol 6000 is used as a dispersing agent, zinc oxide modified hydroxyapatite whisker is prepared by a sol-gel method, zinc nitrate is hydrolyzed and subjected to polycondensation reaction to generate nano particles which are deposited on the surface of hydroxyapatite, and the nano particles are calcined at high temperature, so that the generated zinc oxide nano particles are connected with the hydroxyapatite whisker through covalent bonds, can be firmly loaded on the hydroxyapatite whisker, exert antibacterial activity, and can also improve osteogenesis induction capability of a bone repair material, thereby improving bone formation capability around an implanted part. Compared with nano hydroxyapatite particles, the hydroxyapatite whisker not only can enhance the strength of the bone repair material, but also can enable the material to have higher porosity, and simultaneously has better dispersibility, can avoid stress concentration points caused by hydroxyapatite aggregation, and improves the mechanical property of the material.

The invention screens and optimizes the mass ratio and concentration of the polyvinyl alcohol, the ZnO/HAPw and the I type collagen when the bone repair material is prepared, so that the ZnO/HAPw with relatively consistent degradation speed and bone defect repair speed of the material has good bioactivity and bone conductivity, the defect of insufficient mechanical strength retention time of the polyvinyl alcohol is overcome after the ZnO/HAPw is compounded with the polyvinyl alcohol, the defects of brittle hydroxyapatite and low porosity are also avoided, and the prepared bone repair material has good comprehensive performance. The bone repair material main body is prepared by adopting a freeze-drying method twice, and all components are uniformly distributed, high in porosity and stable in property.

The self-assembly on the plane is easy, and the difficulty of self-assembly on the surface of the porous structure is high, so that the bone repair material is firstly subjected to amination modification, the adsorption capacity of the graphene oxide is increased by utilizing the mutual attraction of the amino and oxygen-containing groups on the surface of the graphene oxide, the graphene oxide layer obtained by self-assembly is more compact and ordered, and conditions are provided for the growth of a subsequent self-assembly layer.

In the preparation process of the self-assembly layer, polypyrrole and polydopamine are respectively synthesized in situ under two pulse voltages. Under the first pulse voltage of 1V, the vicinity of the working electrode, namely the graphene oxide bone repair material, is an oxidation reaction: firstly, polymerizing dopamine into polydopamine nanoparticles, and then aggregating the polydopamine nanoparticles on the surface of the graphene oxide bone repair material to form a polydopamine coating; at a second pulse voltage of-0.8V: poly dopamine nano particles in the electrolyte are used as spherical templates, and the poly dopamine nano particles are adsorbed to form pyrrole monomers by utilizing amino and phenolic hydroxyl on the surfaces of the poly dopamine nano particles near the working electrode; and then-OH is electrolyzed by water near the working electrode, so that the surrounding-OH concentration is supersaturated, and finally polypyrrole is formed on the surface of the graphene oxide bone repair material. After multiple pulse cycles, the polypyrrole-polydopamine alternating self-assembly coating is prepared on the surface of the graphene oxide. The doping agent sulfosalicylic acid used in the invention has a sulfonic acid group and a carboxylic acid group, and the introduction of the doping agent with strong negative electricity groups can effectively improve the surface potential of the conductive polymer. Comparing the surface potentials of the polypyrrole/sulfosalicylic acid self-assembled layer and the contrast group thereof shows that the strong electronegativity dopant sulfosalicylic acid promotes the surface potential of polypyrrole, and the polypyrrole self-assembled layer with high surface potential has strong mercapto oxidation capability, can effectively destroy the mercapto on the surface of bacteria, causes the bacteria to die, and enables the surface of the bone repair material to have good antibacterial performance.

According to the invention, the bone morphogenetic protein-2 is adsorbed in the polypyrrole-polydopamine self-assembly layer through electrostatic interaction between amino groups of the bone morphogenetic protein-2, amino groups of polypyrrole and phenolic hydroxyl groups of polydopamine, due to the existence of adsorption sites, the bone morphogenetic protein-2 can be well controlled and released, the porous structure of the polypyrrole-polydopamine is more beneficial to immobilizing the bone morphogenetic protein-2, and the immobilization amount can be adjusted by controlling the number of the self-assembly layers.

Compared with the prior art, the invention has the following beneficial effects:

(1) the antibacterial collagen-based bone repair material provided by the invention has good antibacterial effect on the surface and the inside, inflammation and infection in the bone grafting process can be avoided to the maximum extent, the used raw materials are nontoxic and harmless, the biocompatibility is good, the zinc oxide/hydroxyapatite whisker used by the main body of the bone repair material has strong osteogenesis activity and antibacterial performance, the polyvinyl alcohol has good water solubility, the degradation product does not cause anaphylactic reaction, the polyvinyl alcohol serving as the bone repair material does not have toxic or side effect, and the type I collagen has low immunogenicity and strong tissue affinity; the polypyrrole self-assembled layer has good stability, can promote cell adhesion and proliferation, wherein the doped biomolecule sulfosalicylic acid can improve the surface potential of the polypyrrole self-assembled layer and has a strong bactericidal effect, the polydopamine has excellent cell adhesion and good hydrophilicity, the composition makes up the respective defects, the respective advantages are fully exerted, and a good effect is obtained in bone defect repair;

(2) the EDC-NHS cross-linking agent is adopted for cross-linking, so that the collagen is promoted to be cross-linked to form an amido bond, and the collagen I, the polyvinyl alcohol and the ZnO/HAPw are integrated through the cross-linking effect, therefore, the prepared bone repair material has good mechanical strength and high porosity, the mass ratio of each component is further optimized, and the degradation speed of the material is relatively consistent with the bone defect repair speed;

(3) the antibacterial collagen-based bone repair material provided by the invention can firmly load the bone morphogenetic protein-2 by self-assembling the polypyrrole-polydopamine coating on the surface of the bone repair material, and is a stable release system due to the electrostatic adsorption among groups, so that sudden release or incapability of releasing can not occur, the utilization rate of the bone morphogenetic protein-2 is improved, and complications caused by overhigh concentration of the bone morphogenetic protein-2 can be greatly avoided.

Drawings

FIG. 1 is a bar graph of BMP-2 immobilization for each set of specimens.

FIG. 2 is a graph showing the amount of BMP-2 released over time for each group of samples.

FIG. 3 shows Ca values of the respective tests²⁺And (4) concentration.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following detailed description is given with reference to specific embodiments.

Example 1

Preparing zinc oxide/hydroxyapatite crystal whisker: adding zinc nitrate and polyethylene glycol 6000 into anhydrous ethanol, stirring, heating in constant temperature water bath for 2 hr to dissolve completely to obtain clarified solution; according to the proportion of zinc nitrate: weighing zinc nitrate and polyethylene glycol according to the mass ratio of 1:2, weighing a proper amount of hydroxyapatite whisker (HAPw) dried in a constant-temperature drying oven at 80 ℃ for 24 hours, adding the weighed hydroxyapatite whisker (HAPw) into an absolute ethyl alcohol solution, and performing ultrasonic dispersion to obtain a dispersion liquid of the HAPw; slowly and dropwise adding the prepared zinc nitrate and polyethylene glycol 6000 clear solution into the HAPw dispersion solution by using a digital display constant flow pump under magnetic stirring, adjusting the pH value by dropwise adding ammonia water and glacial acetic acid together, carrying out water bath reaction for 6h at a set temperature, carrying out ultrasonic dispersion for 20s after the reaction is finished, heating to 80 ℃, evaporating the solvent to dryness, and completely drying in a constant temperature drying oven; placing the obtained product in a resistance furnace, heating to 800 ℃, calcining at constant temperature for 1h, and naturally cooling to room temperature to obtain the zinc oxide/hydroxyapatite whisker (ZnO/HAPw).

Example 2

Preparing a bone repair material: weighing 15g of PVA, adding the PVA into 200mL of distilled water, and heating the PVA in a water bath for 20min at the temperature of 100 ℃ to prepare a PVA solution with the mass fraction of 7%; weighing 2g of I type collagen, dissolving the I type collagen in 8mL of glacial acetic acid with the mass fraction of 1% to obtain an I type collagen solution, adding 1g of ZnO/HAPw into the I type collagen solution after the I type collagen is completely dissolved, uniformly stirring to obtain a mixture, respectively adding 4mL of the prepared 7% PVA solution into the mixture, uniformly stirring, then placing into a mold, storing at-20 ℃ overnight, and carrying out freeze vacuum drying. Dissolving 5.7682g EDC and 1.7314g NHS in 100mL 95% ethanol to obtain cross-linking agent, taking out lyophilized material, placing in cross-linking agent, cross-linking for 24h, taking out material, and soaking in 1M Na₂HPO₄And (3) taking out the materials from the NaCl solution after 24 hours, putting the materials into a glass container filled with a large amount of distilled water, repeatedly shaking and cleaning for 24 hours, replacing the distilled water every 4 hours until the NaCl is completely separated out, freeze-drying the materials again, and performing radiation sterilization to obtain the bone repair material sample.

Example 3

Amination modification of the bone repair material: the bone repair material prepared in example 2 was immersed in a 1mg/mL polyethyleneimine solution for amination modification, taken out after 3 hours, washed, dried, immersed in a 5% hydrochloric acid solution for 2 minutes, taken out, washed, and dried for future use.

Example 4

Preparing a self-assembled layer: immersing the aminated and modified bone repair material into graphene oxide dispersion liquid of 0.5mg/mL for 5min to enable the surface of the material to be self-assembled with a layer of graphene oxide, taking out the graphene oxide, washing with water, and then placing the graphene oxide into an electrode system, wherein the concentration of calcium nitrate, pyrrole, dopamine and sulfosalicylic acid in electrolyte is 10mM, 10mM and 5mM respectively. Carrying out polypyrrole-polydopamine self-assembly under the alternating action of two pulse voltages, wherein the first pulse voltage is 1V and the time is 50 s; the second pulse voltage is-0.8V, the time is 80s, and the two pulses are circulated for 8 times to prepare (PPy-PDA)₈A self-assembled layer.

Example 5

Immobilization of bone morphogenetic protein-2: the bone repair material assembled in the embodiment 5 is washed by deionized water, dried in vacuum, immersed in a bone morphogenetic protein-2 solution with the concentration of 5mg/mL, and dried in an oven at 37 ℃ after 12 hours, so as to obtain the antibacterial collagen-based bone repair material.

Mechanical Strength detection

The mechanical properties of the bone repair material were tested according to ISO527-2-2012 standard. Sample 1 is the antimicrobial collagen-based bone repair material prepared in example 5; sample 2 is a bone repair material made by replacing zinc oxide/hydroxyapatite whiskers with nano-hydroxyapatite particles. Each group of samples was 6, and the average was taken as the test result.

The test results are shown in Table 1.

TABLE 1 mechanical Property test results of bone repair materials

Item	Compressive strength/MPa	Flexural strength/MPa	Elongation at break/%	Modulus of elasticity/GPa
					Sample
1	169.32±7.91	138.27±4.46	15.34±2.85	3.64±1.05
					Sample 2	82.51±8.63	76.39±2.24	8.48±2.52	2.17±1.60

As can be seen from Table 1, the antibacterial collagen-based bone repair material prepared by the invention has excellent mechanical properties. Sample 2 uses nano hydroxyapatite particles to replace zinc oxide/hydroxyapatite whiskers, and because the nano particles are easy to aggregate, stress concentration is caused, so that the compressive strength and the bending strength of the bone repair material are greatly reduced.

Cytotoxicity assays

The test method comprises the following steps: vero cells were seeded on 24-well cell culture plates at a number of 5X 10 cells per well⁵After incubation for 1, 3, and 5 days with the antibacterial collagen-based bone repair material prepared in example 5, a negative control group and an experimental group of the antibacterial collagen-based bone repair material were set, and each group had 3 more wells at each time point. 1. The culture solution was collected 3 and 5 days later, and the supernatant was centrifuged to determine the cytotoxicity of the LDH activity evaluation material according to the instructions of the Lactate Dehydrogenase (LDH) cytotoxicity assay kit.

And (3) test results: the results of detecting LDH activity of Vero cells incubated with bone repair materials for 1, 3 and 5 days are shown in Table 2.

TABLE 2 LDH Activity assay results

LDH is a stable protein existing in the cytoplasm of normal cells, and when the cell membrane is damaged, LDH in cytoplasm is released to the outside of the cells, so that the LDH activity in a culture medium is in direct proportion to the death number of the cells, and the OD value read by an enzyme-labeling instrument of the detection kit is in positive linear correlation with the LDH activity. As can be seen from Table 2, the activity of the experimental group was close to that of the negative control group after 1 day of incubation, and there was no significant difference. The LDH activity of the co-incubation group of the antibacterial collagen-based bone repair material after 3 days and 5 days is obviously lower than that of the negative control group. The test result shows that the antibacterial collagen-based bone repair material has no cytotoxicity, can improve the cell activity and reduce the release of LDH.

BMP-2 immobilization and Release test

Sample preparation: group 1 is the one prepared in example 5 (PPy-PDA)₈A bone repair material; group 2 bone repair Material without self-assembled coating (PPy-PDA) prepared in example 2₀A bone repair material; group 3 is PDA prepared by the electrolyte only containing dopamine monomer in the preparation process of the self-assembled layer₈Self-assembled layer bone repair material; group 4 is PPy prepared by using the electrolyte only containing pyrrole monomer in the preparation process of the self-assembled layer₈Self-assembled layer bone repair materialFeeding; group 5 is prepared by two pulse cycles 6 times during the preparation of the self-assembled layer (PPy-PDA)₆Self-assembled layer bone repair material; group 6 is prepared by two pulse cycles 10 times during the preparation of the self-assembled layer (PPy-PDA)₁₀Self-assembled layer bone repair material.

Immobilization of BMP-2: and (3) cleaning the bone repair materials of the groups by using deionized water, drying in vacuum, immersing in a bone morphogenetic protein-2 solution with the concentration of 5mg/mL, and after 12 hours, placing the sample loaded with the bone morphogenetic protein-2 in a 37 ℃ drying oven for drying to obtain the sample.

Release of BMP-2: each set of samples was set up in 6 replicates. All samples were soaked in an equal amount (2mL) of PBS buffer, placed in a constant temperature environment at 37 ℃ and at 1 day, 3 days, 7 days, 15 days and 25 days of soaking, the PBS solution of each sample was collected separately and then replaced with a new one. The concentration of BMP-2 in the collected PBS solution was determined by BMP kit.

And (3) test results: the BMP-2 immobilization amounts of the respective groups of samples are shown in FIG. 1; the amount of BMP-2 released over time for each set of samples is shown in FIG. 2.

As can be seen from FIG. 1, the BMP-2 loading on the surface of the sample with the assembly coating is higher than that of the sample without the self-assembly coating, because BMP-2 can be adsorbed in the self-assembly coating by electrostatic interaction between its amino group and the phenolic hydroxyl group of PDA or between the amino group of PPy. In the PPy-PDA self-assembled layer, since the alternate coating has high porosity and is more favorable for BMP-2 immobilization, the BMP-2 immobilization amounts of group 1, group 5 and group 6 are higher than those of group 3 and group 4, and the BMP-2 immobilization amount increases as the number of self-assembled layers increases.

As can be seen from FIG. 2, BMP-2 showed a slow release process on the surface of the bone repair material with the self-assembly coating. The alternating self-assembled layers have better sustained release properties than the self-assembled layers alone. Group 2 the release rate of the bone repair material without the self-assembling coating appeared to be faster, with a release rate of 99% already at day 7, since the absence of the self-assembling coating resulted in no site for fixation of BMP-2. The release rate of BMP-2 at 25 days was 92% and 97% for the PDA-only self-assembled layer and the PPy-only self-assembled layer, respectively. The BMP-2 release rates of group 1, group 5 and group 6 were significantly lower than the self-assembling layer bone repair material alone, and the release rates decreased as the number of self-assembling layers increased.

Antibacterial property test

Sample 1 is the bone repair material prepared in example 5; the sample 2 is a bone repair material prepared without zinc oxide and by the same operations as the sample 1; sample 3 is a bone repair material prepared in the same manner as sample 1 except that sulfosalicylic acid was not added. Cutting the sample into the same size, taking a certain amount of fresh Escherichia coli, Staphylococcus aureus and Candida albicans from the solid culture medium by using an inoculating loop, adding into the liquid culture medium, and diluting to the same concentration, namely 1.5 × 10⁶cfu/ml; respectively dripping 1ml of the bacterial liquid on a sample, covering the sample with a sterilization covering film, and culturing for 24 hours at the temperature of 37 ℃ and the humidity of more than 90%; repeatedly cleaning the covering film and the sample by using 24ml of eluent, dripping 0.2ml of eluent on a solid agar culture medium, culturing for 1-48 h at 37 ℃, counting viable bacteria, measuring the number of the viable bacteria, and calculating the sterilization rate, wherein the result is shown in table 3.

Table 3 antibacterial property test results

Sample(s)	Rate of sterilization
		1	99.7％
2	85.4％
		3	90.2％

As can be seen from the test results in table 3, the bone repair material prepared in example 5 of the present invention has good antibacterial properties. The sterilization rate of the sample 2 is only 85.4% because the material does not contain zinc oxide, and the sterilization rate of the sample 3 is 90.2% because the self-assembled layer does not contain sulfosalicylic acid, which indicates that the zinc oxide not only has the sterilization effect on the material, but also has a synergistic effect with the sulfosalicylic acid on the self-assembled layer in terms of antibiosis.

Bone induction test

The capacity of the bone repair material for inducing ectopic bone formation is detected through a mouse femoral muscle bag implantation experiment.

Sample preparation: group 1 is the bone repair material (G1) prepared in example 6; group 2 is a bone repair material prepared by using common nano hydroxyapatite particles according to the subsequent method of the present invention, and the BMP-2 immobilization amount is the same as that of the group 1 sample (G2).

The test method comprises the following steps: taking 30 healthy and clean KM mice, dividing into two groups, each group comprises 15 mice, injecting barbital sodium into abdominal cavity for anesthesia, exposing thigh muscle on right side, respectively implanting bone repair materials of group 1 and group 2 into hind leg muscle of KM mice for implantation experiment, taking out implant after 2 weeks, and measuring new bone Ca²⁺And (4) horizontal.

And (3) test results: ca for each set of experiments²⁺The concentrations are shown in FIG. 3.

As can be seen from fig. 3, under the condition that the BMP-2 immobilization amounts are the same, the ectopic bone induction capability of the bone repair material prepared in example 5 of group 1 is far better than that of the bone repair material prepared in group 2 by using common nano hydroxyapatite particles, which indicates that the zinc oxide/hydroxyapatite whisker used in the present invention has good bone induction performance.

In vivo degradation test

The test method comprises the following steps: taking 40 adult New Zealand white rabbits, dividing the adult New Zealand white rabbits into four groups of A, B, C and D, each group of 10 rabbits, performing general anesthesia on each group of white rabbits, performing conventional disinfection on both upper limbs, performing radial exposure treatment on the forearms of the upper limbs, cutting bones at 1/3 positions in the radius, and removing the radius and periosteum to obtain the bilateral radius segmental bone defect model. Implanting different bone repair materials, wherein the first group is the bone repair material prepared in the embodiment 5 of the invention, the second group is the bone repair material prepared in the way that the mass ratio of PVA to ZnO/HAPw to I collagen is 10:1:2, the third group is the bone repair material prepared in the way that the mass ratio of PVA to ZnO/HAPw to I collagen is 20:1:2, and the third group is the bone repair material prepared in the way that the mass ratio of PVA to ZnO/HAPw to I collagen is 15:2:1, collecting tissue sections at 4 weeks and 16 weeks after operation, and calculating the degradation condition of the bone repair material.

And (3) test results: the residual rates at 4 and 16 weeks after implantation of the bone repair material are shown in table 4 below.

TABLE 4 degradation of bone repair materials

Group of	n	4 weeks	For 16 weeks
				First group	10	83.61±2.27	22.09±1.56
Group B	10	90.24±4.32	62.85±5.38
				C group	10	86.52±3.34	37.63±4.16
Group D	10	88.73±32.95	40.89±3.54

As can be seen from Table 4, the degradation rates at 4 weeks were comparable but the difference was large at 16 weeks, the mass ratio of PVA: ZnO/HAPw: type I in group A was 15:1:2, the material residual rate was only about 22%, the mass ratio of PVA: ZnO/HAPw: type I in group B was 10:1:2, the PVA: ZnO ratio was much higher than that in group A, the material residual rate after 16 weeks was about 62%, the mass ratio of PVA: ZnO/HAPw: type I in group C was 10:1:2, the PVA: ZnO ratio was low, and the material residual rate at 16 weeks was about 37%, which is probably because the mesoporous structure of PVA: ZnO promoted the material degradation process, the mass ratio of PVA: ZnO/HAPw: I in group B was 15:2:1, the collagen ratio was low, the degradation rate was lower than that in example 5, and only at a reasonable ratio, the degradation rate of the bone repair material can be ensured to be consistent with the bone defect repair rate, and an ideal repair effect is achieved.

The above description is only for the specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto, and other modifications or equivalent substitutions made by the technical solution of the present invention by the ordinary skilled in the art should be covered within the scope of the claims of the present invention without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. An antibacterial collagen-based bone repair material is characterized by comprising a bone repair material main body, a graphene oxide, polydopamine, a polypyrrole self-assembly layer doped with sulfosalicylic acid and bone morphogenetic protein-2 fixedly carried in the self-assembly layer, wherein the bone repair material main body is composed of zinc oxide/hydroxyapatite whiskers, type I collagen, polyvinyl alcohol and a cross-linking agent.

2. The antibacterial collagen-based bone repair material according to claim 1, wherein the zinc oxide/hydroxyapatite whiskers are prepared by a method comprising: dispersing zinc nitrate and polyethylene glycol 6000 in anhydrous ethanol, and heating in water bath to dissolve to obtain clear solution; weighing zinc nitrate and polyethylene glycol according to the mass ratio of 1:2, adding the zinc nitrate and the polyethylene glycol together with hydroxyapatite whisker (HAPw) dried for more than 24 hours at constant temperature into absolute ethyl alcohol, ultrasonically dispersing, slowly dropping the clarified liquid into the HAPw dispersion liquid under magnetic stirring, adjusting the pH, carrying out water bath reaction, ultrasonically dispersing after the reaction is finished, evaporating the solvent to dryness, drying, placing in a resistance furnace, heating to 800 ℃ by a program, calcining for 1 hour, and naturally cooling to room temperature to obtain the zinc oxide/hydroxyapatite whisker (ZnO/HAPw).

3. A method for preparing an antibacterial collagen-based bone repair material according to claim 1, comprising the steps of:

step three, preparing a self-assembled layer: immersing the bone repair material modified by the triamination in the step into graphene oxide dispersion liquid, enabling the surface of the bone repair material to be self-assembled with a layer of graphene oxide, taking out the graphene oxide, washing with water, then placing the bone repair material into a three-electrode system of calcium nitrate solution electrolyte added with doping agents of sulfosalicylic acid, pyrrole monomers and dopamine monomers, carrying out polypyrrole-polydopamine self-assembly under the alternating action of two pulse voltages, and controlling the layer number of polypyrrole-polydopamine by setting the number of times of two pulse cycles;

4. The method for preparing an antibacterial collagen-based bone repair material according to claim 3, wherein the mass ratio of the polyvinyl alcohol, ZnO/HAPw and type I collagen in the mixed solution of the first step is 15:1: 2.

5. The method for preparing an antibacterial collagen-based bone repair material according to claim 3, wherein the polyvinyl alcohol solution in the first step is PVA-2400 solution with a mass fraction of 7%.

6. The method of claim 3, wherein the crosslinking agent solution in the first step is EDC-NHS in 95% ethanol, and the crosslinking time is 24 h.

7. The method for preparing an antibacterial collagen-based bone repair material according to claim 3, wherein the step one of impregnating to form the pores specifically comprises the following steps: first, the material was taken out and immersed in Na₂HPO₄And (3) taking the material out of the solution and immersing the material into NaCl solution for 18-30 hours, wherein the NaCl solution needs to be replaced periodically, and then shaking and washing the material with a large amount of distilled water until NaCl is completely separated out.

8. The method for preparing an antibacterial collagen-based bone repair material according to claim 3, wherein the electrolyte solution of step three contains calcium nitrate 10mM, pyrrole 10mM, dopamine 5mM and sulfosalicylic acid 20 mM.

9. The method for preparing an antibacterial collagen-based bone repair material according to claim 3, wherein the first pulse voltage in the third step is 1V and the time is 50 s; the second pulse voltage was-0.8V for 80 s.