CN113209383B - BMP-2 gene sustained-release coating loaded PEEK composite biological implantation material and preparation method thereof - Google Patents

BMP-2 gene sustained-release coating loaded PEEK composite biological implantation material and preparation method thereof Download PDF

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CN113209383B
CN113209383B CN202110532614.4A CN202110532614A CN113209383B CN 113209383 B CN113209383 B CN 113209383B CN 202110532614 A CN202110532614 A CN 202110532614A CN 113209383 B CN113209383 B CN 113209383B
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bmp
peek
plga
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CN113209383A (en
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刘红
秦爽
姜玲玲
路政宽
陈天杰
刘称称
高蕴波
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Jilin University
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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    • A—HUMAN NECESSITIES
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    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/258—Genetic materials, DNA, RNA, genes, vectors, e.g. plasmids
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    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
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    • A61L2420/00—Materials or methods for coatings medical devices
    • A61L2420/06—Coatings containing a mixture of two or more compounds
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    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Abstract

A PEEK composite biological implantation material loaded with a BMP-2 gene slow release coating and a preparation method thereof, belonging to the technical field of biomedical materials. The invention utilizes a double emulsion solvent volatilization method to prepare PLGA-NH carrying BMP-2 gene2Sustained release microspheres; simultaneously, preparing PEEK with nanometer topological appearance and nitrogen-containing functional groups by adopting a nitrogen low-temperature plasma radiation technology, constructing a PDA bionic coating on the surface of the PEEK by depending on the 'wet bonding characteristic' of the PDA, and grafting PLGA-NH carrying BMP-2 genes by taking the PDA as a secondary reaction platform2The microspheres are slowly released, so that a controlled-release gene slow-release system is constructed on the surface of the PEEK, and the composite biological implant material is obtained. The invention introduces PLGA microspheres as a slow release coating, prolongs the effective release time of the bone growth factor, is matched with the key period of bone defect healing, realizes a stable and long bone formation process, and has higher drug loading rate and encapsulation rate.

Description

BMP-2 gene sustained-release coating loaded PEEK composite biological implantation material and preparation method thereof
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a polyether-ether-ketone (PEEK) composite biological implant material loaded with a bone morphogenetic protein-2 (BMP-2) gene sustained-release coating and a preparation method thereof.
Background
As a high-performance implant material, Polyetheretherketone (PEEK) and composite materials thereof have excellent performances of good biocompatibility, high strength, high hardness, corrosion resistance, wear resistance, high temperature resistance, similar elastic modulus with bone tissues and the like, and are one of nonmetal bioprosthetic materials with wide application prospects. The PEEK composite material is used as an orthopedic implant and is applied to the fields of spinal surgery, joint surgery, wound repair and the like, and a good repair effect is achieved. However, PEEK is a bio-inert material, and its hydrophobic surface and high reaction inertia result in low surface energy, poor biological activity, and difficulty in obtaining a very desirable osseointegration effect after being implanted into a human body. If the biological activity of PEEK can be obviously improved on the premise of not influencing the mechanical property of the body, the osseointegration effect of PEEK can be expected to be improved, and a novel medical implant material with more excellent performance is developed.
With the cross-infiltration development of subjects such as materials science, scale chemistry, modern life science and the like, and the continuous breakthrough of relevant theories and technologies such as cell biology, molecular biology and the like, a target gene with the effect of promoting bone is loaded on the surface of an implant material, the interaction between the target gene and cells of an implanted area is accurately regulated and controlled from the gene level, the faster osseous integration (osseointegration) of an implant body and bone tissues is realized, and a brand new thought is provided for the effective modification of the surface of the implant material. A great deal of literature has proved that BMP-2 protein coded by BMP-2 gene can positively regulate the regeneration and repair process of bone tissue, and is a key action factor for forming osseointegration between implant and bone tissue in the implanted area. Therefore, a BMP-2 gene slow release system is constructed on the surface of the PEEK implant material, so that the BMP-2 gene is further transfected into bone mesenchymal stem cells of peripheral tissues of an implant, the secretion of therapeutic BMP-2 protein beneficial to osteogenesis is promoted, the effect of promoting the regeneration of bone tissues is continuously exerted, and the method is a feasible way for improving the biological activity of the PEEK.
Polydopamine (PDA) is formed by Dopamine (DA) which is in contact with air under the condition of alkalescence and automatically generates oxidative polymerization, is rich in catechol groups and amino functional groups of lysine, and has super-strong adhesion property. The PDA has good biocompatibility, is a biological adhesive with great advantages and potentials, can be adhered to the surfaces of almost all materials, including organic, inorganic and biological materials, and forms a functionalized nano thin layer on the surface of the material. Because the o-phenol group contained in PDA is very easy to be oxidized into cyclohexadienedione structure, the structure can be combined with the compound containing amino (-NH)2) And sulfhydryl (-SH) or imino (-NH-) organic polymer to generate Schiff base or Michael addition reaction to form stable chemical bonding force, and lay the foundation for grafting sulfhydrylation or amination bioactive molecules and realizing functional modification of biological materials. Therefore, by using PDA as a secondary reaction platform, it is possible to firmly combine the sulfhydrylation or amination bioactive molecules on the surface of the biomaterial through graft polymerization, thereby changing the microenvironment of the bone implant material and significantly improving the bone combination effect.
Disclosure of Invention
The invention aims to provide a polyether ether ketone (PEEK) composite biological implant material loaded with a bone morphogenetic protein-2 (BMP-2) gene sustained-release coating and a preparation method thereof.
The invention aims to construct an osteogenic slow-release coating on the surface of PEEK, and firstly utilizes a double emulsion solvent volatilization method to prepare PLGA-NH carrying BMP-2 gene2Sustained release microspheres (PLGA: polylactic-co-glycolic acid); simultaneously, preparing PEEK with nanometer topological appearance and nitrogen-containing functional groups by adopting a nitrogen low-temperature plasma radiation technology, constructing a PDA bionic coating on the surface of the PEEK by depending on the 'wet bonding characteristic' of the PDA, and grafting PLGA-NH carrying BMP-2 genes by taking the PDA as a secondary reaction platform2The sustained release microspheres are used for constructing a controlled release gene sustained release system on the surface of PEEK, thus obtaining the inventionThe polyether-ether-ketone (PEEK) composite biological implant material loaded with the bone morphogenetic protein-2 (BMP-2) gene slow release coating.
The invention relates to a preparation method of a PEEK composite biological implantation material loaded with a BMP-2 gene slow release coating, which comprises the following steps as shown in figure 7:
1) dissolving 4-6 g of PLGA containing free carboxyl terminal into 40-60 mL of dichloromethane, adding 40-50 mg of dicyclohexylcarbodiimide and 25-26 mg of N-succinimide, stirring for 11-13 h, separating a reaction product from a precipitate in anhydrous ether, and drying in vacuum to obtain carboxyl activated PLGA;
2) Adding 370-380 mg of hydrazine hexaethylene glycol and carboxyl activated PLGA obtained in the step 1) into 40-60 mL of anhydrous DMSO, stirring for 11-13 h, adding a reaction product into 0-4 ℃ of anhydrous ethanol, separating and precipitating, and drying in vacuum to obtain the aminated PLGA slow release microspheres marked as PLGA-NH2Sustained release microspheres;
3) mixing the following components in a mass ratio of 1: 1, mixing BMP-2 plasmid and Polyethyleneimine (PEI), adding deionized water to prepare a PEI/DNA complex aqueous solution, wherein the concentration of the BMP-2 plasmid in the complex aqueous solution is 50-70 mu g/mL;
4) adding 0.4-0.6 mL of PEI/DNA compound aqueous solution obtained in the step 3) into 1-3 mL of PLGA-NH with the mass fraction of 2%2In a dichloromethane solution of the sustained-release microspheres, stirring for 7-10 s under the condition of 4000-5000 rpm in an ice bath, then quickly pouring into 8-10 mL of polyvinyl alcohol (PVA) aqueous solution with the mass fraction of 3%, stirring for 5-10 s under the condition of 8000-10000 rpm, then pouring into 15-25 mL of polyvinyl alcohol (PVA) aqueous solution with the mass fraction of 0.5%, stirring for 2-4 h, then centrifuging for 10-15 min under the condition of 4000-6000 rpm, washing and precipitating for 2-4 times by deionized water, and then freeze-drying the precipitate to obtain PLGA-NH carrying BMP-2 gene2Slow-release microspheres, denoted PLGA-NH2@ pBMP-2 microspheres, stored at 4 ℃;
5) Grinding and polishing a PEEK test piece (phi is 10mm, and delta is 1mm), sequentially ultrasonically cleaning the PEEK test piece for 20-40 min by using acetone, absolute ethyl alcohol and deionized water, drying the PEEK test piece, and treating the surface of the PEEK test piece for 10-15 min by using nitrogen low-temperature plasma under the conditions of 400-500V voltage, 400-600W power and 10-20 Pa pressure to obtain a test piece NPEEK;
6) dissolving Dopamine (DA) in 10mmol/L Tris-HCl buffer solution to prepare a dopamine solution with the pH value of 8.5 and the concentration of 2 g/L; soaking the test piece NPEEK obtained in the step 5) in the test piece NPEEK, placing the test piece NPEEK in a constant-temperature shaking table at 37 ℃, taking out the test piece NPEEK after the light-shielding reaction is carried out for 20-24 hours, and repeatedly washing the test piece NPEEK by using deionized water to obtain a test piece NPEEK with Polydopamine (PDA) deposited on the surface, and marking the test piece NPEEK-PDA as a test piece NPEEK-PDA;
7) taking 80-100 mg of PLGA-NH obtained in the step 4)2Mixing the @ pBMP-2 microspheres in 10mL of deionized water uniformly to prepare a high-concentration suspension of the microspheres; placing the test piece NPEEK-PDA obtained in the step 6) in a high-concentration suspension of microspheres for reacting for 4-6 h, and freeze-drying to obtain the test piece with PLGA-NH loaded on the surface2NPEEK-PDA test piece of microsphere, mark as NPEEK-PDA- (PLGA-NH)2@ pBMP-2), namely the PEEK composite biological implantation material loaded with the BMP-2 gene sustained release coating.
In the method, the molar ratio of the lactic acid monomer to the glycolic acid monomer in the PLGA in the step 1) is 50: 50, Mw 10000;
Step 3) firstly, digesting pCMV6-XL4 by Not I, then treating the digestion product by T4 DNA polymerase, and then digesting the digestion product by Eco RI to obtain a linear DNA fragment containing a BMP-2 target gene, wherein the DNA fragment has a cohesive end and a flat end at the same time; digesting the pACCMV-plpA shuttle plasmid of adenovirus type 5 by BamH I, flattening the viscous end by T4 DNA polymerase, digesting by Eco RI, and connecting the digested plasmid with a DNA fragment containing a BMP-2 target gene by using DNA ligase to obtain a BMP-2 plasmid (pBMP-2 for short); mw of PEI 25,000;
and step 5), the plasma generator is a nitrogen plasma reactor, the reaction pressure is 20pa, the voltage is 500V, the power is 500W, and the time is 15 min.
Step 6) the pH of the dopamine solution was adjusted using sodium hydroxide (0.1 mol/L).
Compared with the prior art, the invention has the following advantages:
(1) PLGA-NH prepared by multiple emulsion solvent volatilization method2The @ pBMP-2 microspheres and the W/O/W type multiple emulsion can effectively prevent the water-soluble medicine from entering a continuous phase so as to improve the encapsulation efficiency;
(2) the PEI nano particles with positive charges and the BMP-2 plasmids with negative charges are used for electrostatic attraction, the maximal aggregation of the plasmids is realized, the encapsulation efficiency is improved, and on the other hand, the formed compound protects the BMP-2 plasmids from being damaged in the preparation process of the microspheres.
(3) The nitrogen low-temperature plasma radiation is convenient to operate and has obvious effect. When the particles with high energy impact the material, the original appearance can be damaged, an uneven nano-scale structure is formed, and the surface area of the substrate material is increased; meanwhile, macromolecular chains on the surface of the substrate material are broken and bonded with active particles in plasma or active gas molecules in air, and new elements and functional groups are introduced on the surface, which are both beneficial to the deposition of PDA and the biological related activity of the surface of the material.
(4) The PDA coating is introduced, not only the chemical bonding factor of the fixed microspheres is considered, but also the good cell biological activity of the PDA coating is considered, the calcium ion deposition can be promoted, and the osteogenic differentiation of cells can be induced;
(5) three surface modification strategies of nano-matrix composite material preparation, plasma surface modification and gene-loaded sustained-release microsphere preparation are comprehensively applied to achieve a composite effect, and the method belongs to cross innovation research in the fields of materials science, supermolecule chemistry and medicine.
The preparation method of the invention obtains the PEEK biological implantation material loaded with the BMP-2 gene slow release coating, the surface successfully constructs the nano topological morphology, and the microspheres are uniformly dispersed on the surface of the substrate material; the microspheres synthesized by the multiple emulsion solvent volatilization method have uniform particle size, complete form and higher drug loading rate and encapsulation rate. The release of BMP-2 plasmid of the whole material is relatively stable within 40 days, and the accumulated release amount reaches 82%. As is well known, BMP-2 is the most potential bone growth stimulating factor in the bone morphogenetic protein family, can induce mesenchymal stem cells to differentiate into osteoblasts, thereby promoting the healing and repair of bone defects, and the effect can make up the biological inertia of PEEK materials. PLGA microspheres are introduced as a slow release coating, so that the effective release time of the bone growth factor is prolonged, the effective release time is matched with the key period of bone defect healing, and a stable and long-term bone formation process is realized. The surface appearance, functional groups, roughness and hydrophilicity of the material are improved, and a high-quality environment is provided for cell adhesion and osteogenic differentiation.
Drawings
FIG. 1 shows PLGA-NH obtained in step 4) of example 12The particle size distribution of the microspheres, wherein the inset is a scanning electron microscope picture of the microspheres.
It can be seen that PLGA-NH2The microspheres are regular spheres, full in shape, smooth in surface and almost free of damage or adhesion. The particle sizes of the microspheres are basically uniform, and 90% of the particle sizes of the microspheres are found to be distributed in the range of 0.8-5.6 microns after a plurality of SEM pictures are selected and subjected to the statistical analysis of Nano measurer 1.2. The content of unencapsulated DNA in the microsphere supernatant is measured by a double-stranded DNA quantitative detection kit and a fluorescence microplate reader, and the drug loading capacity and the encapsulation efficiency are further evaluated. The drug loading rate and the encapsulation rate are respectively calculated according to the following formulas:
Figure BDA0003068499340000041
Figure BDA0003068499340000042
PLGA-NH synthesized by the method2The drug loading rate and the encapsulation rate of the microspheres are respectively 1.6% + -0.6% and 42.7% + -6.9%.
FIGS. 2A, 2B, 2C, and 2D are test pieces of PEEK, NPEEK-PDA, and NPEEK-PDA- (PLGA-NH), respectively2@ pBMP-2) surface microtopography.
Therefore, the surface of the untreated PEEK test piece is relatively flat and smooth, and grinding traces can be seen. Strip-shaped irregular bulges are formed on the surface of a PEEK test piece (NPEEK) subjected to nitrogen low-temperature plasma treatment, and each bulge surface is composed of a plurality of small bulges which are closely arranged. After dopamine is deposited on the surface of a test piece (NPEEK-PDA), the original convex-concave appearance becomes unobvious and only shows a strip-shaped structure. After microspheres are loaded (NPEEK-PDA- (PLGA-NH) 2@ pBMP-2)), it can be seen that the microspheres are successfully dispersed on the surface of the base material, most of the microspheres can still maintain the full spherical state, and onlyA small fraction of them are broken during the microsphere immobilization process.
FIGS. 3A, 3B, 3C, 3D and Table 1 show test pieces PEEK, NPEEK-PDA and NPEEK-PDA- (PLGA-NH), respectively2@ pBMP-2) and roughness data.
TABLE 1 test pieces PEEK, NPEEK-PDA- (PLGA-NH)2@ pBMP-2) roughness data (Ra for mean, RMS for root mean Square value)
Figure BDA0003068499340000051
It can be seen that pure PEEK has a smooth surface with minimal roughness compared to the other groups. The NPEEK surface is generally formed with volcano-like elevations and is doped with many small protrusions, and the roughness is significantly increased due to the large surface relief. Granular protrusions with dopamine aggregation can be seen on the surface of NPEEK-PDA, and the roughness is slightly reduced. As the maximum particle size of the microspheres can reach about 5.6 mu m, AFM can only represent NPEEK-PDA- (PLGA-NH)2@ pBMP-2), this group having the greatest roughness.
FIGS. 4A, 4B, 4C and Table 2 show the element characterization curves and assay data of the test pieces PEEK, NPEEK and NPEEK-PDA, respectively.
It can be seen that PEEK is composed of C and O elements, and therefore there are corresponding characteristic peaks at 284.6eV and 532.5 eV. After the PEEK was treated with nitrogen low-temperature plasma, a large amount of nitrogen-containing elements were grafted, and thus NPEEK showed a characteristic peak of nitrogen at 400eV, while detecting a nitrogen content of 4.42%. After PDA modification, C, O, N element peak is detected on the surface of the test piece, the nitrogen content is changed to 6.98%, and it is noted that the NPEEK-PDA group is very similar to the theoretical ratio of corresponding dopamine content no matter the oxygen-carbon content ratio or the nitrogen-carbon content ratio, so that the PDA coating is successfully formed on the surface of the test piece.
Table 2: element content data of test pieces PEEK, NPEEK and NPEEK-PDA
Grouping C(％) O(％) N(％) O/C N/C
PEEK 76.96 21.23 - 0.276 -
NPEEK 63.12 23.69 4.42 0.375 0.07
NPEEK-PDA 73.88 19.14 6.98 0.259 0.945
FIG. 5 shows test pieces of PEEK, NPEEK-PDA- (PLGA-NH)2@ pBMP-2).
It can be seen that the PEEK without any treatment has the largest contact angle, about 83.1 °, which is hydrophobic relative to the other groups. After plasma treatment, NPEEK became hydrophilic and the contact angle decreased to 58.2 °. After coating on PDA, the contact angle of the material surface is further reduced, making the material have excellent hydrophilicity. Whereas the water contact angle increased slightly to 47.1 ° after addition of the microspheres to NPEEK-PDA, mainly due to micron-scale topography hindering the diffusion of water molecules.
FIG. 6 is a graph showing the effect of NPEEK-PDA- (PLGA-NH2@ pBMP-2) on sustained release.
Therefore, the pBMP-2 is released at a high speed in the first 7 days, and the release of the pBMP-2 is accelerated after the microspheres are fixed due to the inevitable dissolution reaction in the process of loading part of the microspheres on the surface of the PEEK test piece. After 7 days, the release rate of the pBMP-2 is gradually slowed down, finally the pBMP-2 is released more stably within 40 days, the cumulative release amount reaches 82 percent, and the aim of releasing the pBMP-2 for a long time by the material is fulfilled.
FIG. 7 is a synthetic flow chart of a PEEK biological implantation material loaded with a BMP-2 gene slow release coating.
Detailed Description
Example 1:
1) 5g of PLGA containing free carboxyl ends (wherein the molar ratio of lactic acid to glycolic acid monomers was 50: 50, Mw 10000) is dissolved in 50mL of dichloromethane, then 45.4mg of dicyclohexylcarbodiimide and 25.3mg of N-succinimide are added, after stirring for 12 hours, the reaction product is separated from the precipitate in anhydrous ether, and after vacuum drying, carboxyl activated PLGA is obtained;
2) adding 375mg of hydrazine hexaethylene glycol and carboxyl activated PLGA obtained in the step 1) into 50mL of anhydrous DMSO, stirring for 12h, adding a reaction product into anhydrous ethanol at 4 ℃, separating precipitates, and drying in vacuum to obtain the aminated PLGA slow release microspheres marked as PLGA-NH2Sustained release microspheres;
3) digesting pCMV6-XL4 by Not I, treating the digested pCMV6-XL4 by T4 DNA polymerase, and digesting the digested pCMV6-XL4 by Eco RI to obtain a linear DNA fragment containing a BMP-2 target gene, wherein the fragment has a cohesive end and a blunt end at the same time; digesting the pACCMV-plpA shuttle plasmid of adenovirus type 5 by BamH I, flattening the viscous end by T4 DNA polymerase, digesting by Eco RI, and connecting the digested plasmid with a DNA fragment containing a BMP-2 target gene by using DNA ligase to obtain a BMP-2 plasmid (pBMP-2 for short); the mass ratio of BMP-2 plasmid to Polyethyleneimine (PEI) is 1: 1, preparing 0.6mL PEI/DNA complex aqueous solution containing 36 mu g BMP-2 plasmid by using deionized water;
4) 0.6mL of PEI/DNA complex aqueous solution obtained in the step 3) is added into 2mL of PLGA-NH with the mass fraction of 2%2In dichloromethane solution of slow-release microspheres, stirring for 10s under ice bath and 5000rpm, quickly pouring into 10mL of polyvinyl alcohol (PVA) aqueous solution with the mass fraction of 3%, stirring for 10s under 10000rpm, then pouring into 20mL of PVA aqueous solution with the mass fraction of 0.5%, stirring for 4h, centrifuging for 15min under 5000rpm, washing and precipitating with deionized water for 3 times, and freeze-drying the precipitate to obtain PLGA-NH2@ pBMP-2 microsphere powder, storing at 4 deg.C;
5) grinding and polishing a PEEK test piece (phi is 10mm, and delta is 1mm), sequentially ultrasonically cleaning the PEEK test piece for 30min by using acetone, absolute ethyl alcohol and deionized water, drying the PEEK test piece, placing the PEEK test piece into a plasma processor, and treating the surface of the PEEK test piece for 15min by using nitrogen low-temperature plasma under the conditions of 500V voltage, 500W power and 20Pa pressure to obtain a test piece NPEEK;
6) dissolving DA in 10mmol/L Tris-HCl buffer solution to prepare a dopamine solution with the pH value of 8.5 and the concentration of 2 g/L; soaking the test piece NPEEK obtained in the step 5) in the test piece NPEEK, placing the test piece NPEEK in a constant-temperature shaking table at 37 ℃, taking out the test piece NPEEK after the light-shielding reaction is carried out for 24 hours, and repeatedly washing the test piece NPEEK by using deionized water to obtain the test piece NPEEK with the surface deposited with Polydopamine (PDA), and marking the test piece NPEEK as NPEEK-PDA;
7) Taking 100mg of PLGA-NH obtained in the step 4)2Mixing the @ pBMP-2 microspheres in 10mL of deionized water uniformly to prepare a high-concentration suspension of the microspheres; placing the test piece NPEEK-PDA obtained in the step 6) in the prepared microsphere suspension for reacting for 6h, and freeze-drying to obtain the test piece with the surface loaded with PLGA-NH2NPEEK-PDA test piece of microsphere, mark as NPEEK-PDA- (PLGA-NH)2@ pBMP-2), namely the PEEK composite biological implantation material loaded with the BMP-2 gene sustained release coating.

Claims (7)

1. A preparation method of a PEEK composite biological implantation material loaded with a BMP-2 gene slow release coating comprises the following steps:
1) dissolving 4-6 g of PLGA containing free carboxyl terminal into 40-60 mL of dichloromethane, adding 40-50 mg of dicyclohexylcarbodiimide and 25-26 mg of N-succinimide, stirring for 11-13 h, separating a reaction product from a precipitate in anhydrous ether, and drying in vacuum to obtain carboxyl activated PLGA;
2) adding 370-380 mg of hydrazine hexaethylene glycol and carboxyl activated PLGA obtained in the step 1) into 40-60 mL of anhydrous DMSO, stirring for 11-13 h, adding a reaction product into 0-4 ℃ of anhydrous ethanol, separating and precipitating, and drying in vacuum to obtain the aminated PLGA slow release microspheres marked as PLGA-NH2Sustained release microspheres;
3) mixing the following components in a mass ratio of 1: 1, mixing BMP-2 plasmid and PEI, adding deionized water to prepare a PEI/DNA complex aqueous solution, wherein the concentration of the BMP-2 plasmid in the complex aqueous solution is 50-70 mu g/mL;
4) Adding 0.4-0.6 mL of PEI/DNA compound aqueous solution obtained in the step 3) into 1-3 mL of PLGA-NH with the mass fraction of 2%2In a dichloromethane solution of the sustained-release microspheres, stirring for 7-10 s under the condition of ice bath and 4000-5000 rpm, then quickly pouring into 8-10 mL of PVA aqueous solution with the mass fraction of 3%, stirring for 5-10 s under the condition of 8000-10000 rpm, then pouring into 15-25 mL of PVA aqueous solution with the mass fraction of 0.5%, stirring for 2-4 h, centrifuging for 10-15 min under the condition of 4000-6000 rpm, washing and precipitating with deionized water for 2-4 times, and freeze-drying the precipitate to obtain PLGA-NH-carrying BMP-2 gene2Slow-release microspheres, denoted PLGA-NH2@ pBMP-2 microsphere powder, storing at 4 deg.C;
5) polishing a PEEK test piece, sequentially ultrasonically cleaning the PEEK test piece for 20-40 min by using acetone, absolute ethyl alcohol and deionized water, drying the PEEK test piece, and treating the surface of the PEEK test piece for 10-15 min by using nitrogen low-temperature plasma under the conditions of 400-500V voltage, 400-600W power and 10-20 Pa pressure to obtain a test piece NPEEK;
6) dissolving dopamine in a Tris-HCl buffer solution of 10mmol/L to prepare a dopamine solution with the pH value of 8.5 and the concentration of 2 g/L; soaking the test piece NPEEK obtained in the step 5) in the test piece NPEEK, placing the test piece NPEEK in a constant-temperature shaking table at 37 ℃, taking out the test piece NPEEK after the light-shielding reaction is carried out for 20-24 hours, and repeatedly washing the test piece NPEEK by using deionized water to obtain the test piece NPEEK with polydopamine deposited on the surface, and marking the test piece NPEEK-PDA as a test piece NPEEK-PDA;
7) Taking 80-100 mg of PLGA-NH obtained in the step 4)2Mixing the @ pBMP-2 microspheres in 10mL of deionized water uniformly to prepare a high-concentration suspension of the microspheres; placing the test piece NPEEK-PDA obtained in the step 6) in a high-concentration suspension of microspheres for reacting for 4-6 h, and freeze-drying to obtain a PEEK composite biological implant material loaded with a BMP-2 gene slow release coating; wherein, PLGA is polylactic acid-glycolic acid copolymer, PEEK is polyether ether ketone, PEI is polyethyleneimine, PVA is polyvinyl alcohol.
2. The method for preparing the PEEK composite biological implant material loaded with the BMP-2 gene slow-release coating of claim 1, wherein the preparation method comprises the following steps: step 1), in PLGA, the molar ratio of lactic acid monomer to glycolic acid monomer is 50: 50, Mw 10000.
3. The method for preparing the PEEK composite biological implant material loaded with the BMP-2 gene slow-release coating of claim 1, wherein the preparation method comprises the following steps: in the step 3), firstly, after the pCMV6-XL4 is digested by Not I, the digestion is carried out by T4 DNA polymerase, and then the digestion is carried out by Eco RI to obtain a linear DNA fragment containing a BMP-2 target gene, wherein the DNA fragment has a cohesive end and a flat end at the same time; then digesting the pACCMV-plpA shuttle plasmid of adenovirus type 5 by BamH I, then flattening the viscous end by T4 DNA polymerase, digesting by Eco RI, and connecting the DNA fragment containing the BMP-2 target gene by using DNA ligase to obtain the BMP-2 plasmid.
4. The method for preparing the PEEK composite biological implant material loaded with the BMP-2 gene slow-release coating of claim 1, wherein the preparation method comprises the following steps: in step 3), Mw of PEI was 25,000.
5. The method for preparing the PEEK composite biological implant material loaded with the BMP-2 gene slow-release coating of claim 1, wherein the preparation method comprises the following steps: and 5) treating the nitrogen plasma at the pressure of 20pa, the voltage of 500V, the power of 500W and the time of 15 min.
6. The method for preparing the PEEK composite biological implant material loaded with the BMP-2 gene slow-release coating of claim 1, wherein the preparation method comprises the following steps: step 6) the pH of the dopamine solution is adjusted with 0.1mol/L sodium hydroxide.
7. A PEEK composite biological implantation material loaded with a BMP-2 gene slow release coating is characterized in that: is prepared by the method of any one of claims 1 to 6.
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