CN113354786B

CN113354786B - Method for preparing difunctional polyether-ether-ketone by using mussel derived peptide and biological orthogonal reaction and application thereof

Info

Publication number: CN113354786B
Application number: CN202110766346.2A
Authority: CN
Inventors: 耿德春; 李蒙; 柏家祥; 陶华强; 葛高然; 肖龙; 王志荣; 李文明; 张巍; 潘国庆
Original assignee: First Affiliated Hospital of Suzhou University
Current assignee: First Affiliated Hospital of Suzhou University
Priority date: 2021-07-07
Filing date: 2021-07-07
Publication date: 2023-07-04
Anticipated expiration: 2041-07-07
Also published as: CN113354786A

Abstract

The invention discloses a method for preparing difunctional polyether-ether-ketone by utilizing mussel derived peptide and biological orthogonal reaction and application thereof, wherein the preparation method comprises the following steps: a. synthesis of clickable mussel-derived peptides; b. clickable mussel-derived peptide-modified polyetheretherketone; preparation of DBCO-AMP and DBCO-OGP; d. and (3) preparing the difunctional polyether-ether-ketone. The preparation method is simple and convenient, the preparation cost is low, the prepared difunctional polyether ether ketone (PEEK-AO) has excellent performance, and in vitro cell experiments prove that the PEEK-AO has the functions of promoting the adhesion, proliferation and osteogenic differentiation of BMSCs, and has antibacterial performance on staphylococcus aureus and escherichia coli; in vivo experiments prove that PEEK-AO promotes implant-bone interface integration by using a rat femur distal end implant rod model, has antibacterial capability and has better application prospect in clinic.

Description

Method for preparing difunctional polyether-ether-ketone by using mussel derived peptide and biological orthogonal reaction and application thereof

Technical Field

The invention belongs to the technical field of application of medical biological materials, and particularly relates to a method for preparing bifunctional polyether ether ketone (PEEK) by utilizing mussel derived peptides and biological orthogonal reaction and application thereof.

Background

Polyetheretherketone (PEEK) is a special thermoplastic engineering plastic that has been widely used in orthopaedics and oral maxillofacial surgery because of its excellent mechanical properties approaching cortical bone and extra-osseous stability in physiological environments. However, conventional PEEK materials, due to their bio-inertia, have problems of insufficient osseointegration and postoperative bacterial infection, often result in loosening of the implant, even failure of implantation, and impose a heavy burden on patients and the whole society. Thus, orthopaedic prostheses made from PEEK need to be further functionalized to improve osteogenic and anti-infective activity around the implant. To achieve anti-infective function, various antimicrobial agents, such as antibiotics, quaternary Ammonium Compounds (QACs), and certain metals (e.g., ag and Cu) and their oxides, are commonly used to modify the surface of bone implants. However, all of these functional components may present potential toxicity to humans, and abuse of antibiotics may even exacerbate the development of new drug resistant bacteria (MDRB). Antibacterial peptides (AMPs) are an emerging antibacterial agent that can target and concentrate on the cell membrane of a pathogen, translocating membrane proteins, thereby impeding cell wall synthesis and cell respiration, ultimately leading to pathogen death. However, as noted above, AMPs alone is still not perfect, as lack of osteogenic properties is another major problem with PEEK implants. As an endogenous peptide present in serum of mammals, OGP has been studied well in a free state or a bound state to regulate osteogenic differentiation of bone marrow mesenchymal stem cells and further promote matrix mineralization, and thus can be used to promote bone regeneration after bone surgery implantation. However, PEEK materials have higher chemical resistance than most other polymeric biomaterials, which presents a significant barrier to their functionalization at the surface of biomolecules. The clickable mussel heuristic peptides designed in our study are capable of bio-orthogonal reactions (i.e., dibenzylcyclooctyne (DBCO) -azido cycloaddition chemistry), showing various advantages, including not only mussel-like surface adhesion, but also specificity, rapidity, and reaction thoroughness. It is conceivable that the optimal performance of the two bone implants could synergistically promote firm osseointegration of the PEEK implant, even in the face of bacterial invasion. Furthermore, this work may provide a promising solution for surface bioengineering of other inert biological materials, in particular for rational design of multifunctional surfaces for diverse clinical needs.

Disclosure of Invention

The invention aims to research and particularly endow polyether ether ketone (PEEK) with antibacterial and osteogenic dual functions through surface chemical modification of mussel derived peptide combined biological orthogonal reaction, so as to meet the clinical requirements on biocompatibility and osseointegration of PEEK. The invention enhances biocompatibility, osseointegration capability and anti-infective capability by reasonably integrating and optimizing anti-infective and osteoinductive properties on the surface of PEEK by using mussel foot protein (Mfps) -mimetic peptides with clickable azide ends. The treated PEEK surface promotes adhesion and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).

In order to achieve the above object, the present invention provides the following technical solutions:

a method for preparing bifunctional polyether-ether-ketone by using mussel derived peptide and biological orthogonal reaction comprises the following steps:

a. synthesis of clickable mussel-derived peptides

3, 4-dihydroxy-L-phenylalanine DOPA was introduced into peptide sequences using pyruvic acid protected Fmoc-DOPA (acetone) -OH; specifically, hexavalent DOPA units with one lysine spacer are integrated with azido groups linked to polyethylene glycol to obtain clickable mussel-derived peptides (DOPA) ₆ -PEG ₅ -Azido；

(DOPA) ₆ -PEG ₅ Azido has the structural formula shown in formula A:

b. clickable mussel derived peptide modified polyether ether ketone

Polyether-ether-ketone (PEEK) is synthesized with the step a (DOPA) ₆ -PEG ₅ -Azido is immersed in PBS to obtain polyetheretherketone-Azido;

preparation of DBCO-AMP and DBCO-OGP

The active sequences of the antibacterial peptide AMP and the osteogenic growth peptide OGP are selected, and are respectively coupled through N-hydroxysuccinimide-amine and maleimide-mercaptan and blocked by DBCO to prepare DBCO-AMP and DBCO-OGP, wherein the structural formulas of the DBCO-AMP and the DBCO-OGP are respectively shown as a formula B and a formula C;

the sequence of the antimicrobial peptide AMP is: RWRWRWRW;

the active sequence of the osteogenic growth peptide OGP is as follows: YGGFGG;

d. preparation of difunctional polyether-ether-ketone

And c, performing biological orthogonal clicking on the DBCO-AMP and/or DBCO-OGP prepared in the step c and the azido modified polyether-ether-ketone prepared in the step b to synthesize the difunctional polyether-ether-ketone.

Further, the polyether-ether-ketone is treated with plasma prior to use.

Further, the operation steps of the step d are as follows: DBCO-AMP and/or DBCO-OGP are incubated with polyetheretherketone-Azido in PBS.

Further, the molar ratio of DBCO-AMP to DBCO-OGP reaction is 4: 0. 3: 1. 2: 2. 1:3 or 0:4.

further, the material after the reaction in step d is thoroughly rinsed with ultra pure water and then dried with nitrogen gas for further use.

The invention also discloses the difunctional polyether-ether-ketone prepared by the method for preparing the difunctional polyether-ether-ketone.

The invention further discloses application of the difunctional polyetheretherketone in preparing a bone implant with antibacterial and osteogenic functions.

The beneficial effects are that: the invention discloses a method for preparing difunctional polyether-ether-ketone by utilizing mussel derived peptide and biological orthogonal reaction and application thereof, compared with the prior art, the invention has the following advantages:

(1) The preparation method of the difunctional polyether-ether-ketone adopts a surface chemical modification technology, not only comprises mussel-like surface adhesion, but also has specificity, rapidness and reaction thoroughness of click chemistry.

(2) After the PEEK matrix is modified by the method disclosed by the invention, the properties of the PEEK matrix are not greatly changed, and elements harmful to human bodies are not introduced, so that the excellent mechanical properties of PEEK are maintained.

(3) The preparation method is simple and convenient, the preparation cost is low, the prepared difunctional polyether ether ketone (PEEK-AO) has excellent performance, and in vitro cell experiments prove that the PEEK-AO has the functions of promoting the adhesion, proliferation and osteogenic differentiation of BMSCs, and has antibacterial performance on staphylococcus aureus and escherichia coli; in vivo experiments prove that PEEK-AO promotes implant-bone interface integration by using a rat femur distal end implant rod model, has antibacterial capability and has better application prospect in clinic.

Drawings

FIG. 1 is a graph showing XPS analysis results;

FIG. 2 is a graph of AFM analysis results;

FIG. 3 is a graph of water contact angle results for materials;

FIG. 4 is a graph of CCK-8 results;

FIG. 5 is a morphology of BMSC cells under electron microscopy;

FIG. 6 is a graph of in vitro bacterial plating results;

FIG. 7 is a graph of the result of the cytoskeleton;

FIG. 8 is a diagram showing the expression of an osteogenic related gene;

FIG. 9 is a graph showing ALP and ARS staining results;

FIG. 10 is a graph showing the quantitative results of ALP kit detection and extracellular matrix mineralization;

FIG. 11 is a graph of three-dimensional reconstruction results of a rat femur;

FIG. 12 is a graph of CT quantitative analysis;

FIG. 13 is a graph of results of femur HE staining;

FIG. 14 is a graph showing the results of a mechanical push-out experiment;

FIG. 15 is a graph showing the results of in vivo antibacterial staining;

FIG. 16 is a schematic diagram of the preparation of a bifunctional polyetheretherketone.

Detailed Description

The invention is further described below in connection with specific embodiments, which are exemplary only and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

Example 1 preparation of bifunctional polyetheretherketone

1. Materials: peptides were prepared using standard Fmoc solid phase synthesis. PEEK discs (15 mm diameter, 1.0 mm thickness) were purchased from Waston Medical Appliance Co (Changzhou, china) and PEEK rods (1.5 mm diameter, 10mm length) were purchased from Tianzhu Chang medical technology Co., ltd (Beijing, china).

2. Characterization of the instrument used: HPLC was performed on a liquid chromatography mass spectrometer (Ekspert TM, eksig, usa). ESI-MS was performed on an electrospray ionization mass spectrometer (API 4000+, SCIEX, usa). XPS was performed on an X-ray photoelectron spectrometer (K-Alpha, american thermoelectric Co.) and AFM images were obtained on an atomic force microscope (Dimension ICON, bruce Co.). Static contact angle measurements were performed on contact angle measuring instruments (OCTA 21, DATAPHYSICS, germany).

3. Preparation of bifunctional polyetheretherketone (schematic diagram is shown in fig. 16): with O ₂ PEEK discs or rods were plasma treated and then combined with a biomimetic peptide (DOPA) ₆ -PEG ₅ Azido (formula A) (0.01 mg mL) was immersed in PBS for 15 minutes to obtain PEEK-Azido. The PEEK-Azido samples were then incubated with DBCO-AMP (formula B), DBCO-OGP (formula C) or a mixture of both in PBS. To obtain PEEK samples functionalized with different concentrations of AMP and/or OGP, five groups of PEEK samples were prepared with DBCO-AMP/DBCO-OGP feed molar ratios of 4: 0. 3: 1. 2: 2. 1:3 and 0:4, and the modified samples were designated PEEK-Azido, PEEK-A, respectively ₄ O ₀ ，PEEK-A ₃ O ₁ ，PEEK-A ₂ O ₂ ，PEEK-A ₁ O ₃ And PEEK-A ₀ O ₄ . After different surface functionalization treatments, the substrate was thoroughly rinsed with ultrapure water and then dried with nitrogen for further use to prepare the bifunctional polyetheretherketone.

EXAMPLE 2 Performance Studies of bifunctional polyetheretherketones

1. Cell culture: BM-MSCs were obtained from the Shanghai cell Bank of the national academy of sciences and cultured in Dulbecco's modified Eagle Medium (DMEM, hyClone, USA) supplemented with 10% fetal bovine serum (FBS, gibco, USA) and 1% penicillin/streptomycin (Gibco, USA) was added. During cell culture, the medium was changed every three days until 70% to 80% of the cell coverage on the cell culture dish was reached. Cell seeding density was 2×10 for each sample using 24 well plates for seeding ⁴ Individual cells.

2. Cell compatibility: after culturing BM-MSCs on different PEEK samples for 24 hours, cell staining was performed using a live/dead cell staining kit (AmyJet Scientific, chinese marchantia). Calcein AM (2×10) ^-6 M) and EthD-1 (10X 10) ^-6 M) were used to stain live cells green, dead cells red, and observed using a laser confocal microscope (TCS SP8, lycra, germany), respectively. LDH and CCK-8 kits (chinese, beijing) were also used to determine proliferation and cytotoxicity of BM-MSCs cultured on different samples. Briefly, BM-MSCs were cultured on different PEEK samples for 1, 3 and 5 days, after which the medium was collected, centrifuged and 10% LDH solution was added, and after 2 hours the absorbance at 490nm wavelength was measured on a full wavelength plate reader (SuPerMax 3000al, flash, china) to determine LDH activity. At the same time point, 200. Mu.L of 10% CCK-8 solution was added to each sample, and after incubation for 2 hours, absorbance at 450nm was measured on a full wavelength plate reader (SuPerMax 3000AL, FLASH, china).

3. Cell adhesion and morphology: BM-MSC cultured on different samples was assayed by fluorescent staining. Briefly, after 48 hours of cell culture, cells on different samples were fixed with 4% paraformaldehyde for 30 minutes, then permeabilized with 0.1% triton X-100 (Beyotime, china) for 2 minutes. Subsequently, the cells were washed 3 times with PBS and non-specific binding sites were blocked using 10% bovine serum albumin (beyotidme, china). Each well was then incubated with 250. Mu.LF-actin (1:200 dilution, guangdong, china) for 1 hour at room temperature, washed 3 times with PBS, and then alexa FluorTM 488 phalloidin (Thermo Fisher, USA) was added. Incubate for an additional 1 hour in the dark. Thereafter, the cells were counterstained with 4', 6-dimid-2-phenylindole hydrochloride (DAPI, beyotime, china) for 5 minutes, rinsed 3 times with PBS, and then observed on a laser confocal microscope (TCS SP8, germanica). In addition to fluorescent staining, SEM analysis was performed to investigate cell adhesion and morphology. After 1 and 3 days of incubation, cells on different samples were fixed with glutaraldehyde (2.5% v/v) for 2 hours, then dehydrated in gradient ethanol (20%, 40%,60%,80% and 100%) for 15 minutes each. Subsequently, the different samples were vacuum-dried, sprayed with gold, sputter-coated, and then observed under FE-SEM (HITACHI, S-4700, japan).

4. Antibacterial activity in vitro: staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were used for the antibacterial assay. Both bacterial strains were grown in LB medium (Sigma-Aldrich, USA). Briefly, 500. Mu.L of bacterial suspension (1X 10 per ml) ⁶ ) Inoculated onto different substrates in 24-well plates and incubated statically at 37℃for 6 hours. After 3 gentle washes with PBS, bacteria attached to the different samples were fixed with paraformaldehyde at 4 ℃ for 30 min and treated with gradient ethanol (20%, 40%,60%,80%, and 100%) for 15 min each and further dehydrated with t-butanol for 30 min. Subsequently, the different samples were vacuum dried, sprayed with gold, sputter coated, and the morphology of the adherent bacteria was observed under FE-SEM (S-4700, HITACHI, japan). The in vitro antibacterial efficiency was further determined by plate counting. Specifically, bacteria attached to different samples were sonicated in 1mL sterile PBS. After 10-fold dilution, 100. Mu.L of the bacterial suspension was inoculated onto agar plates and incubated at 37℃for 12 hours. Counting Colony Forming Units (CFU)Number and imaging, and determining the antibacterial rate by the following formula: antibacterial ratio = (AB)/a×100%, where a refers to the average CFU of the PEEK control group and B refers to the average CFU of the different experimental groups. The membrane permeability of bacteria in the different groups was further assessed using ONPG (Sigma-Aldrich, usa). Specifically, 500. Mu.L of bacterial suspension (1X 10 per ml) ⁶ ) The attached bacteria were then treated with 500. Mu. LONPG solution (0.75M in NaH2PO4 buffer) for 2 hours after incubation on different substrates at 37℃for 6 hours. The absorbance of the yellow supernatant in each well was measured at 405nm on a spectrophotometer microplate reader (Bio-Rad 680, USA). Furthermore, ATP levels of bacteria in the different groups were assessed using an ATP kit (Abcam, usa) according to the manufacturer's instructions.

5. In vitro osteogenic differentiation: for osteogenic differentiation, BM-MSC was cultured on different PEEK substrates for 3 days, and then the medium was replaced with osteogenic medium (DMEM supplemented with 0.1. Mu.M dexamethasone, 50. Mu.M ascorbic acid, 10 mM. Beta. -glycerol). Phosphate and 10% fbs were used for osteogenic culture. ALP activity of BM-MSC on different samples was measured on

days

4, 7 and 14 after osteoinduction using ALP kit (Beyotime, china) and normalized to total protein content. For ALP staining, cells were rinsed 3 times with PBS after 14 days of osteoinduction, fixed with paraformaldehyde (4%) for 30 min at 4℃and then left for 20 min in the dark with the addition of (BCIP/NBT) working solution (Beyotime, china).

6. Mineralization of matrix: after 7, 14 and 21 days of osteogenic culture, cells cultured on different samples were fixed with 95% ethanol for 15 minutes, then stained with 40mM RS (Beyotidme, china) at room temperature for 30 minutes. Subsequently, the cells were washed 3 times with deionized water and observed by light microscopy (DM 750M, leica, germany). In the quantitative determination, 10% (v/w) cetylpyridinium chloride (Sigma-Aldrich, USA) (pH 7.0) was added for 15 minutes, and the absorbance at 570nm of the solutions in the different groups was measured.

7. Immunofluorescent staining: after 7 days of osteogenic culture, BM-MSC cultured on different dishes were fixed with paraformaldehyde (4%) for 30 minutes at 4 ℃. Subsequently, 0.2% Triton X-100 (Beyotime, china) was added to infiltrate the cells and all samples were sealed with blocking buffer (Beyotime, china). Thereafter, a primary antibody was added to each well (anti-OCN, 1:200,Cell Signaling Technology, U.S.) and incubated overnight at 4 ℃. The cells were then rinsed 3 times with PBS and the corresponding fluorescent secondary antibodies (Alexa Fluor 488, ab150077, abcam, UK) were added and incubated in the dark for 1 hour. All samples were counterstained with DAPI (Beyotime, china), placed on a microscope slide with fluorescent anti-quenching medium (Beyotime, china) and observed under a laser confocal microscope (TCS SP8, lecaka, germany).

8. qRT-PCR: after 4, 7 and 14 days of osteogenic culture, total RNA was extracted from the different groups of BM-MSCs by adding Trizol reagent (Beyotime, china). dNTP reagent (TaKaRa, japan) and RNase-free H were also used ₂ The gene levels of ALP, runx2, COL1A1 and OCN were analyzed by qRT-PCR (S1000, biorad, U.S.A.) on mixtures of O (Abcam, cambridge, UK). Specific forward and reverse primers are listed below. Gene expression data was analyzed using the 2- ΔΔCT method and normalized using the glyceraldehyde phosphate dehydrogenase (GAPDH) gene.

ALP forward: 5'-GGGGTCAAAGCCAACTACAA-3' the number of the individual pieces of the plastic,

ALP reversal: 5'-CTTCCCTGCTTTCTTTGCAC-3';

runx2 forward: 5'-GCCGGGAATGATGAGAACTA-3' the number of the individual pieces of the plastic,

runx2 reverse: 5 'GGACCGTCCACTGTCTTT-3';

COL1A1 forward: 5'-AATGGTGCTCCTGGTATTGC-3' the number of the individual pieces of the plastic,

COL1A1 reverse: 5'-GGTTCACCACTGTTGCCTTT-3';

OCN forward direction: 5'-GAGGGCAGTAAGGTGGTGAA-3' the number of the individual pieces of the plastic,

OCN reverse: 5'-GTCCGCTAGCTCGTCACAAT-3';

GAPDH forward: 5'-TGACCTCAACTACATGGTCTACA-3' the number of the individual pieces of the plastic,

GAPDH reversal: 5'-CTTCCCATTCTCGGCCTTGTACA-3'.

9. Modeling in vivo: based on the use of different implants, 65 Sprague Dawley rats (male, 6 weeks old, average body weight=300±30 g) were randomly divided into five groups: PEEK, PEEK-Azido, PEEK-A ₄ O ₀ ，PEEK-A ₀ O ₄ And PEEK-A ₂ O ₂ (13 rats per group). Animal protocols were approved by the first hospital animal ethics committee attached to the university of su zhou (su zhou, china). To construct an implant-related infection model, staphylococcus aureus (2×10 in 20 μlpbs) ³ Bacteria) were uniformly coated on different PEEK implants, and then the implants were left in a wet environment at 37 ℃ for 4 hours to allow bacteria to attach. Thereafter, all experimental rats were placed with implants at the distal ends of the left and right femur under sterile conditions. First, the experimental rats were subjected to general anesthesia by intraperitoneal injection of 2% sodium pentobarbital (2 mL/kg). Subsequently, a 10mm longitudinal incision was made along the medial side of the knee joint, pulling the extensor device with the patella laterally. In the case of knee flexion, a bony canal (1.5 mm in diameter and 12 mm in length) was drilled from the femoral intercondylar notch using a k-wire. Next, a different set of PEEK implants were inserted into the medullary canal of the femur through the distal end of the femur until the implant ends were below the articular surface. The patella is repositioned and the knee extension structure is reconstructed. Finally, soft tissues were sutured and animals were injected intramuscularly with analgesia 3 days post-surgery. Troponin (10 mg/kg) was injected into the thigh muscle of different groups of experimental rats 28 and 35 days after implantation to mark new bone formation.

10. In vivo anti-infection evaluation: 2 weeks after surgery, 20 rats (4 per group) were euthanized with an excess of pentobarbital and the femur with different PEEK implants was harvested for the following experiments. First, to determine the number of surviving bacteria in the different groups, the harvested implants were immersed in 1mL of sterile PBS, sonicated for 10 minutes to isolate the attached bacteria, and diluted 10-fold with PBS. Thereafter, 100. Mu.L of the different dilutions were distributed on agar plates and then incubated at 37℃for 12 hours. Visible bacterial colonies in the different groups were imaged and recorded and the antimicrobial ratio calculated as described above. In addition, the implants were immersed in 5mL LB medium (Sigma-Aldrich, USA), incubated overnight at 37℃and then turbidity was determined. The soft tissue surrounding the different groups of implants was carefully obtained and fixed in 4% formaldehyde solution for 72 hours. At the same time, all the femur collected was fixed in formalin (10%) for 48 hours and calcified in 10% ethylenediamine tetraacetic acid (EDTA, sigma-Aldrich, usa) for 4 weeks. After removal of the PEEK rods bone tissue surrounding the implant was obtained, after 3 washes with PBS, the different groups of soft and bone tissue were dehydrated with stepwise ethanol solutions (50%, 70%,90%,100% and 100%), filled with xylene, paraffin embedded and cut into slices. Sections cut to 6 μm were used for H & E and Giemsa staining. Finally, the stained sections were observed and photographed by fluorescence microscopy (Axio Imager 2, zeiss, germany).

11. Osseointegration assessment: 6 weeks post-surgery, another 45 rats (9 per group) were euthanized with an excess of pentobarbital and the femur with different PEEK implants was collected for the following experiments. First, the scan parameters were set to 9 μm per layer at a voltage of 50kV, a current of 500 μa, and bone tissue surrounding the implant (n=6 per group) was evaluated by high resolution micro CT (SkyScan 1176, bargish Aartselaar). A cylinder with a diameter of 1.7mm and a length of 10mm, located near the femoral growth plate, was defined as the target volume for CT analysis. 3D image reconstruction was performed and the system evaluated the morphological parameters of BMD, conn.D, BV/TV, BS/BV, tb.N, tb.Th and Tb.Sp. In addition, the collected femur (n=4 per group) was evaluated by a biomechanical push-out test using a material mechanics test system (procine, zwick, germany). Initially, a 1mm resection was performed on the distal femur to expose the PEEK rod. The femoral samples containing the implants were fixed using dental bone cement prior to performing the biomechanical push-out test. The fixed sample is perpendicular to the bottom surface to ensure that the pushing direction is parallel to the long axis of the implant. Thereafter, the implant was continuously pushed in the loading direction at a speed of 1mm per minute. During the push-out test, the load of the force is recorded to identify the maximum fixation strength. Histological immunofluorescent staining: all harvested femur were fixed in formalin (10%) for 48 hours prior to histological immunofluorescent staining. Without decalcification, a portion of the femur (n=6 per group) was cut into 1 millimeter sections. The calcein markers in the different groups were then visualized by fluorescence microscopy (Axio Imager 2, zeiss, germany) and MAR calculated. Sections that were not decalcified were stained with toluidine blue and visualized using a laser confocal microscope (TCS SP8, lycra, germany). The remaining femur (n=6 per group) was calcified in 10% edta (Sigma-Aldrich, usa) for 4 weeks, dehydrated with gradient ethanol, embedded in paraffin after PEEK implant removal, and then cut into 6 μm sections for H & E staining. The H & E stained sections were visualized by fluorescence microscopy (Axio image 2, zeiss, germany). In addition, immunofluorescent staining was performed to visualize bone-related markers. First, primary antibodies to RunX2 (ab 192256, england Abcam, england) and Osterix (ab 22552, england Abcam, england) were added, incubated at 4 ℃ for 12 hours, after which the sections were washed and incubated in the dark for an additional 1 hour, followed by the addition of the corresponding fluorescent secondary antibodies (Alexa Fluor 647 and Alexa Fluor 488). All sections were counterstained with DAPI (Beyotime, china) and then observed under a fluorescence microscope (Axio Imager 2, zeiss, germany). Fluorescence intensity was assessed using image J software (bescens da, usa).

12. Statistical analysis: values are expressed as mean ± standard deviation. A t-test was performed to compare the differences between the two groups, followed by one-way analysis of variance, and then a Tukey test was performed to make multiple comparisons. * Differences of p <0.05 were considered significant, while differences of #p <0.01 were considered highly significant.

EXAMPLE 3 experimental results

1. Characterization detection

XPS was used to detect and analyze the chemical composition of the sample surface, C and O were the dominant elements in XPS spectra of PEEK samples, and after treatment with mussel-derived click peptides, N was introduced, indicating that N was successfully loaded onto the surface of the samples (FIG. 1).

AFM results showed: the PEEK surface is relatively flat and the PEEK-AO surface becomes very dense, exhibiting uniform undulations, which greatly increases the roughness of the material, thereby increasing the contact area of the material with the cells, providing a better adhesion condition (fig. 2).

The water contact angle results show that: the water contact angle of PEEK is about 80 °, whereas the water contact angle detected on the modified sample is below 40 °. The significant decrease in water contact angle after surface modification is attributable to(DOPA) ₆ -PEG ₅ Azido and DBCO blocked peptides of high hydrophilicity (FIG. 3).

2. In vitro results:

cell proliferation experiments (CCK-8) show that after modification, cell proliferation is obviously increased compared with that of a control group. Scanning electron microscope can see that PEEK-A ₀ O ₄ BMSCs cells spread more fully on the material. Whereas cells of the PEEK group had significant shrinkage. RT-PCR results show that PEEK-AO can better promote ALP, COL1A1 and OCN (osteoblast marker differentiation), runx2 (osteoblast differentiation main transcription factor). ALP staining and ARS staining were observed to be the darkest staining color PEEK-A ₀ O ₄ Group (fig. 4-10).

The antibacterial experiments demonstrated that all AMP-containing samples (PEEK-A) compared to bare PEEK ₄ O ₀ ，PEEK-A ₃ O ₁ ，PEEK-A ₂ O ₂ And PEEK-A ₁ O ₃ ) Can inhibit the growth of bacteria. Furthermore, the antibacterial efficacy of the different groups appears to be dependent on the manner of AMP, with higher AMP feed ratios resulting in better bacterial inhibition. When the AMP/OGP feed ratio is 4:0 or 3:1 (PEEK-A) ₄ O ₀ And PEEK-A ₃ O ₁ ) In the process, the peptide modified sample has antibacterial rate to Escherichia coli and Staphylococcus aureus>90% (FIG. 6).

3. In vivo results:

Micro-CT detection: obviously, around PEEK-A ₄ O ₀ And PEEK-A ₂ O ₂ The newly formed bone of the implant is much denser than that observed in the other groups. Quantitative analysis of Bone Mineral Density (BMD), connection density (conn.d), bone volume/total volume (BV/TV), bone surface/bone volume (BS/BV), and other parameters, such as small Liang Shu (tb.n), trabecular thickness (tb.th) and trabecular separation (tb.sp), all indicate that AMP-modified implants are more beneficial for bone regeneration around the implant than other implants. The mechanical push-out experiments demonstrated the difference in osseointegration between the different groups. HE staining results: in PEEK, PEEK-Azido and PEEK-A ₀ O ₄ A fibrous layer is formed around the implant and the bone mass around the implant is limited, possibly due toNegative effects of bacterial infection in these groups. In contrast, PEEK-A ₄ O ₀ And PEEK-A ₂ O ₂ The implant will introduce more and more dense surrounding trabecular bone because the decorated AMP eliminates most of the bacteria surrounding the implant that interfere with osteogenesis. (FIGS. 11-14).

The antibacterial experiment shows that: h&E-staining showed that neutrophils could penetrate the peri-implant soft tissue in the AMP-free group (PEEK, PEEK-Azido and PEEK-A) ₀ O ₄ ) Many bacteria were observed in the corresponding images after giemsa staining. At the same time, there was many neutrophil infiltrates around the implants in these groups, and bacteria were detected around bone tissue and bone marrow cavities (fig. 15).

To sum up: since bioorthogonal reactions can achieve precise coordination by varying the feed molar ratio of AMP and OGP, the best PEEK surface is ultimately achieved in this study, which can inhibit bacterial growth for long periods, stabilize bone homeostasis and promote interfacial bone regeneration.

Sequence listing

<110> university of Suzhou affiliated first hospital

<120> method for preparing bifunctional polyether-ether-ketone by using mussel derived peptide and biological orthogonal reaction and application thereof

<141> 2021-07-07

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<170> SIPOSequenceListing 1.0

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<213> Artificial sequence (chemical Synthesis)

<400> 1

Arg Trp Arg Trp Arg Trp

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<213> Artificial sequence (chemical Synthesis)

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Tyr Gly Phe Gly Gly

1 5

Claims

1. The method for preparing the bifunctional polyether-ether-ketone by utilizing mussel derived peptides and combining biological orthogonal reaction is characterized by comprising the following steps of:

a. synthesis of clickable mussel-derived peptides

3, 4-dihydroxy-L-phenylalanine DOPA was introduced into peptide sequences using pyruvic acid protected Fmoc-DOPA (acetone) -OH; specifically, hexavalent DOPA units with one lysine spacer are integrated with azido groups linked to polyethylene glycol to obtain clickable mussel-derived peptides (DOPA) ₆ -PEG ₅ -Azido；(DOPA) ₆ -PEG ₅ Azido has the structural formula shown in formula A:

b. clickable mussel derived peptide modified polyether ether ketone

preparation of DBCO-AMP and DBCO-OGP

the sequence of the antimicrobial peptide AMP is: RWRWRW as shown in SEQ ID NO:1 is shown in the specification;

the active sequence of the osteogenic growth peptide OGP is as follows: YGGFGG as shown in SEQ ID NO:2 is shown in the figure;

d. preparation of difunctional polyether-ether-ketone

2. A method for preparing bifunctional polyetheretherketone using mussel-derived peptides in combination with bioorthogonal reaction according to claim 1, wherein the polyetheretherketone is treated with plasma prior to use.

3. The method for preparing the bifunctional polyetheretherketone by using mussel-derived peptides in combination with bio-orthogonal reaction according to claim 1, wherein the operation step of the step d is as follows: DBCO-AMP and/or DBCO-OGP are incubated with polyetheretherketone-Azido in PBS.

4. The method for preparing bifunctional polyetheretherketone by mussel-derived peptides in combination with bioorthogonal reaction of claim 1, wherein the molar ratio of DBCO-AMP to DBCO-OGP reaction is 4: 0. 3: 1. 2: 2. 1:3 or 0:4.

5. the method for preparing bifunctional polyetheretherketone by mussel-derived peptide combined biological orthogonal reaction of claim 1, wherein the material after the reaction in step d is thoroughly rinsed with ultra pure water and then dried with nitrogen gas for further use.

6. A bifunctional polyetheretherketone prepared by the method of any one of claims 1-5.

7. Use of the bifunctional polyetheretherketone of claim 6 for the preparation of an antibacterial and osteogenic bone implant.