WO2021243979A1

WO2021243979A1 - Polyether-ether-ketone composite implant, preparation method therefor and application thereof

Info

Publication number: WO2021243979A1
Application number: PCT/CN2020/131209
Authority: WO
Inventors: 王怀雨; 谢灵霞; 童丽萍
Original assignee: 深圳先进技术研究院
Priority date: 2020-06-03
Filing date: 2020-11-24
Publication date: 2021-12-09
Also published as: CN113750290A

Abstract

Disclosed are a polyether-ether-ketone composite implant, a preparation method therefor and an application thereof. The composite implant comprises: a polyether-ether-ketone substrate, a degradable polymer coating wrapped on the surface of the polyether-ether-ketone substrate, a functional substance loaded and/or grafted on the degradable polymer coating. The functional substance comprises a substance having an immune regulation function, and/or a substance having an osteogenesis regulation function. The preparation method comprises: constructing a degradable polymer coating loaded with a functional substance on the surface of a polyether-ether-ketone substrate; modifying and activating the surface of the degradable polymer coating loaded with the functional substance, and introducing an active group on the surface of the degradable polymer coating loaded with the functional substance; and grafting the functional substance by means of the active group. Also disclosed is an application of the polyether-ether-ketone composite implant in the preparation of drugs for treating bone defects. The polyether-ether-ketone composite implant prepared in the present invention not only maintains the excellent mechanical properties of the polyether-ether-ketone substrate, but also adds the immune activity and osteogenic activity on the surface.

Description

Polyetheretherketone composite implant and preparation method and application thereof

Technical field

The invention belongs to the technical field of biomedical polymer materials, and relates to a polyetheretherketone composite implant with bone immune regulation function, and a preparation method and application thereof.

Background technique

Bone defect is a common clinical disease, and implantation of filler materials is a common method for treating bone defects. The performance requirements for traditional bone implant materials include: excellent physical and chemical properties, such as good corrosion resistance, non-wearing surfaces, and non-toxic effects of wear debris on the body; mechanical properties matching the bone tissue, and good biological properties Such as histocompatibility. With the development of implant materials and further understanding of the interaction between implant materials and the human body, especially the in-depth study of the interaction between implants and the immune system, it indicates that the early immune environment after the material is implanted in the body is important for the later osseointegration It is found that the implant material that exists as a foreign body will damage the healing of bones if it continues to cause local or systemic inflammation ^[1] . Modern clinical medicine puts forward higher performance requirements for implant materials, such as low immunogenicity, and even better induction of bone tissue regeneration by actively adjusting immune response, that is, regulating the early bone immune environment through materials is beneficial to Better bone regeneration.

Poly-ether-ether-ketone (PEEK) is a semi-crystalline, thermoplastic linear aromatic polymer with light weight, good biological stability and non-toxicity, and its mechanical properties are close to that of human bones. FDA approved and used for clinical use. Compared with traditional metal materials, PEEK has a lower modulus of elasticity, which is close to that of human cortical bone. This similarity can reduce the stress shielding effect caused by elastic mismatch and avoid possible bone damage. In addition, PEEK has natural radiolucency, excellent mechanical properties and chemical resistance. However, the PEEK material itself is biologically inert, lacks immunological activity and osteogenic activity, and its osseointegration properties limit its clinical application.

Because of its unusually stable chemical inertness, the ability of chemical methods to modify its surface is very limited. It is often modified through physical methods such as blending, physical coating of functional coatings on the surface, and physical-mediated grafting. . Common modification methods of PEEK include blending with active material (hydroxyapatite) or constructing a hydroxyapatite coating on its surface ^[2] ; after sulfonating the surface of PEEK, plasma implantation of zinc ions ^[3] ; By wet chemical grafting, PEEK surface is coated with BMP-2 to improve the osteogenic performance ^[4] ; plasma immersion ion implantation is used to build a titanium dioxide or diamond-like coating on the surface of PEEK to increase the surface roughness, chemical modification, and other combinations. Biologically active particles ^[5,6] . Modified by blending with active substances, its mechanical properties will often lead to mismatch with adjacent bone tissues, which affects the osteogenic effect; the PEEK surface is constructed with calcium phosphate or hydroxyapatite coating, which can improve its surface Biologically active, but the bonding strength between the prepared coating and the PEEK surface is limited, peeling may occur, the hydroxyapatite layer on the substrate is easily broken, and particle wear debris may be generated, which can change the immune response , Secrete inflammatory factors and cause pathological bone resorption. Wet chemical grafting of BMP-2 can improve the bone formation performance, but the reaction generates a series of oxygen-containing groups on the surface, which increases the uncertainty of subsequent experiments, is not conducive to the directional immobilization of proteins, the reaction is not precise enough, and the reaction repeatability is poor , The experimental results are highly random. Plasma immersion ion implantation builds titanium dioxide or diamond-like coating on the surface of PEEK, which can increase the surface roughness, chemical modification and combination with other biologically active particles; after sulfonating the PEEK surface, plasma implants zinc ions to increase the surface roughness However, the performance of the above-mentioned coating or the factors attached to the surface is single, and the influence of inflammation on osteogenesis after implantation of the material is not considered, which may lead to inconsistent results in vivo and in vitro, and implant failure.

In summary, in view of the lack of immunological activity and osteogenic activity on the surface of PEEK materials, and poor osseointegration characteristics, the invention of a PEEK composite implant with bone immunomodulatory function on the existing basis has important application value.

references

[1] Wei F, Xiao Y. Modulation of the Osteoimmune Environment in the Development of Biomaterials for Osteogenesis[M]//Novel Biomaterials for Regenerative Medicine. Springer, Singapore, 2018: 69-86.

[2] S.-W. Ha, J. Mayer, B. Koch, and E. Wintermantel, “Plasma-sprayed hydroxylapatite coating on carbon fibre reinforced thermoplastic composite materials,” Journal of Materials Science: Materials in Medicine, vol. 5, no. 6-7, pp. 481–484, 1994.

[3] Liu, Wei, et al. "Zinc‐modified sulfonatedpolyetheretherketone surface with immunomodulatory function for guiding cell fate and bone regeneration." Advanced Science 5.10 (2018): 1800749.

[4] Liu L ,Zheng Y, Zhang Q, et al. Surface phosphonation treatment shows dose-dependent enhancement of the bioactivity of polyetheretherketone[J]. RSC Advances, 2019, 9.

[5] T. Lu, X. Liu, S. Qian et al., “Multilevel surface engineering of nanostructured TiO2 on carbon-fiber-reinforced polyetheretherketone," Biomaterials, vol. 35, no. 22, pp. 5731–5740, 2014.

[6] Dufils J, Faverjon F, Heau C, et al. Combination of laser surface texturing and DLC coating on PEEK for enhanced tribological properties[J]. Surface & Coatings Technology, 2017: 29-41.

technical problem

In order to solve the technical problems raised in the above background art, the purpose of the present invention is to provide a polyetheretherketone composite implant and its preparation method and application. The polyetheretherketone composite implant of the present invention not only maintains the excellent mechanical properties of PEEK, but also increases the osteogenic activity on the surface, and can actively regulate bone immunity in the early stage of implantation to achieve better bone regeneration, and the preparation method is simple to operate convenient.

Technical solutions

In order to achieve the above objective, the technical solution adopted by the present invention is as follows: In the first aspect, the present invention provides a polyether ether ketone composite implant, comprising: a polyether ether ketone substrate, wrapped on the polyether ether ketone substrate Functional substances supported and/or grafted on the surface of the degradable polymer coating and the degradable polymer coating;

The functional substance includes a substance that has a function of regulating immunity, and/or a substance that has a function of regulating osteogenesis; preferably, the functional substance includes a substance that has a function of regulating immunity and a substance that has a function of regulating osteogenesis;

The substance with regulating immune function includes a biological molecule with regulating immune function, and/or a drug with regulating immune function; said substance with regulating osteogenic function includes a biological molecule with regulating osteogenic function, and/or , A drug that regulates osteogenic function.

Further, it includes: a polyether ether ketone substrate, a degradable polymer coating wrapped on the surface of the polyether ether ketone substrate, and a functional substance supported and grafted on the degradable polymer coating;

Preferably, the functional substance grafted by the degradable polymer coating includes a substance having a function of regulating immunity, and the substance having a function of regulating immunity includes a biological molecule with a function of regulating immunity, and/or, having a function of regulating immunity. Functional medicine; the functional substance carried by the degradable polymer coating includes a substance with a function of regulating bone formation, and the substance with a function of regulating bone formation includes a biomolecule with a function of regulating bone formation, and/or, It is a medicine that regulates the function of bone formation.

Further, the biomolecules with regulating immune function include one or a combination of at least two of the biomolecules with regulating inflammation function. Preferably, the biomolecules with regulating immune function include IL-4, IL- 6. IL-10, a precursor of NO, one or a combination of at least two of IL-12 and TGF; more preferably, the biomolecule with immune function is the cytokine IL-10;

The drug with regulating immune function includes one or a combination of at least two immunosuppressive agents capable of inhibiting inflammatory response, inhibiting inflammatory cell proliferation and activating autophagy response. Preferably, the drug with regulating immune function includes One or a combination of at least two of glucocorticoids, tacrolimus, rapamycin, thalidomide, triptolide, infliximab, adalimumab, and mycophenolate mofetil.

Further, the medicine with the function of regulating osteogenesis includes one or a combination of at least two medicines with anti-inflammatory, osteogenesis-promoting effect or osteoclast inhibitory effect. Preferably, the medicine with regulating osteogenesis includes One or a combination of at least two of dexamethasone, alendronate, fluoride, statins, teriparatide, and teronidine; more preferably, the osteogenesis-regulating drug is ground Semisone (DEX);

The biomolecules with the function of regulating bone formation include one or a combination of at least two of the biomolecules capable of promoting angiogenesis, or promoting the differentiation of stem cells into osteoblasts, or having the function of promoting the proliferation of osteoblasts. Preferably, The biomolecules capable of regulating osteogenesis include one or a combination of at least two of BMP-2, VEGF, OPG, PDGF, TGF, and insulin-like growth factor-1.

Further, the degradable polymer coating includes one or a combination of at least two of the following polymers wrapped on the surface of a polyetheretherketone substrate, and the polymer includes polytrimethylene carbonate (PTMC), polylactic acid (PLA), polycaprolactone (PCL), polylactic acid and polyglycolic acid (PLGA), aliphatic polyester polymer, aromatic-aliphatic copolyester;

Preferably, the molecular weight of the polymer is 5,000 to 500,000 Daltons.

In the second aspect, the present invention provides a method for preparing any one of the above-mentioned polyetheretherketone composite implants, including the following steps:

Construct a degradable polymer coating with functional substances on the surface of the polyetheretherketone substrate;

The functional substance includes a substance that has a function of regulating immunity, and/or a substance that has a function of regulating bone formation;

Or 1) Build a degradable polymer coating on the surface of the polyetheretherketone substrate;

2) Modify the surface of the activated degradable polymer coating, and introduce active groups on the surface of the degradable polymer coating;

3) Graft functional substances through active groups;

Or 1) Construct a degradable polymer coating with functional substances on the surface of the polyetheretherketone substrate;

2) Modify the surface of the degradable polymer coating with activated functional substances, and introduce active groups on the surface of the degradable polymer coating with functional substances;

3) Graft functional substances through active groups;

Preferably, it includes the following steps:

1) Build a degradable polymer coating on the surface of the polyetheretherketone substrate with substances that can regulate bone formation;

2) Modified activation of the degradable polymer coating surface loaded with substances capable of regulating osteogenesis, and introducing active groups on the surface of the degradable polymer coating loaded with substances capable of regulating osteogenesis;

3) Grafting substances with immune function through active groups;

Further, the method for constructing a degradable polymer coating loaded with a substance capable of regulating osteogenesis includes a solvent volatilization method, a pulling extraction method or an atomizing spraying method;

Preferably, the mass ratio of the bone-regulating substance to the polymer in the degradable polymer coating constructed on the surface of the polyetheretherketone substrate and loaded with the substance that regulates bone formation is 1:1-30 . When it is not in this range, too little drug content has no effect, and too high drug content is toxic.

Further, the method for introducing active groups on the surface of the biodegradable polymer coating loaded with substances capable of regulating osteogenesis is gas plasma immersion ion implantation;

Preferably, the gas used for the gas plasma immersion ion implantation includes one or a combination of at least two of argon, nitrogen, ammonia, oxygen, hydrogen and other gases;

Preferably, the background vacuum degree used for the gas plasma immersion ion implantation is 1×10 ^-3 to 9×10 ^-3 Pa;

Preferably, the gas introduction flow rate used for the gas plasma immersion ion implantation is 20-100 SCCM;

Preferably, the negative bias applied to the sample plate used for the gas plasma immersion ion implantation is 0-10kV;

Preferably, the implantation pulse width used in the gas plasma immersion ion implantation is 20-200 microseconds;

Preferably, the injection pulse frequency used for the gas plasma immersion ion implantation is 50~500 Hz;

Preferably, the radio frequency power used for the gas plasma immersion ion implantation is 100~1000 W;

Preferably, the implantation time used for the gas plasma immersion ion implantation is 10 to 120 minutes.

Further, the method for grafting a substance with an immune function through an active group is to immerse the surface of a degradable polymer coating containing a substance with a function of regulating osteogenesis into a material containing a substance with a function of regulating immunity. Incubate in the solution for a certain period of time to covalently graft a substance that has an immune-regulating function. ；

Preferably, the concentration of the solution containing the substance with the immune function is 10ng/mL-10μg/mL;

Preferably, the grafting time is 6 to 72 hours.

In the third aspect, the present invention provides the application of any one of the above-mentioned polyetheretherketone composite implants in the preparation of implants for filling parts of bone defects.

Beneficial effect

Compared with the prior art, the drug-loaded modified coating constructed on the PEEK surface of the present invention has the following advantages:

1) With bone immune regulation function;

2) The drug-loaded modified coating constructed on the surface of PEEK has a time sequence for the controlled release of the drug, which simulates the self-repair process after a human fracture or bone defect occurs. It first regulates inflammation and then regulates osteogenesis, and regulates inflammation and regulation of formation. The double-layer effect of bone is superimposed to improve the immune activity and osteogenic activity of PEEK materials;

3) PEEK composite implants implanted in the body present the characteristics of sequential release of regulatory factors, that is, in the early inflammatory reaction stage, as the coating degrades, the most surface grafted immunomodulatory molecules are preferentially released in the bone immune environment for regulation Immune response, as the biodegradable polymer coating gradually degrades in the middle and late stages of osteogenesis, the internally loaded osteogenic drugs, such as dexamethasone, are continuously released to regulate osteogenesis, making the material possess both immunological activity and osteogenic activity , So as to better apply in the clinic;

4) The drug-loaded coating constructed on the surface of PEEK can control the long-term stable release of the drug, without cytotoxicity caused by excessive local drug concentration, and the concentration of the released drug can be adjusted by controlling the ratio of the drug to the polymer, and The degradation cycle of the coating can be controlled by adjusting the thickness of the coating and the molecular weight of the polymer (for example, the higher the molecular weight of polytrimethylene carbonate, the faster the degradation speed); the content of grafted biomolecules can be adjusted by adjusting the solution of grafted biomolecules To control the concentration;

5) The polymer used in the drug-carrying coating constructed on the surface of PEEK is biodegradable, can be hydrolyzed or enzymatically in the body, and the degradation products are neutral, have little harm, have no toxic side effects to the body, and will not cause the body Compared with the construction of inorganic and metal non-degradable coatings on the surface of PEEK, this application does not change the properties of the PEEK material itself, maintains the excellent mechanical properties of the substrate PEEK, and the surface coating is tightly combined with the substrate;

6) In the present invention, biomolecules can be directly grafted after the surface of the drug-carrying polymer is treated by plasma immersion ion implantation, without the use of chemical cross-linking agents, which is safe and convenient.

7) The drug-loaded coating constructed on the surface of PEEK can greatly improve the physical and chemical properties of the surface, such as hydrophilicity and hydrophobicity, surface energy, surface chemical composition, and increase the surface biological activity without affecting the performance of the PEEK material matrix.

8) The method of constructing the drug-loaded coating on the surface of PEEK is simple and easy to operate.

Description of the drawings

Figure 1a is a scanning electron micrograph of unmodified polyetheretherketone (PEEK) in Example 1 of the present invention; Figure 1b is a 5% DEX coating prepared on the surface of polyetheretherketone by solution volatilization in Example 1 of the present invention Scanning electron micrograph of the surface of the layer;

2 is a scanning electron micrograph of a 5% DEX surface treated by nitrogen plasma immersion ion implantation in Example 2 of the present invention;

3 is a scanning electron micrograph of the surface of 2KV-IL-10 with a concentration of 40ng/ml IL-10 after being implanted by nitrogen plasma immersion ion in Embodiment 3 of the present invention;

Figure 4a is the result of the static water contact angle of the surface before and after modification of the PEEK material in Example 4 of the present invention; Figure 4b is the result of the water contact angle of the sample after the plasma surface treatment of Example 2 in Example 4 of the present invention after being placed for one month Test result diagram;

Fig. 5 is a full spectrum diagram of surface elements measured by an X-ray electron spectrometer after modification of the PEEK material in Example 5 of the present invention;

Fig. 6a is a graph showing the results of the proliferation of macrophages RAW264.7 on the surface of each sample in Example 6 of the present invention; Fig. 6b is a result of the use of Mouse TGF-beta1 Valukine in Example 6 of the present invention ELISA kit (R&D system) and Mouse TNF-A Valukine ELISA kit (R&D) detect the expression results of inflammation-related factors TNF-α and TGF-β1; Figure 6c is the detection of M1 by real-time fluorescent quantitative PCR in Example 6 of the present invention The results of expression of genes related to M2;

Figure 7 is the result of real-time fluorescent quantitative PCR detecting the expression of osteogenic-related genes in the conditioned medium in Example 7 of the present invention; wherein RAW264.7 (+) represents the conditioned medium, and RAW264.7 (-) represents the non-conditioned medium;

Fig. 8a is a result of the proliferation of MC3T3-E1 cells on the surface of each sample in Example 8 of the present invention; Fig. 8b is the detection of bone formation related to MC3T3-E1 cells on the surface of the sample by real-time fluorescent quantitative PCR in Example 8 of the present invention A result map of gene expression and mineralization on the surface of the sample;

Figure 9 is a graph showing the immunofluorescence results of the macrophage phenotype of each sample implanted subcutaneously in rats in Example 9 of the present invention;

Figure 10a is the 3D model and 2D image obtained by Micro-CT after each sample implanted in the rat femur in Example 10 of the present invention. The result of tissue staining with VG.

Embodiments of the present invention

In order to better understand the content of the present invention, the content of the present invention will be further described below with reference to the drawings and specific implementation methods, but the protection content of the present invention is not limited to the following embodiments.

Example 1 Preparation of PEEK and 5% DEX samples

The polyether ether ketone discs with a diameter of 15 mm and a thickness of 1 mm were polished with sand with meshes of 600, 800, 1000, 1200, and 2000 in turn. The polished polyether ether ketone was successively polished with acetone, alcohol, and deionized water. Ultrasonic clean. The sample after this pretreatment is labeled PEEK.

A drug-carrying coating was constructed on the surface of PEEK by solvent evaporation. The selected polymer coating is polytrimethylene carbonate (PTMC), the solvent is dichloromethane, the drug is dexamethasone, and the mass ratio of drug to polymer is 1:5. The sample after this pretreatment is labeled 5% DEX. The specific preparation method is as follows: first add PTMC and dexamethasone in a dichloromethane solution at a mass ratio of 5:1 to form a uniform solution, and then pour the homogeneous solution containing PTMC and dexamethasone on the surface of the PEEK sample. The solvent methylene chloride evaporates cleanly, forming a uniform coating on the surface of PEEK.

The surface of the polyether ether ketone before and after the treatment was observed by scanning electron microscope, and the surface micro morphology as shown in FIG. 1 was obtained. It can be seen from Figure 1a that the surface of PEEK without coating can be traced by sandpaper. Figure 1b shows that the entire surface is smooth and flat after the drug-loaded coating is constructed on the surface, and no drug is concentrated on the surface.

Example 22KV sample preparation

The gas plasma immersion ion implantation technology was used to process the 5% DEX in Example 1. The specific treatment process is as follows: background vacuum degree is 2×10 ^-3 Pa, gas introduction flow rate is 60 SCCM, negative bias voltage applied to the sample plate is 2 kV, injection pulse width is 50 microseconds, and injection pulse frequency is 50 Hz , The RF power is 1000 W. Among them, the injection time of nitrogen is 60 minutes, and the processed sample is called 2KV. The 5% DEX surface after the nitrogen plasma immersion ion implantation treatment was observed by a scanning electron microscope, and the surface micro-topography picture shown in Fig. 2 was obtained. It can be seen from Fig. 2 that there is no obvious difference between the surface of 5% DEX and 2KV samples, indicating that the nitrogen plasma immersion ion implantation treatment does not significantly change the surface morphology of 5% DEX.

Example 32KV-IL-10 sample preparation

The modified 2KV sample in Example 2 was immersed in a phosphate buffer solution (PBS) containing IL-10, where the concentration of IL-10 was 40 ng/ml, and stored at 4 °C for 24 Hour. Then the sample was taken out of the IL-10 solution, and the sample was rinsed with PBS without IL-10 to remove the ungrafted IL-10, and the sample was labeled 2KV-IL-10. The microscopic morphology of the sample grafted with IL-10 was observed through a scanning electron microscope, and the result is shown in Figure 3. It can be seen from Figure 3 that there is no obvious change in the surface of the coating after grafting IL-10.

Example 4 Test of Hydrophilicity and Hydrophobicity of the Samples Obtained in Example 1, Example 2, and Example 3

The samples obtained in Example 1, Example 2, and Example 3 were dried in a vacuum drying oven for 24 hours and then measured with a water contact angle meter to measure their surface hydrophilicity and hydrophobicity. The results are shown in Figure 4a. It can be seen from Figure 4a that the water contact angle of PEEK is 95 degrees, which is a hydrophobic surface. After constructing 5% dexamethasone on its surface, the contact angle of 5% DEX surface is 85 degrees, which is hydrophilic. Obviously improved, after further nitrogen plasma treatment, the surface hydrophilic groups increased, the surface contact angle of 2KV decreased to 56 degrees, after grafting IL-10, the surface water contact angle of 2KV-IL-10 was 38 degrees, It can be seen that the hydrophilicity of PEEK surface has been significantly improved after modification. The water contact angle of the sample after plasma surface treatment in Example 2 was tested again after being placed for one month. The test result is shown in Figure 4b. The result shows that the hydrophilic and hydrophobic properties of the sample surface did not change after one month, which improved the hydrophilicity. The effect is long-lasting.

Example 5 X-ray photoelectron spectroscopy test of the samples obtained in Example 1, Example 2, and Example 3

The samples obtained in Example 1, Example 2, and Example 3 were analyzed by X-ray photoelectron spectroscopy (XPS) to analyze the changes of surface elements, and the full spectrum is shown in FIG. 5. It can be seen from Figure 5 that only the characteristic peaks of C1s (285.14 eV) and O1s (532.03 eV) appear in the polytrimethylene carbonate (PTMC) and 5% DEX spectra. After nitrogen plasma treatment, the 2KV sample newly appeared The characteristic peak of N1s (400.00 eV), after grafting IL-10, a new characteristic peak of Sp2 (164.02 eV) appeared, and the N signal in this sample was significantly enhanced. This is due to the grafting of IL-10 protein, IL-10 protein Contains a lot of N elements.

Example 6 The samples obtained in Example 1, Example 2, and Example 3 were tested in vitro to regulate inflammatory response

After UV sterilization of each sample, 40,000 macrophages of RAW264.7 were inoculated on the surface. After 1, 3, and 5 days of culture, the growth of the macrophages was measured by CCK-8. The results are shown in Figure 6a. A result of the increase in the surface of the sample. From the results in the figure, it can be seen that the surface of PEEK loaded with dexamethasone has an inhibitory effect on the proliferation of macrophages. After plasma treatment, the surface is grafted with IL-10 to inhibit macrophages. It is more obvious that the coating prepared on the surface of PEEK can inhibit the proliferation of macrophages.

After UV sterilization of each sample, 40,000 macrophages RAW264.7 were inoculated on the surface. After 6 hours of inoculation, the cell culture supernatant of each sample was collected, centrifuged at 2000 rpm for 5 minutes, and then used Mouse TGF-beta1 Valukine ELISA kit (R&D system) and Mouse TNF-A Valukine ELISA kit (R&D), according to the kit instructions to detect the expression of inflammation-related factors TNF-α and TGF-β1, the detection results are shown in Figure 6b, followed by the PEEK group In contrast, the other three groups all inhibited the release of macrophage pro-inflammatory factor TNF-α and promoted the release of macrophage anti-inflammatory factor TGF-β1. Among them, the 2KV-IL-10 group had the most significant effect.

After UV sterilization of each sample, 40,000 macrophages RAW264.7 were inoculated on the surface. After the cells were cultured on the sample surface for 1 and 3 days, the macrophage RNA was extracted with Trizol, and the macrophage was further detected by real-time fluorescent quantitative PCR after reverse transcription. The relative expression of M1 phenotype genes CCR-7, TNF-a, IL1-β gene and the relative expression of M2 phenotype genes CD206, IL-10, TGF-β1, and the relative expression of M1 phenotype genes CD206, IL-10, and TGF-β1 when phages were cultured on the surface of each sample for 1 day and 3 days. The result is shown in Figure 6c. It can be seen from the figure that the other three groups of macrophages cultured for 1 day and 3 days after the relative expression of M1 type genes CCR-7, TNF-a, IL-1b were lower than the PEEK group, M2 type genes CD206, IL-10, The relative expression of TGF-b was higher than that of the PEEK group, indicating that it can inhibit the expression of M1 type genes and promote the expression of M2 type genes after PEEK constructs a drug-loaded coating.

In summary, the results of Figure 6b and Figure 6c show that the modified coating 2KV-IL-10 on the surface of PEEK can better inhibit the polarization of macrophages to M1 type and promote the polarization of macrophages to M2 type compared with other groups.

Example 7 Exploring the effect of PEEK surface modification layer regulating macrophages on osteogenesis

Culture the macrophages on the samples in Example 1, Example 2, and Example 3 for 1 to 3 days. The culture medium was collected every day to obtain the culture supernatant, and the macrophage culture supernatant and DMEM high glucose were cultured. Prepare the conditioned medium based on the volume ratio of 2:1; the processing method of each sample is the same as the processing method of inoculating macrophages, and it is placed in a 24-well plate, and an equal volume of serum-free, cell-free, high-glucose culture is added to each well Base, collect 1 to 3 days of medium as non-conditioned medium, inoculate 30,000 mouse embryonic osteoblast precursor cells (MC3T3-E1) in a 24-well plate, remove the medium after 12 hours, and replace it every day Corresponding days of conditioned medium and non-conditioned medium, after three days of culture, the expression of osteogenic genes ALP, OCN, OPN, RUNX2 was detected by real-time fluorescent quantitative PCR, and the effect of PEEK surface modification layer on macrophages was explored. The result is shown in Figure 7. It can be seen from the expression of bone formation-related genes in Figure 7 that the macrophage conditioned medium group has higher expression than the non-conditioned medium group. Compared with the PEEK group, the expression of each gene in the other three groups Both are higher, and the difference between the 2KV-IL-10 group and the PEEK group is the largest, indicating that PEEK can regulate macrophages to promote osteogenesis after modification.

Example 8 The samples obtained in Example 1, Example 2, and Example 3 were tested in vitro to regulate and control osteogenesis

The samples in Example 1, Example 2, and Example 3 were subjected to the experiment of regulating osteogenesis in vitro. MC3T3-E1 cells were inoculated on the surface of each sample. After 1, 3, and 7 days, the cells were detected on the surface by CCK-8 reagent. Proliferation effect; when the surface planted cells grow to 80%, replace the normal medium with osteoinductive liquid and re-culture for 7, 14, 21 days, and then use real-time fluorescence quantitative PCR to detect the expression of osteogenic related genes ALP, OCN, OPN, and pass Alizarin red staining detects the degree of cell mineralization on the sample surface, and the results are shown in Figure 8. Figure 8a is a result of the proliferation of MC3T3-E1 cells on the surface of each sample; Figure 8b is the detection of the expression of the osteogenic genes ALP, OCN, OPN of MC3T3-E1 cells on the surface of the sample and on the surface of the sample by real-time fluorescent quantitative PCR A result of mineralization. It can be seen from the proliferation results of MC3T3-E1 cells in Figure 8a that after 1 day, 3 days, and 7 days in culture, the cell proliferation of the 5% DEX group and the 2KV group was lower than that of PEEK due to the effect of the drug dexamethasone. The 2KV-IL-10 group The proliferation of the cells was higher than that of the PEEK group, indicating that the modified coating 2KV-IL-10 on the surface of PEEK can promote the proliferation of MC3T3-E1 cells in vitro. From Figure 8b, the relative expression of the osteogenic genes ALP, OCN, and OPN can be It can be seen that the expression of 5% DEX, 2KV and 2KV-IL-10 is higher than that of PEEK group. Among them, the expression of 2KV-IL-10 group is the highest. From the 21-day mineralization result, it can be seen that 5% The surface mineralization of the three groups of DEX, 2KV and 2KV-IL-10 was more obvious than that of the PEEK group, and the effect of the 2KV-IL-10 group was the most significant. The above cell proliferation and osteogenic experiments show that the surface preparation of the modified coating 2KV-IL-10 can promote the proliferation and osteogenic differentiation of MC3T3-E1 cells.

Example 9 explores whether the sample PEEK, 5% DEX, and 2KV-IL-10 in Example 1 and Example 3 can regulate inflammation in vivo

The samples obtained in Example 1, Example 3, PEEK, 5% DEX, and 2KV-IL-10 were implanted into the subcutaneously of SD rats. The specific process was as follows: 9 SD female rats about 12 weeks old were randomly divided For the three groups, 2% sodium pentobarbital (2.3ml/kg) was injected anesthetized before the operation. After the anesthesia was properly anesthetized, the rats were fixed on the operating table on their stomachs. Skin preparation, conventional iodophor, and ethanol disinfection, cut at symmetrical positions on both sides of the rat's back, implant each group of samples under the skin and suture, and iodophor disinfection. Rats were sacrificed 3 days after material implantation. Take the tissue with implant and fix it in 4% paraformaldehyde. After fixation, perform immunofluorescence staining to explore whether PEEK can modulate inflammation in vivo and regulate the immunofluorescence of macrophage phenotype. The result is shown in Figure 9. In Figure 9, DAPI stains the nucleus, INOS stains macrophages expressing M1 type, and CD163 is M2 type. It can be seen from the figure that the expression of iNOS in 5% DEX and 2KV-IL-10 groups is low. In PEEK, the expression of CD163 is higher than that of PEEK group, among which 2KV-IL-10 group is more significant than 5% DEX group. The results show that modified PEEK samples 5% DEX and 2KV-IL-10 can also inhibit inflammation in vivo , Regulates the polarization of macrophages to M2 type.

Example 10 explores the osteogenesis effect of the sample PEEK, 5% DEX, 2KV-IL-10 in Example 1 and Example 3 in vivo

The samples in Example 1, Example 3, PEEK, 5% DEX, and 2KV-IL-10 were implanted into the femur of SD rats. The specific operation is as follows: 9 SD female rats about 12 weeks old were randomly divided into three In the group, 2% sodium pentobarbital (2.3ml/kg) was injected anesthetized before the operation. After the anesthesia was proper, the rats were fixed in the supine position on the operating table. Skin preparation, conventional iodophor, and ethanol disinfection. Fix the knee joint in the most flexed position, then cut a longitudinal incision about 10mm along the lateral side of the knee joint, bluntly separate the muscles, and shift the knee joint to expose the distal femur, at the intercondylar notch of the distal femur Drill a cylindrical hole with a diameter of 2 mm parallel to the long axis of the femur (use normal saline while drilling to prevent osteonecrosis caused by high temperature), after the hole is drilled, rinse the remaining debris with normal saline, and implant For the prepared samples, each rat had bilateral distal femoral defects. Suture the wound carefully. Postoperative iodophor disinfects the wound. In order to characterize the formation and mineralization of new bone, a multicolor sequential fluorescent labeling method was used. At 2 weeks, 4 weeks and 6 weeks after surgery, the fluorescent dye Alizarin Red S (purchased from Sigmae-Aldrich, USA) was injected into the abdominal cavity sequentially. ) 30 mg/kg, tetracycline hydrochloride 25 mg/kg and 20 mg/kg calcein (Sigmae-Aldrich). The rats were sacrificed 8 weeks after the material implantation. Take the femur with the implant and fix it in 4% paraformaldehyde. After the fixation, perform Micro-CT detection and histomorphological observation to explore its osteogenic effect in the body; use the bone containing the sample with 4 % Paraformaldehyde fixation, gradient ethanol dehydration (80%, 90%, 100%), PMMA embedding, and VG staining after sectioning to characterize new bone formation. The results are shown in Figure 10. Figure 10a is the 3D model and 2D image obtained by Micro-CT. The results show that 8 weeks later, the formation of new bone around the material is 5% DEX and 2KV-IL-10 group samples are significantly higher than the PEEK group samples, indicating these two groups The samples can stimulate more new bone to grow in the bone marrow cavity, showing better osseointegration, especially 2KV-IL-10 has a significant effect; Figure 10b is the result of VG staining of new bone tissue after sectioning, showing 5 New bone was formed around the samples of %DEX and 2KV-IL-10 groups and the bone layer was intact, which was thicker than the PEEK group, indicating that the modified PEEK has good osteogenic activity. The 2KV-IL-10 group has better osteogenic activity. The %DEX group performed better.

In summary, the PEEK composite implant of the present application can simulate the human bone regeneration process, and promote the rapid regeneration of bone at the bone defect site by adjusting the bone immune system and bone healing growth factors. In the early stage of implantation, cytokines can be used to regulate the phenotype of macrophages, making them polarized from the pro-inflammatory phenotype M1 to the anti-inflammatory M2, that is, regulating the immune system from inflammatory response to promoting Bone repair; and further promote osteogenic differentiation through subsequent drug release, thereby endowing the prepared PEEK composite implant with active bone immune regulation and achieving better bone regeneration. The preparation method of the composite implant is simple to operate, safe and harmless.

The above are only specific implementations of the present invention, not all implementations. Any equivalent changes made by those of ordinary skill in the art to the technical solutions of the present invention by reading the specification of the present invention are covered by the claims of the present invention. .

Claims

A polyether ether ketone composite implant, which is characterized by comprising: a polyether ether ketone substrate, a degradable polymer coating wrapped on the surface of the polyether ether ketone substrate, a degradable polymer coating carried and / Or grafted functional substances;

The functional substance includes a substance that has a function of regulating immunity, and/or a substance that has a function of regulating bone formation;

The substance with regulating immune function includes a biological molecule with regulating immune function, and/or a drug with regulating immune function; said substance with regulating osteogenic function includes a biological molecule with regulating osteogenic function, and/or , A drug that regulates osteogenic function.
The polyetheretherketone composite implant according to claim 1, characterized by comprising: a polyetheretherketone substrate, a degradable polymer coating on the surface of the polyetheretherketone substrate, and a degradable polymer Functional substances supported and grafted on the coating;

Preferably, the functional substance grafted by the degradable polymer coating includes a substance having a function of regulating immunity, and the substance having a function of regulating immunity includes a biological molecule with a function of regulating immunity, and/or, having a function of regulating immunity. Functional medicine; the functional substance carried by the degradable polymer coating includes a substance with a function of regulating bone formation, and the substance with a function of regulating bone formation includes a biomolecule with a function of regulating bone formation, and/or, It is a medicine that regulates the function of bone formation.
The polyetheretherketone composite implant according to claim 1 or 2, characterized in that the biomolecules with regulating immune function comprise one or a combination of at least two of the biomolecules with regulating inflammation function, Preferably, the biomolecules with immune regulation function include IL-4, IL-6, IL-10, precursors of NO, IL-12, TGF, or a combination of at least two; more preferably, The biomolecule with immune function is the cytokine IL-10;

The drug with regulating immune function includes one or a combination of at least two immunosuppressive agents capable of inhibiting inflammatory response, inhibiting inflammatory cell proliferation and activating autophagy response. Preferably, the drug with regulating immune function includes One or a combination of at least two of glucocorticoids, tacrolimus, rapamycin, thalidomide, triptolide, infliximab, adalimumab, and mycophenolate mofetil.
The polyetheretherketone composite implant according to claim 1 or 2, characterized in that, the drug with the function of regulating osteogenic function comprises one of drugs with anti-inflammatory, osteogenic effect or osteoclast inhibitory effect Or a combination of at least two, preferably, the drugs with regulating osteogenic formation include dexamethasone, alendronate sodium, fluoride, statins, teriparatide, teronidine or at least one of A combination of the two; more preferably, the drug with regulating osteogenesis is dexamethasone;

The biomolecules with the function of regulating bone formation include one or a combination of at least two of the biomolecules capable of promoting angiogenesis, or promoting the differentiation of stem cells into osteoblasts, or having the function of promoting the proliferation of osteoblasts. Preferably, The biomolecules capable of regulating osteogenesis include one or a combination of at least two of BMP-2, VEGF, OPG, PDGF, TGF, and insulin-like growth factor-1.
The polyether ether ketone composite implant according to claim 1 or 2, wherein the degradable polymer coating comprises one or a combination of at least two of the following polymers wrapped in polyether ether ketone The coating formed on the surface of the substrate, the polymer includes polytrimethylene carbonate, polylactic acid, polycaprolactone, polylactic acid, polyglycolic acid, aliphatic polyester polymer, aromatic-aliphatic copolyester .
The preparation method of polyetheretherketone composite implant according to any one of claims 1 to 5, characterized in that it comprises the following steps:

Construct a degradable polymer coating with functional substances on the surface of the polyetheretherketone substrate;

The functional substance includes a substance that has a function of regulating immunity, and/or a substance that has a function of regulating bone formation;

The substance with regulating immune function includes a biological molecule with regulating immune function, and/or a drug with regulating immune function; said substance with regulating osteogenic function includes a biological molecule with regulating osteogenic function, and/or , Drugs that can regulate osteogenic function;

Or 1) Build a degradable polymer coating on the surface of the polyetheretherketone substrate;

2) Modify the surface of the activated degradable polymer coating, and introduce active groups on the surface of the degradable polymer coating;

3) Graft functional substances through active groups;

The functional substance includes a substance that has a function of regulating immunity, and/or a substance that has a function of regulating bone formation;

The substance with regulating immune function includes a biological molecule with regulating immune function, and/or a drug with regulating immune function; said substance with regulating osteogenic function includes a biological molecule with regulating osteogenic function, and/or , Drugs that can regulate osteogenic function;

Or 1) Construct a degradable polymer coating with functional substances on the surface of the polyetheretherketone substrate;

2) Modify the surface of the degradable polymer coating with activated functional substances, and introduce active groups on the surface of the degradable polymer coating with functional substances;

3) Graft functional substances through active groups;

The functional substance includes a substance that has a function of regulating immunity, and/or a substance that has a function of regulating bone formation;

The substance with regulating immune function includes a biological molecule with regulating immune function, and/or a drug with regulating immune function; the substance with regulating osteogenic function includes a biological molecule with regulating osteogenesis function, and/or , Drugs that can regulate osteogenic function;

Preferably, it includes the following steps:

1) Constructing a degradable polymer coating on the surface of the polyetheretherketone substrate with substances that can regulate bone formation;

2) Modified activated degradable polymer coating surface loaded with substances capable of regulating osteogenesis, and active groups are introduced on the surface of the degradable polymer coating loaded with substances capable of regulating osteogenesis;

3) Grafting substances with immune function through active groups;

The substance with regulating immune function includes a biological molecule with regulating immune function, and/or a drug with regulating immune function; the substance with regulating osteogenic function includes a biological molecule with regulating osteogenesis function, and/or , A drug that regulates osteogenic function.
The method for preparing a polyetheretherketone composite implant according to claim 6, wherein the method for constructing a degradable polymer coating loaded with a substance capable of regulating osteogenesis includes solvent volatilization, lifting Extraction method or atomization spraying method;

Preferably, the mass ratio of the bone-regulating substance to the polymer in the degradable polymer coating constructed on the surface of the polyetheretherketone substrate and loaded with the substance that regulates bone formation is 1:1-30 .
The method for preparing a polyetheretherketone composite implant according to claim 6, wherein the method for introducing active groups on the surface of the biodegradable polymer coating loaded with substances capable of regulating osteogenesis is gas Plasma immersion ion implantation;

Preferably, the gas used for the gas plasma immersion ion implantation includes one or a combination of at least two of argon, nitrogen, ammonia, oxygen, hydrogen and other gases;

Preferably, the background vacuum degree used for the gas plasma immersion ion implantation is 1×10 -3 to 9×10 -3 Pa;

Preferably, the gas introduction flow rate used for the gas plasma immersion ion implantation is 20-100 SCCM;

Preferably, the negative bias applied to the sample plate used for the gas plasma immersion ion implantation is 0-10kV;

Preferably, the implantation pulse width used in the gas plasma immersion ion implantation is 20-200 microseconds;

Preferably, the injection pulse frequency used for the gas plasma immersion ion implantation is 50~500 Hz;

Preferably, the radio frequency power used for the gas plasma immersion ion implantation is 100~1000 W;

Preferably, the implantation time used for the gas plasma immersion ion implantation is 10 to 120 minutes.
The method for preparing a polyetheretherketone composite implant according to claim 6, wherein the method for grafting a substance with an immune-regulating function through an active group is that the modified activated load has the function of regulating osteogenesis The surface of the degradable polymer coating of the substance is immersed in a solution containing the substance with the immune function and incubated for a certain period of time to covalently graft the substance with the immune function;

Preferably, the concentration of the solution containing the substance with the immune function is 10ng/mL-10μg/mL;

Preferably, the grafting time is 6 to 72 hours.
Use of the polyetheretherketone composite implant according to any one of claims 1 to 5 in the preparation of implants for filling parts of bone defects.