CN112546292A

CN112546292A - Raw material composition, polyether ether ketone based composite material, preparation method and application

Info

Publication number: CN112546292A
Application number: CN202011474342.9A
Authority: CN
Inventors: 魏杰; 王雪红; 赵君; 王帆; 潘泳康; 王德强; 钱军; 郑晨; 严思清
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2020-12-14
Filing date: 2020-12-14
Publication date: 2021-03-26

Abstract

The invention discloses a raw material composition, a polyether ether ketone-based composite material, a preparation method and an application. It contains the following components: the volume ratio of niobium (Nb) powder and polyetheretherketone powder is 1:4-2:3, and the particle size of the niobium powder is 10-120 nm. In the present invention, by incorporating Nb into PEEK, a nanostructure is constructed on the surface of the material and the surface properties are improved, thereby enhancing cell response. The nanofilament structure was constructed on the surface of the material by lye etching and the surface properties were improved, thereby enhancing the cellular response. The replacement of functional element Ca ²⁺ was completed, and the nanofilament structure was retained for loading functional small molecules. The composite material has good roughness and hydrophilicity, and builds a nanostructure on the surface of the material; it enhances the ability of protein adsorption, the response of osteogenic, fibroblast, and epithelial cells; it has good biological activity and biocompatibility, Can promote cell adhesion, proliferation and differentiation.

Description

Raw material composition, polyether ether ketone based composite material, preparation method and application

Technical Field

The invention relates to a raw material composition, a polyether-ether-ketone-based composite material, a preparation method and application.

Background

Polyetheretherketone (PEEK) is a special plastic material, has excellent properties of high temperature resistance, corrosion resistance, fatigue resistance, irradiation resistance, self-flame retardance, self-lubrication and the like, is expected to replace traditional materials such as metal, ceramics and the like in a plurality of fields such as machinery, petrifaction, nuclear power, rail transit, electronics, medical treatment and the like, and becomes one of the most popular special plastic materials at present. PEEK has been frequently used in the direction of dental implants, joint rivets, maxillofacial reconstruction, and interbody fusion cages.

However, years of research have also found that the hydrophobic, hydrophobin of PEEK, the inherent bioinert properties of which impair early bone regeneration and osseointegration at the bone implant interface, may even have an adverse effect on long-term implantation. Therefore, improving the biological properties of PEEK implants (e.g., promoting osteogenesis and osteointegration) is becoming a research focus for their applications.

Blending modification is often used to combine the advantages of different materials, and the application of organic-inorganic composite materials as implants in orthopedics has been widely studied in recent decades. For the biological modification of polyetheretherketone, the most common method is blending modification, which enhances the osteogenic activity by incorporating bioactive components.

In the prior art, polyether-ether-ketone and niobate are blended to prepare the composite material. For example, chinese patent application CN201910498305.2 discloses a polyetheretherketone/niobate composite material and a preparation method thereof, which is obtained by blending polyetheretherketone and niobate, and then sintering at a high temperature or melting and co-extruding to sinter the composite material to form a biomedical material. Chinese patent application No. CN201910544002.X discloses an antibacterial piezoelectric polyetheretherketone composite material and a preparation method thereof, wherein polyetheretherketone and niobate are dried and then mixed, a polyetheretherketone/niobate composite fiber material is prepared by a molding method at 350-450 ℃, the surface of the composite fiber material is treated, and then the composite fiber material is soaked in a solvent containing antibacterial drugs for loading, and then the composite fiber material is dried in vacuum. However, the blending modification uses niobate which has a certain degradation in the implant, and the degraded product and metabolic condition are subject to further experimental investigation, so that uncertainty and risk exist.

Niobium (Nb) is used as a metal material, has higher elastic modulus (80GPa), and has the advantages of wear resistance, corrosion resistance and higher strength. The traditional biomedical metal materials mainly comprise Ti, titanium alloy, tantalum and the like, the research on Nb is very little, the research on Nb mainly focuses on adding a small amount of Nb into the titanium alloy, and the research on titanium/niobium alloy (which can be doped with other elements and is not always Nb) is carried out. Nb has the defects of higher density and over high elastic modulus (80GPa) compared with the human autologous bone and is not matched with bone tissues, so that the stress shielding phenomenon is generated when the Nb is implanted into a human body, and the bone absorption is caused. And the nanoscale Nb powder is easy to self-agglomerate and difficult to be uniformly mixed with other powder materials, so that the application of the Nb powder in the field of biomedicine is limited.

Disclosure of Invention

The invention aims to solve the technical problem that niobate is used for blending modification of PEEK in the prior art, the niobate has certain degradation uncertainty and risk in an implant, or is not matched with bone tissues, the stress shielding phenomenon is generated in the implant, bone absorption is caused, or niobium powder is easy to agglomerate, and the defect that the niobate is easy to absorb and agglomerate in comparison with bone tissues in the prior art is overcomeIs difficult to be uniformly mixed with other powder materials, limits the defect of application of the powder materials in the biomedical field, and provides a raw material composition, a polyether-ether-ketone-based composite material, a preparation method and application. According to the invention, a nano structure is constructed on the surface of the material and the surface performance is improved by doping Nb into PEEK, so that the cell response is enhanced. And a nano-wire structure can be constructed on the surface of the material in an alkali liquor etching mode, the surface performance is improved, and the cell response is further enhanced. Can also mildly complete the functional element Ca²⁺And the nano-filament structure is reserved for loading functional small molecules.

In view of the problems in the background art, through continuous research and experiments of the inventor, the inventor finds that the niobium powder and the PEEK powder are compounded, so that the defects of over-high niobium elastic modulus and over-high density can be effectively overcome, and the defects of PEEK in the aspects of insufficient bioactivity, poor osteogenic performance, incapability of promoting new bone regeneration, formation of osseointegration and the like can be overcome. When the inventor compounds niobium powder and PEEK powder, the following problems are mainly overcome: 1) in the prior art, the research and report of bioactive materials mainly focus on bioactive fillers such as bioglass, bioceramic and the like, and because the nano niobium powder has poor dispersion uniformity and is easy to agglomerate, the niobium powder is not involved at all. 2) The niobium powder dosage is as follows: if the volume fraction of the niobium powder in the composite material is too small, on one hand, the niobium powder cannot be well and uniformly mixed, and on the other hand, the composite material has poor bone-promoting effect; if the volume fraction of the niobium powder in the composite material is too large, the niobium powder is poor in dispersion uniformity and easy to agglomerate, at the moment, the niobium powder in the composite material cannot be uniformly dispersed, and the mechanical property of the composite material cannot reach the standard of human bones. 3) Niobium powder particle size: if the niobium powder has an excessively large particle diameter, it cannot be uniformly dispersed in PEEK, and if the powder has an excessively small particle diameter, it is easily agglomerated in the process of forming, particularly in the process of sintering, and therefore, it is difficult to obtain a niobium powder having a good osteogenic property.

The inventor pays creative work to find that under the condition that the dosage ratio of the niobium powder to the PEEK powder is 1: 4-2: 3, and the particle size of the niobium powder is 10 nm-120 nm, the niobium powder has important influence on the surface nanostructure of the composite material and the improvement of the surface performance and cells, so that the composite material has the advantages that the overall strength is matched with human bones, the composite material has good bioactivity, stress shielding is not generated, and the bone performance and the mechanical performance are promoted.

The invention solves the technical problems through the following technical scheme.

The invention provides a raw material composition, which comprises the following components: niobium (Nb) powder and polyether ether ketone (PEEK) powder, wherein the volume ratio of the Nb powder to the PEEK powder is 1: 4-2: 3, and the particle size of the Nb powder is 10-120 nm.

In the present invention, the particle size of the polyether ether ketone powder is preferably 10 to 40 μm.

In the present invention, the polyether ether ketone (PEEK) generally refers to a high polymer composed of a repeating unit having a main chain structure containing one ketone bond and two ether bonds. Preferably, the polyetheretherketone has a melting point of 330 to 340 ℃, a glass transition temperature of 140 to 150 ℃, and a density of 1.0 to 1.5g/cm³The polymerization degree is 150-250, and the molecular weight is 40000-60000; the melting point of the polyether-ether-ketone is 334 ℃, the glass transition temperature is 143 ℃, and the density is 1.30g/cm ³200 and 50000 molecular weight, for example type 450G polyetheretherketone (available from VICTREX, uk).

In the present invention, the niobium powder preferably has a particle size of 20nm to 100 nm.

In the present invention, the volume ratio of the niobium powder to the polyetheretherketone powder is preferably 20:80, 30:70, 40:60, or 50: 50. More preferably, the niobium powder is 20 to 50% by volume, the polyetheretherketone powder is 50 to 80% by volume, for example, the niobium powder is 40% by volume, and the polyetheretherketone powder is 60% by volume.

In the present invention, the raw material composition may be prepared by a conventional method in the art, and the niobium powder and the polyetheretherketone powder are generally mixed uniformly. The mixing operation is preferably carried out in a high-speed mixer. The mixing time is preferably 48-55 h, for example 48 h. The rotation speed during mixing is preferably 800-1200 rpm, for example 1000 rpm.

The invention provides a preparation method of a PEEK-based composite material, which comprises the following steps: and (3) carrying out cold pressing, sintering and molding on the raw material composition.

The invention can only be prepared by a cold pressing sintering forming method, and if the hot pressing forming method is adopted, bubbles are more easily generated in the material and remain in the material, thus having adverse effect on the overall mechanical property of the material. If the melt blending molding is adopted, the process is relatively more complicated.

In the present invention, the operation and conditions of the cold press sintering may be conventional in the art. Generally comprising the steps of: and (3) pressing and forming the raw material composition, then heating, and sintering and forming.

The compression molding operation and conditions may be conventional in the art, and are typically performed in a powder tablet press at ambient temperature conditions.

The dimensions of the press-formed sample may be conventional in the art, for example a sheet cylinder, Φ 11 × 2 mm.

The sintering and forming are generally carried out in a muffle furnace, and the temperature rise speed of the muffle furnace is preferably 0.5-2 ℃/min, for example 1-2 ℃/min.

The sintering forming temperature is preferably 340-380 ℃, for example 350 ℃. If the sintering temperature is lower than 340 ℃, the obtained composite material has low strength and cannot be sintered into blocks. If the sintering temperature is higher than 380 ℃, the PEEK in the obtained composite material is easy to break and degrade molecular chains.

The time for the sintering and forming is preferably 2h to 5h, for example 3 h. If the sintering molding time is less than 2 hours, the strength of the obtained composite material is low, and the composite material cannot be sintered into blocks. If the sintering time is more than 5 hours, the obtained composite material is easy to deform.

In the present invention, the PEEK-based composite material is preferably subjected to an immersion treatment in an alkali solution. The impregnation treatment is preferably carried out by the following steps: soaking the PEEK-based composite material in alkali liquor at the temperature of 55-65 ℃; the alkali liquor is NaOH solution.

In the prior art, surface treatment is mainly carried out on various metals (such as titanium, tantalum and niobium) by using an alkali solution, an amorphous structure layer is constructed on the surface, and the biological performance of the material is improved under the condition of not changing the property of a matrix. At present, the surface treatment of the composite material surface by using NaOH solution is not performed. After dipping treatment in alkali liquor, the surface of the composite material constructs a nano-wire structure; the alkali liquor concentration is different, and the appearance of the material surface is different. The nano-filament structure is constructed on the surface of the material in an alkali liquor etching mode, the surface performance is improved, and further the cell response is enhanced.

Wherein, the impregnation is generally static impregnation.

Wherein the soaking time is preferably 22-26 h, such as 24 h.

Wherein the impregnation temperature is preferably 58 to 62 ℃, for example 60 ℃.

The concentration of the NaOH solution is preferably 0.1mol/L to 1mol/L, for example, 0.2mol/L, 0.3mol/L, 0.5mol/L, 0.7mol/L, 0.8mol/L, or 0.9 mol/L.

If the time of the dipping treatment is not within the range of 22-26 h, the temperature is not within 58-62 ℃, and the type and concentration of the alkali liquor are not within 0.1-1 mol/L, the surface appearance of the obtained product is changed, the nano-filaments with uniform surfaces cannot be obtained, and the excellent effects of promoting cell proliferation and differentiation cannot be realized.

Wherein, after the impregnation, the obtained product is generally washed and dried.

Preferably, the alkali treated PEEK-based composite material is subjected to the following treatment: in the presence of Ca⁺²Dipping in the solution to obtain the Ca-PEEK-based composite material.

Wherein the Ca is contained⁺²The solution may be conventional in the art and is capable of providing Ca⁺²Preferably CaCl₂And (3) solution.

Wherein the Ca is contained⁺²The concentration of the solution is preferably 0.1 to 1mol/L, for example 0.2mol/L, 0.3mol/L, 0.5mol/L, 0.7mol/L, 0.8mol/L or 0.9 mol/L.

Wherein, the impregnation is generally static impregnation.

Wherein the soaking time is preferably 22-26 h, such as 24 h.

If the time of the dipping treatment is not in the range of 22 to 26 hours, the temperature is not in the range of 58 to 62 ℃, and the Ca content is⁺²The solution and the Ca with the concentration of 0.1mol/L to 1mol/L are grafted on the surface of the composite material²⁺Has a great influence on the content of Ca, and cannot complete the functional element Ca²⁺And the nano-filament structure is reserved for loading functional small molecules. For example, the surface appearance of the obtained product is changed, the nano-filaments with uniform surfaces cannot be obtained, and excellent effects of promoting cell proliferation and differentiation and the like cannot be realized.

Further preferably, the surface of the Ca-PEEK-based composite material is loaded with antibacterial and anti-inflammatory molecules. The content of the loaded antibacterial and anti-inflammatory molecules has obvious influence on the antibacterial performance and the anti-inflammatory performance of the composite material.

The antibacterial and anti-inflammatory molecules can be small molecules which are conventional in the field and can simultaneously realize antibacterial and anti-inflammatory functions, such as Ber molecules (Berberine, Berberine, for short Ber).

The loading operation and conditions can be conventional in the art, and for example, the loading can be performed by a method of dropping a solution containing the antibacterial and anti-inflammatory molecules in portions and then drying.

When the anti-bacterial and anti-inflammatory molecule is Ber, the solution of the anti-bacterial and anti-inflammatory molecule may be a Ber-ethanol solution.

The loading amount of the Ca-PEEK-based composite material surface can be conventional in the field, for example, the loading amount can be selected according to the concentration and release rate of the drug which plays the role of antibiosis and anti-inflammation, and the required concentration and release rate of the drug determine the loading amount of the drug.

In a preferred embodiment, when the anti-bacterial and anti-inflammatory molecule is Ber, the Ber loading may be 200 μ g/379mm²The PEEK-based composite material.

The invention also provides a composite material prepared by the preparation method.

The invention also provides an application of the composite material in bone repair.

Wherein, the bone repair body is preferably a spine bone repair body or a dental implant. The spine bone prosthesis is also called an interbody fusion cage and comprises a cervical interbody fusion cage and a thoracic/lumbar interbody fusion cage.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

1. in the PEEK-based composite material, the niobium powder and the PEEK powder are compounded, so that the defects of overhigh niobium elastic modulus and overlarge density can be effectively overcome, the composite material has better roughness and hydrophilicity, and a nano structure is constructed on the surface of the material; these improved surface characteristics enhance the protein adsorption capacity of the material and, in this way, the ligament enhances the osteogenic, fibroblastic, epithelial cell response; has good bioactivity and biocompatibility, and can promote cell adhesion, proliferation and differentiation.

In a preferred embodiment, PNC40 roughness 2.46 μm, water contact angle 81.75 °, protein adsorption rate 25.41% (BSA) and 21.49% (Fn), all three cellular responses were optimized.

2. After the surface of the PEEK-based composite material is modified by low-concentration alkali liquor, an amorphous Na-Nb-O nanowire structure can be constructed on the surface of the material (in a preferred embodiment, the surface of 0.2Na-PNC40 is partially covered by an amorphous Na-Nb-O nanowire layer, and the surface of 0.5Na-PNC40 is almost completely covered by an amorphous Na-Nb-O nanowire layer). The appearance of the amorphous Na-Nb-O nano-wire improves the roughness and the hydrophilicity of the material (the wider the nano-wire covering, the more obvious the improvement), does not change the pH value of the surrounding environment greatly, enhances the protein adsorption capacity of the material, and enhances the response of osteogenesis, fibroblasts and epithelial cells for the ligament (the wider the nano-wire covering, the better the response).

Combining all factors, in a preferred embodiment, the roughness of 0.5Na-PNC40 is 3.27 μm, the water contact angle is 41.75 degrees, the protein adsorption rate is 38.78% (BSA) and 37.15% (Fn), and all three cell responses are optimal, so that the method has great application potential in the field of percutaneous bone implantation.

3. In a preferred embodiment, CaCl is used₂When the solution is used for further modifying the material with the amorphous Na-Nb-O nano-wire structure, the functional element Ca is completed²⁺For Na⁺Then, the natural antibacterial and anti-inflammatory small-molecule berberine (Ber) is loaded by utilizing the loose and porous appearance of the surface of the material. Through CaCl₂After the solution treatment, the amorphous Na-Nb-O nanowire layer on the surface of the material is converted into an amorphous Ca-Nb-O nanowire layer, the appearance and the roughness are not changed, the hydrophilicity is reduced slightly, but the Ca²⁺The protein adsorption capacity is greatly improved due to the existence of the protein, so that the responses of osteogenesis, fibriform and epithelial cells are also greatly enhanced; after the Ber is further loaded, the shape, roughness, hydrophilicity and protein adsorption capacity of the material are not obviously changed compared with those of the material without the Ber, the responses of osteogenesis, fibroblasts and epithelial cells are similar to those of Ca-PNC40, and meanwhile, the anti-inflammatory and antibacterial effects are obviously improved.

Combining all factors, in a preferred embodiment, the roughness of Ca-PNC40@ Ber is 3.29 mu m, the water contact angle is 44.75 degrees, the protein adsorption rate is 51.42 percent (BSA) and 48.74 percent (Fn), the three cell responses are optimal, and the cell responses have anti-inflammatory and antibacterial functions, and have great application potential in the field of percutaneous bone implantation.

Drawings

FIG. 1 is an XRD pattern of PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC 40.

FIG. 2 is EDS spectra of PNC40 (FIG. 2a),0.2Na-PNC40 (FIG. 2b),0.5Na-PNC40 (FIG. 2c) and 5.0Na-PNC40 FIG. 2 d).

FIG. 3 is SEM images of PNC40 (FIG. 3a, FIG. 3e),0.2Na-PNC40 (FIG. 3b, FIG. 3f),0.5Na-PNC40 (FIG. 3c, FIG. 3g) and 5.0Na-PNC40 (FIG. 3d, FIG. 3 h). Wherein in FIG. 3g, the top right corner is an SEM image of the same material at a larger magnification.

FIG. 4 shows TMM images and roughness values for PNC40 (FIG. 4a),0.2Na-PNC40 (FIG. 4b),0.5Na-PNC40 (FIG. 4c) and 5.0Na-PNC40 (FIG. 4 d).

FIG. 5 shows water contact angles for PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 ([ p ] p <0.05, vs PNC 40).

FIG. 6 is a graph showing the change in pH of SBF after immersion in SBF of PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC 40.

Figure 7 shows the results of protein adsorption tests on the surface of PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 (. p <0.05, vs PNC 40).

FIG. 8 is an SEM image of BMSCs surface cultured on PNC40 (FIG. 8a, FIG. 8e),0.2Na-PNC40 (FIG. 8b, FIG. 8f),0.5Na-PNC40 (FIG. 8c, FIG. 8g) and 5.0Na-PNC40 (FIG. 8d, FIG. 8h) for 3d (FIG. 8a, FIG. 8b, FIG. 8c, FIG. 8d) and 7d (FIG. 8e, FIG. 8f, FIG. 8g, FIG. 8 h).

FIG. 9 is a CLSM image of BMSCs cultured on the surface of 1d (FIG. 9a, FIG. 9b, FIG. 9c, FIG. 9g, FIG. 9d),3d (FIG. 9e, FIG. 9f, FIG. 9g, FIG. 9h), and 7d (FIG. 9i, FIG. 9j, FIG. 9k, FIG. 9l) at PNC40 (FIG. 9a, FIG. 9e, FIG. 9i),0.2Na-PNC40 (FIG. 9b, FIG. 9f, FIG. 9j),0.5Na-PNC40 (FIG. 9c, FIG. 9g, FIG. 9k), and 5.0Na-PNC40 (FIG. 9d, FIG. 9h, FIG. 9 l).

FIG. 10a is a graph of the relative adhesion of BMSCs cultured for 6,12 and 24h on PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC 40; FIG. 10b is a graph of the OD of BMSCs cultured on samples for 1,3, and 7 d; fig. 10c shows ALP activity when BMSCs were cultured on

samples

7,10 and 14d (ip <0.05, vs PNC 40).

FIG. 11 is an SEM image of L929 surface cultured 3d (FIG. 11a, FIG. 11b, FIG. 11c, FIG. 11d) and 7d (FIG. 11e, FIG. 11f, FIG. 11g, FIG. 11h) at PNC40 (FIG. 11a, FIG. 11e),0.2Na-PNC40 (FIG. 11b, FIG. 11f),0.5Na-PNC40 (FIG. 11c, FIG. 11g) and 5.0Na-PNC40 (FIG. 11d, FIG. 11 h).

FIG. 12 is a CLSM image of L929 surface cultured on PNC40 (FIG. 12a, FIG. 12e),0.2Na-PNC40 (FIG. 12b, FIG. 12f),0.5Na-PNC40 (FIG. 12c, FIG. 12g) and 5.0Na-PNC40 (FIG. 12d, FIG. 12h) for 1d (FIG. 12a, FIG. 12b, FIG. 12c, FIG. 12d) and 3d (FIG. 12e, FIG. 12f, FIG. 12g, FIG. 12 h).

FIG. 13a is the relative adhesion rate of L929 when cultured on PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 for 6,12 and 24 h. FIG. 13b is OD values of L929 when cultured on PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 for 1,3 and 7d (. p <0.05, vs PNC 40).

FIG. 14 is SEM images of Hacat surface cultured on PNC40 (FIG. 14a, FIG. 14e),0.2Na-PNC40 (FIG. 14b, FIG. 14f),0.5Na-PNC40 (FIG. 14c, FIG. 14g) and 5.0Na-PNC40 (FIG. 14d, FIG. 14h) for 3d (FIG. 14a, FIG. 14b, FIG. 14c, FIG. 14d) and 7d (FIG. 14e, FIG. 14f, FIG. 14g, FIG. 14 h).

FIG. 15 is a CLSM image of Hacat on the surface of PNC40 (FIG. 15a, FIG. 15e, FIG. 15i),0.2Na-PNC40 (FIG. 15b, FIG. 15f, FIG. 15j),0.5Na-PNC40 (FIG. 15c, FIG. 15g, FIG. 15k) and 5.0Na-PNC40 (FIG. 15d, FIG. 15h, FIG. 15l) for 1d (FIG. 15a, FIG. 15b, FIG. 15c, FIG. 15d),3d (FIG. 15e, FIG. 15f, FIG. 15g, FIG. 15h) and 7d (FIG. 15i, FIG. 15j, FIG. 15k, FIG. 15 l).

FIG. 16a shows the relative adhesion rates of Hacat when cultured for 6,12 and 24h on PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC 40. FIG. 16b shows the OD values of Hacat when cultured on PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 for 1,3 and 7d (. p <0.05, vs PNC 40).

FIG. 17 is an XRD pattern of PNC40, Na-PNC40 and Ca-PNC 40.

FIG. 18 is EDS maps of PNC40 (FIG. 18a), Na-PNC40 (FIG. 18b), and Ca-PNC40 (FIG. 18 c).

FIG. 19 is an SEM image of PNC40 (FIG. 19a, FIG. 19d, FIG. 19g), Na-PNC40 (FIG. 19b, FIG. 19e, FIG. 19h) and Ca-PNC40 (FIG. 19c, FIG. 19f, FIG. 19 i). Wherein in FIG. 19h, the top right corner is an SEM image of the same material at a larger magnification. In fig. 19i, the top right corner is an SEM image of the same material at a larger magnification.

FIG. 20a is the UV absorption curve of Ber. FIG. 20b is the measured Ber standard curve. FIG. 20c is a plot of the Ber release profile of @ Ca-PNC40@ Ber in PBS.

FIG. 21 shows TMM images and roughness values for the surfaces of PNC40 (FIG. 21a), Na-PNC40 (FIG. 21b), Ca-PNC40 (FIG. 21c) and Ca-PNC40@ Ber (FIG. 21 d).

FIG. 22 shows the results of water contact angle measurements for PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber surfaces ([ p ] 0.05, vs PNC 40).

FIG. 23 is a graph showing the change in pH of SBF after immersion in SBF of PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber.

FIG. 24 shows the results of protein adsorption assays on the surfaces of PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber ([ p ] 0.05, vs PNC 40).

FIG. 25 is an SEM image of BMSCs cultured on the surface of PNC40 (FIG. 25a, FIG. 25e), Na-PNC40 (FIG. 25b, FIG. 25f), Ca-PNC40 (FIG. 25c, FIG. 25g) and Ca-PNC40@ Ber (FIG. 25d, FIG. 25h) for 1d (FIG. 25a, FIG. 25b, FIG. 25c, FIG. 25d) and 5d (FIG. 25e, FIG. 25f, FIG. 25g, FIG. 25 h).

FIG. 26 is a CLSM image of BMSCs cultured on the surface of 1d (FIG. 26a, FIG. 26b, FIG. 26c, FIG. 26h, FIG. 26d),3d (FIG. 26e, FIG. 26f, FIG. 26g, FIG. 26h) and 5d (FIG. 26i, FIG. 26j, FIG. 26k, FIG. 26l) at PNC40 (FIG. 26a, FIG. 26e, FIG. 26i), Na-PNC40 (FIG. 26b, FIG. 26f, FIG. 26j), Ca-PNC40 (FIG. 26c, FIG. 26g, FIG. 26k) and Ca-PNC40@ Ber (FIG. 26d, FIG. 26h, FIG. 26 l).

FIG. 27a is a graph showing the relative adhesion rates of BMSCs cultured for 6,12 and 24h on PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber; FIG. 27b is a graph of the OD of BMSCs cultured on samples for 1,3, and 5 d; fig. 27c shows ALP activity when BMSCs were cultured on

samples

7,10 and 14d (ip <0.05, vs PNC 40).

FIG. 28 is an SEM image of L929 cultured on the surface of PNC40 (FIG. 28a, FIG. 28e), Na-PNC40 (FIG. 28b, FIG. 28f), Ca-PNC40 (FIG. 28c, FIG. 28g) and Ca-PNC40@ Ber (FIG. 28d, FIG. 28h) for 3d (FIG. 28a, FIG. 28b, FIG. 28c, FIG. 28d) and 5d (FIG. 28e, FIG. 28f, FIG. 28g, FIG. 28 h).

FIG. 29 is a CLSM image of L929 cultured on the surface of 1d (FIG. 29a, FIG. 29b, FIG. 29c, FIG. 29d) and 3d (FIG. 29e, FIG. 29f, FIG. 29g, FIG. 29h) in PNC40 (FIG. 29a, FIG. 29e), Na-PNC40 (FIG. 29b, FIG. 29f), Ca-PNC40 (FIG. 29c, FIG. 29g) and Ca-PNC40@ Ber (FIG. 29d, FIG. 29 h).

FIG. 30a is a graph of the relative adhesion rates of L929 when cultured on PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber for 6,12 and 24 h. FIG. 30b is the OD of L929 when cultured on PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber for 1,3 and 5d (. p <0.05, vs PNC 40).

FIG. 31 is an SEM image of Hacat surface cultured on PNC40 (FIG. 31a, FIG. 31e), Na-PNC40 (FIG. 31b, FIG. 31f), Ca-PNC40 (FIG. 31c, FIG. 31g) and Ca-PNC40@ Ber (FIG. 31d, FIG. 31h) for 3d (FIG. 31a, FIG. 31b, FIG. 31c, FIG. 31d) and 5d (FIG. 31e, FIG. 31f, FIG. 31g, FIG. 31 h).

FIG. 32 is a CLSM image of Hacat on the surface of PNC40 (FIG. 32a, FIG. 32e, FIG. 32i), Na-PNC40 (FIG. 32b, FIG. 32f, FIG. 32j), Ca-PNC40 (FIG. 32c, FIG. 32g, FIG. 32k) and Ca-PNC40@ Ber (FIG. 32d, FIG. 32h, FIG. 32l) for 1d (FIG. 32a, FIG. 32b, FIG. 32c, FIG. 32d),3d (FIG. 32e, FIG. 32f, FIG. 32g, FIG. 32h) and 5d (FIG. 32i, FIG. 32j, FIG. 32k, FIG. 32 l).

FIG. 33a is a graph of the relative adhesion of Hacat when cultured for 6,12 and 24h on PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber; FIG. 33b is the OD of Hacat when cultured on samples for 1,3 and 5 d; FIG. 33c is a western blot of cohesin proteins by Hacat when cultured on samples for 5d (. p <0.05, vs PNC 40).

FIG. 34a shows inflammatory factor expression after RAW 264.7 culture on PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber. FIG. 34b is a western blot of inflammatory proteins after incubation of RAW 264.7 on PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber ([ p ] <0.05, vs PNC 40).

FIGS. 35 a-35 h are photographs of colonies of E.coli and S.aureus isolated from the surface of PNC40 (FIG. 35a, FIG. 35e), Na-PNC40 (FIG. 35b, FIG. 35f), Ca-PNC40 (FIG. 35c, FIG. 35g), Ca-PNC40@ Ber (FIG. 35d, FIG. 35h) grown on agar for 24 h; bacterial inhibition of e.coli (i) with s.aureus (j) (. p <0.05, vs PNC 40).

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Polyetheretherketone (PEEK) powder is available from VICTREX, UK under the model number 450G and has a particle size of 10-40 μm.

Niobium powder is available from nano technology ltd, nano europe, china, with a particle size of 20-100 nm.

Example 1

The preparation method of PNC40 comprises the following steps:

the raw material composition comprises: niobium powder (with the particle size of 20 nm-100 nm) and polyether ether ketone (PEEK) powder (10-40 mu m), wherein the consumption of the niobium powder is 40vt percent, the consumption of the PEEK powder is 60vt percent, and the percentages are volume percentages of the components respectively accounting for the total volume of the niobium powder and the PEEK powder.

And mixing the niobium powder and the PEEK powder in a high-speed mixer at the rotating speed of 1000rpm for 48 hours, and uniformly mixing to obtain the raw material composition.

The raw material composition is firstly pressed and formed in a powder tablet machine (a flaky cylinder with the size phi of 11 multiplied by 2mm), then heated in a muffle furnace and sintered and formed. Wherein the temperature rise speed of the muffle furnace is 1 ℃/min. The temperature for sintering and molding is 350 ℃. The sintering and molding time is 3 h.

The PNC40 surface modification method comprises the following steps:

PNC40 samples were immersed in 10mL NaOH solutions (detailed in Table 1) of different concentrations for 24h at 60 deg.C; and taking out, cleaning and drying to obtain the sample. The parameters and abbreviations in the PNC40 surface modification method are shown in Table 1.

TABLE 1 PNC40 surface modification Process parameters and abbreviations

Example 2

Ca²⁺And preparation of a Ber supporting material:

s1, Na-PNC40 (i.e., 0.5Na-PNC40 in example 1) sample was placed in 10mL CaCl at 60 deg.C₂The solution (concentration: 0.2mol/L) is put for 24 hours; and taking out, cleaning and drying to obtain the Ca-PNC40 sample.

S2, preparing a Ber-ethanol solution with the concentration of 200 mug/mL, dripping 1mL of liquid medicine on the surface of the Ca-PNC40 sample in batches, and drying to obtain the Ca-PNC40@ Ber sample.

The surface modification process parameters and abbreviations of the samples are shown in table 2.

TABLE 2 PNC40 surface modification Process parameters and abbreviations

EXAMPLES example 1 characterization of the physical and chemical Properties of the samples

XRD analysis (detection instrument: D/MAX 2550VB/PC, Rigaku Co., Japan)

The chemical compositions and crystal structures of PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 were analyzed by XRD (range: 10-80 ℃).

FIG. 1 is an XRD pattern of PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC 40. As can be seen from the figure: after being treated by 0.2M NaOH solution, the peak intensity of the material at the corresponding position is obviously reduced compared with that of the material without treatment, which shows that a layer of amorphous substance is generated on the surface of the material by the treatment of the 0.2M NaOH solution; after being treated by 0.5M NaOH solution, the peak intensity of the material at the corresponding position is more obviously reduced than that of 0.2Na-PNC40, which shows that a layer of amorphous substance is generated on the surface of the material by the treatment of 0.5M NaOH solution, and the material is thicker or has wider coverage than that of 0.2Na-PNC 40; ③ after being treated by 5.0M NaOH solution, a plurality of new characteristic peaks appear compared with the untreated one, which shows that a layer of high-crystallinity substance is generated on the surface of the material by the treatment of 5.0M NaOH solution.

SEM and EDS analysis (detection apparatus: S-4800, Hitachi, Japan)

The surfaces of PNC40,0.2Na-PNC40,0.5Na-PNC40, 5.0Na-PNC40 were observed using SEM, and elemental analysis was performed on the surfaces of the samples using EDS.

FIG. 2 is EDS spectra of PNC40 (FIG. 2a),0.2Na-PNC40 (FIG. 2b),0.5Na-PNC40 (FIG. 2c) and 5.0Na-PNC40 FIG. 2 d). The results show that Na elements appear on the surface of the material after the material is treated by NaOH solutions with different concentrations; and along with the increase of the concentration of the NaOH solution, the content of Na and O elements on the surface of the material is increased.

FIG. 3 is SEM images of PNC40 (FIG. 3a, FIG. 3e),0.2Na-PNC40 (FIG. 3b, FIG. 3f),0.5Na-PNC40 (FIG. 3c, FIG. 3g) and 5.0Na-PNC40 (FIG. 3d, FIG. 3 h). Wherein in FIG. 3g, the top right corner is an SEM image of the same material at a larger magnification. As can be seen from the figure: firstly, after being treated by 0.2M NaOH solution, part of Nb particles are ablated and become a plurality of nano-wires; secondly, after being treated by 0.5M NaOH solution, almost all Nb particles are converted into nanowires to cover the surface of the material, and the diameter of the nanowires is about 50nm and is uniform; thirdly, the nano-filaments are transformed into irregular blocky crystals after being treated by 5.0M NaOH solution.

3. Roughness analysis

The roughness of the surface of PNC40,0.2Na-PNC40,0.5Na-PNC40, 5.0Na-PNC40 was measured by using a three-dimensional topography measuring microscope (inspection apparatus: Infine Focus G4, Alicona, Austria).

FIG. 4 shows TMM images and roughness values for PNC40 (FIG. 4a),0.2Na-PNC40 (FIG. 4b),0.5Na-PNC40 (FIG. 4c) and 5.0Na-PNC40 (FIG. 4 d). As can be seen from the figure: roughness of PNC40 is 2.48 μm; ② after being treated by 0.2M and 0.5M NaOH solution, the surface roughness of the material is slightly improved (0.2Na-PNC 40: 2.91 μ M; 0.5Na-PNC 40: 3.27 μ M); ③ after 5.0M NaOH treatment, the surface roughness of the material is greatly improved (5.0Na-PNC 40: 7.43 μ M).

4. Hydrophilicity assays

Surface water contact angles were measured for PNC40,0.2Na-PNC40,0.5Na-PNC40, 5.0Na-PNC40 using a contact angle measuring instrument (detection instrument: XG-CAMB3, Shanghai Bitsuan Kogyo Co., Ltd.). When the contact angle was measured, the volume of the liquid was 10. mu.L.

FIG. 5 shows water contact angles for PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 ([ p ] p <0.05, vs PNC 40). The data obtained show that the hydrophilicity of the material was improved by NaOH treatment, and the higher the NaOH concentration in the treatment, the better the hydrophilicity of the material (PNC 40: 81.25 ℃; 0.2Na-PNC 40: 62.25 ℃; 0.5Na-PNC 40: 41.75 ℃; 5.0Na-PNC 40: 32.00 ℃).

5. In vitro immersion experiment

PNC40,0.2Na-PNC40,0.5Na-PNC40, and 5.0Na-PNC40 were each soaked in 10mL of a simulated body fluid (SBF, Legene, China) and placed in a constant temperature shaking chamber. The pH of the SBF was measured at 1,3, 5, 7,10 and 14d, respectively.

FIG. 6 is a graph showing the change in pH of SBF after immersion in SBF of PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC 40. The data obtained show that: PNC40 can not cause the change of the pH value of the soak solution; ② after being processed by 0.2M and 0.5M NaOH solutions, the material can cause the pH of the soak solution to rise slightly, the pH of the soak solutions of 0.2Na-PNC40 and 0.5Na-PNC40 is 7.71 and 7.89 respectively at 14 days; ③ after being treated by 5.0M NaOH solution, the pH of the soaking solution is greatly increased by the material, and the pH of the soaking solution of 5.0Na-PNC40 is 11.05 at 14 days.

6. Protein adsorption assay

Protein adsorption capacity analysis was performed on the surfaces of PNC40,0.2Na-PNC40,0.5Na-PNC40, and 5.0Na-PNC40 using BSA and Fn as model proteins.

Protein adsorption capacity analysis was performed on the surfaces of PNC40,0.2Na-PNC40,0.5Na-PNC40, and 5.0Na-PNC40 using bovine serum albumin (BSA, Solarbio, China) and fibronectin (Fn, Solarbio, China) as model proteins. Specifically, a BSA solution (100. mu.g/mL) and an Fn solution (25. mu.g/mL) were prepared using a phosphate buffer solution (PBS, China TBD Science). The sample was placed in 2mL of protein solution and left at 37 ℃ for 240 min. Then, it was gently rinsed twice with PBS to wash away unbound proteins, followed by incubation in 2mL of a 2% sodium dodecyl sulfate solution to obtain a supernatant containing dissociated proteins, the protein concentration of which was determined by BCA kit (Beyotime Biotechnology, china) and analyzed at 562nm wavelength with a plate reader (DMN-9602G, puran, china). The protein adsorption rate was calculated according to the following formula:

BSA adsorption ratio＝BSA concentration÷100μg/mL

Fn adsorption ratio＝Fn concentration÷25μg/mL

figure 7 shows the results of protein adsorption tests on the surface of PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 (. p <0.05, vs PNC 40). As can be seen from the figure: the protein adsorption rate of PNC40 is BSA: 25.41% and Fn: 21.49 percent; and secondly, after the materials are treated by 0.2M and 0.5M NaOH solutions, the protein adsorption capacity of the materials is improved, wherein the protein adsorption rate of 0.2Na-PNC40 is BSA: 30.86% and Fn: 29.45%, the protein adsorption rate of 0.5Na-PNC40 was BSA: 38.78% and Fn: 37.15 percent; thirdly, after being treated by 0.5M NaOH solution, the surface of the material has overhigh alkalinity, protein denaturation and white floccule in the solution, and the surface of the material has no protein adsorption capacity.

Effect example 2 example 1 sample in vitro osteoblast response

1. Cell culture

The osteoblasts were BMSC obtained from rat femoral bone marrow. The cells of 3-5 passages were seeded on the surface of the sterilized (ethylene oxide) material at a density of 5X 104 per well in a medium selected from alpha-MEM (Thermo Fisher Scientific, USA) containing serum and penicillin and streptomycin double antibody. Other culture conditions were as follows: humidity 100%, CO₂Content 5%, temperature 37 ℃.

2. Morphology of cells

After culturing BMSCs on the surfaces of PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 for different times, the shapes, adhesion states and quantities of the BMSCs on the surfaces of the materials are observed by using SEM (in 3 and 7d) and CLSM (in 1,3 and 7d), respectively.

After culturing BMSCs on the surfaces of PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 for 3d and 7d, the morphology, adhesion state and amount of BMSCs on the material surface were observed using SEM and confocal laser scanning microscope (detection instrument: CLSM, A1R, Nikon, Japan), respectively.

Specifically, the samples for SEM were prepared as follows: after washing the samples with PBS, they were fixed with 2.5% glutaraldehyde solution for 4h, then dehydrated sequentially with 10%, 30%, 50%, 70%, 90% and 100% strength ethanol; samples for CLSM were prepared as follows: after washing the samples with PBS, the samples were fixed with 2.5% glutaraldehyde solution for 4h, followed by staining with FITC-phaseolin (Beyotime Biotechnology, China) for 30min and DAPI (Beyotime Biotechnology, China) for 12min in sequence, and washing the excess dye with PBS after each staining.

FIG. 8 is an SEM image of BMSCs surface cultured on PNC40 (FIG. 8a, FIG. 8e),0.2Na-PNC40 (FIG. 8b, FIG. 8f),0.5Na-PNC40 (FIG. 8c, FIG. 8g) and 5.0Na-PNC40 (FIG. 8d, FIG. 8h) for 3d (FIG. 8a, FIG. 8b, FIG. 8c, FIG. 8d) and 7d (FIG. 8e, FIG. 8f, FIG. 8g, FIG. 8 h). At 3d, cells were present on the surface of PNC40,0.2Na-PNC40 and 0.5Na-PNC40, with the most cells on the surface of 0.5Na-PNC40 and the best spreading. At 7d, the cells had significant proliferation and growth on all three materials, especially 0.5Na-PNC 40. Furthermore, 5.0Na-PNC40 was devoid of cell growth at both time points and should be attributed to the excessive alkalinity brought about after surface crystal dissolution.

FIG. 9 is a CLSM image of BMSCs cultured on the surface of 1d (FIG. 9a, FIG. 9b, FIG. 9c, FIG. 9g, FIG. 9d),3d (FIG. 9e, FIG. 9f, FIG. 9g, FIG. 9h), and 7d (FIG. 9i, FIG. 9j, FIG. 9k, FIG. 9l) at PNC40 (FIG. 9a, FIG. 9e, FIG. 9i),0.2Na-PNC40 (FIG. 9b, FIG. 9f, FIG. 9j),0.5Na-PNC40 (FIG. 9c, FIG. 9g, FIG. 9k), and 5.0Na-PNC40 (FIG. 9d, FIG. 9h, FIG. 9 l). The number of cells on PNC40,0.2Na-PNC40 and 0.5Na-PNC40 all increased gradually with increasing days of culture. Meanwhile, the spreading and proliferation of cells on the surface of the material treated with 0.2M, 0.5M NaOH solution was improved compared to PNC40, with 0.5Na-PNC40 being the best. Furthermore, 5.0Na-PNC40 was devoid of cell growth at both time points and should be attributed to the excessive alkalinity brought about after surface crystal dissolution.

3. Cell adhesion and proliferation

The activity of BMSCs on the surfaces of PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 at different culture times was measured by the CCK-8 method, and 6 th, 12 th and 24 th hours were selected for evaluating cell adhesion, and 1 st, 3 rd and 7d were selected for evaluating cell proliferation.

Specifically, after incubation for the corresponding time, the samples were washed with PBS and transferred to a new 24-well plate, followed by incubation for 120min with 450 μ L of medium and 50 μ L of CCK-8 solution per well. Supernatants were taken in new 96-well plates and the corresponding OD values were read at 450nm using a microplate reader. The OD value of PNC40 surface cells at 6h was set as 100% for calculation of relative cell adhesion rate. Cell proliferation is directly expressed as the relative trend in OD values.

samples

7,10 and 14d (ip <0.05, vs PNC 40).

FIG. 10a shows the relative adhesion rates of BMSCs when cultured for 6,12 and 24h on PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC 40. The results show that: compared with PNC40, the relative adhesion rate of cells on the surface of a material treated by 0.2M and 0.5M NaOH solution is improved, wherein 0.5Na-PNC40 is optimal; ② 5.0Na-PNC40 surface has no cell adhesion.

FIG. 10b shows the OD values of BMSCs when cultured on PNC40,0.2Na-PNC40,0.5Na-PNC40, and 5.0Na-PNC40 for 1,3, and 7 d. The results show that: the OD values of the cells on the surfaces of 0.2Na-PNC40 and 0.5Na-PNC40 are obviously higher than that of PNC40 in all the culture time, wherein 0.5Na-PNC40 is the highest; ② 5.0Na-PNC40 surface has no cell proliferation. The specific data are shown in Table 3.

TABLE 3

4. Cell differentiation

The ability of PNC40,0.2Na-PNC40,0.5Na-PNC40, 5.0Na-PNC40 to promote osteogenic differentiation of BMSCs was evaluated by analyzing the activity of alkaline phosphatase (ALP) in cells.

Specifically, after

osteogenic induction culture

7,10, 14d, the medium was removed and 1% NP-40 (Beyotime Biotechnology, China) was added per well and lysed for 1h to give a lysate, and the total protein concentration was determined using the BCA kit. In addition, 50. mu.L of the lysate was incubated with 100. mu.L of ALP working solution (containing 1mmol/L of MgCl 2.6H2O and 0.1mol/L of α -aminoacetic acid, pH 9) for 30min, followed by reading the corresponding OD at 405nm using a microplate reader. ALP activity was expressed using an OD value of 405 nm/total protein content/min.

FIG. 10c is ALP activity of BMSCs cultured on PNC40,0.2Na-PNC40,0.5Na-PNC40, and 5.0Na-PNC40 for 7,10, and 14 d. The results show that: ALP activities of 0.2Na-PNC40 and 0.5Na-PNC40 are obviously higher than that of PNC40 in all culture times, wherein 0.5Na-PNC40 is the highest; ② 5.0Na-PNC40 surface without cell differentiation.

Effect example 3 example 1 sample in vitro fibroblast response

1. Cell culture

The fibroblast was selected from L929, purchased from the Chinese double denier IBS cell bank. The cells were seeded onto the surface of the sterilized (ethylene oxide) material at a density of 4X 104 cells per well in RPMI-1640 (Thermo Fisher Scientific, USA) containing serum and cyan and catenaDouble antibody. Other culture conditions were as follows: humidity 100%, CO₂Content 5%, temperature 37 ℃.

2. Morphology of cells

After L929 was cultured on the surfaces of PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 for different times, the morphology, adhesion state and amount of L929 on the material surface were observed by SEM (in 3 and 7d) and CLSM (in 1 and 3d), respectively.

The preparation method of the sample used in SEM was the same as that in the 2 nd cell morphology in Effect example 2, and details thereof are not repeated.

FIG. 11 is an SEM image of L929 surface cultured 3d (FIG. 11a, FIG. 11b, FIG. 11c, FIG. 11d) and 7d (FIG. 11e, FIG. 11f, FIG. 11g, FIG. 11h) at PNC40 (FIG. 11a, FIG. 11e),0.2Na-PNC40 (FIG. 11b, FIG. 11f),0.5Na-PNC40 (FIG. 11c, FIG. 11g) and 5.0Na-PNC40 (FIG. 11d, FIG. 11 h). At 3d, cells were present on the surface of PNC40,0.2Na-PNC40 and 0.5Na-PNC40, with 0.5Na-PNC40 being most preferred. At 7d, there was significant proliferation and growth of cells on all three materials, especially at 0.5Na-PNC40, with cells already covering most of the material. Furthermore, 5.0Na-PNC40 was devoid of cell growth at both time points and should be attributed to the excessive alkalinity brought about after surface crystal dissolution.

FIG. 12 is a CLSM image of L929 surface cultured on PNC40 (FIG. 12a, FIG. 12e),0.2Na-PNC40 (FIG. 12b, FIG. 12f),0.5Na-PNC40 (FIG. 12c, FIG. 12g) and 5.0Na-PNC40 (FIG. 12d, FIG. 12h) for 1d (FIG. 12a, FIG. 12b, FIG. 12c, FIG. 12d) and 3d (FIG. 12e, FIG. 12f, FIG. 12g, FIG. 12 h). The number of cells on PNC40,0.2Na-PNC40 and 0.5Na-PNC40 all increased gradually with increasing days of culture. At the same time, cell proliferation was enhanced on the surface of the material treated with 0.2M and 0.5M NaOH solutions compared to PNC40, with 0.5Na-PNC40 being the best. Furthermore, 5.0Na-PNC40 was devoid of cell growth at both time points and should be attributed to the excessive alkalinity brought about after surface crystal dissolution.

3. Cell adhesion and proliferation

The activities of L929 on the surfaces of PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 were measured at different culture times by the CCK-8 method, and 6 th, 12 th and 24 th hours were selected for evaluating cell adhesion, and 1 st, 3 th and 7 th days were selected for evaluating cell proliferation.

The specific experimental method was the same as that in the adhesion and proliferation of the cells of item 3 of Effect example 2, and the details thereof are not repeated.

FIG. 13a is the relative adhesion rate of L929 when cultured on PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 for 6,12 and 24 h. The results show that: comparing with PNC40, the relative adhesion rate of cell on the surface of material treated by 0.2M and 0.5M NaOH solution is increased, wherein 0.5Na-PNC40 is optimal; ② 5.0Na-PNC40 surface has no cell adhesion.

FIG. 13b is OD values of L929 when cultured on PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 for 1,3 and 7d (. p <0.05, vs PNC 40). The results show that: the OD values of the cells on the surfaces of 0.2Na-PNC40 and 0.5Na-PNC40 are obviously higher than that of PNC40 in all the culture time, wherein 0.5Na-PNC40 is the highest; ② 5.0Na-PNC40 surface has no cell proliferation. The specific data are shown in Table 4.

TABLE 4

Group of	PNC40	0.2Na-PNC40	0.5Na-PNC40	5.0Na-PNC40
					Cell adhesion Rate/% (6h)	100.00±8.22	109.24±12.98	121.02±15.02	0.00
Cell adhesion Rate/% (12h)	141.23±8.19	158.46±14.78	187.41±21.79	0.00
					Cell adhesion Rate/% (24h)	184.13±8.38	218.69±18.29	261.79±31.99	0.00
Absorbance (1 day)	0.22±0.02	0.25±0.02	0.30±0.02	0.00
					Absorbance (3 days)	0.39±0.02	0.42±0.04	0.56±0.06	0.00
Absorbance (7 days)	0.55±0.04	0.62±0.06	0.91±0.10	0.00

Effect example 4 example 1 sample in vitro epithelial cell response

1. Cell culture

Epithelial cells were selected from Hacat, purchased from the Chinese Bekindred IBS cell Bank. Cells were plated at 4X 10 per well⁴The density of (A) was inoculated on the surface of the sterilized (ethylene oxide) material, and the medium was DMEM (Thermo Fisher Scientific Co., U.S.A.) containing serum and double antibody of cyan and catenary antibodies. Other culture conditions were as follows: humidity 100%, CO₂Content 5%, temperature 37 ℃.

2. Morphology of cells

After culturing the Hacat on the surfaces of PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 for different times, observing the morphology, adhesion state and quantity of the Hacat on the surface of the material by using SEM (at 3d and 7d) and CLSM (at 1, 3d and 7d), respectively.

FIG. 14 is SEM images of Hacat surface cultured on PNC40 (FIG. 14a, FIG. 14e),0.2Na-PNC40 (FIG. 14b, FIG. 14f),0.5Na-PNC40 (FIG. 14c, FIG. 14g) and 5.0Na-PNC40 (FIG. 14d, FIG. 14h) for 3d (FIG. 14a, FIG. 14b, FIG. 14c, FIG. 14d) and 7d (FIG. 14e, FIG. 14f, FIG. 14g, FIG. 14 h). At 3d, cells were present on the surface of PNC40,0.2Na-PNC40 and 0.5Na-PNC40, with 0.5Na-PNC40 being most preferred. At 7d, there was significant proliferation and growth of cells on all three materials, especially at 0.5Na-PNC40, with cells already covering most of the material. Furthermore, 5.0Na-PNC40 was devoid of cell growth at both time points and should be attributed to the excessive alkalinity brought about after surface crystal dissolution.

FIG. 15 is a CLSM image of Hacat on the surface of PNC40 (FIG. 15a, FIG. 15e, FIG. 15i),0.2Na-PNC40 (FIG. 15b, FIG. 15f, FIG. 15j),0.5Na-PNC40 (FIG. 15c, FIG. 15g, FIG. 15k) and 5.0Na-PNC40 (FIG. 15d, FIG. 15h, FIG. 15l) for 1d (FIG. 15a, FIG. 15b, FIG. 15c, FIG. 15d),3d (FIG. 15e, FIG. 15f, FIG. 15g, FIG. 15h) and 7d (FIG. 15i, FIG. 15j, FIG. 15k, FIG. 15 l). The number of cells on PNC40,0.2Na-PNC40 and 0.5Na-PNC40 all increased gradually with increasing days of culture. Meanwhile, the proliferation of cells on the surface of the material treated with 0.2M and 0.5M NaOH solution was improved compared to PNC40, with 0.5Na-PNC40 being the best. Furthermore, 5.0Na-PNC40 was devoid of cell growth at both time points and should be attributed to the excessive alkalinity brought about after surface crystal dissolution.

3. Cell adhesion and proliferation

The CCK-8 method was used to determine the Hacat activity of the surfaces of PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 at different culture times, with 6 th, 12 th and 24 th hours being selected for the evaluation of cell adhesion and 1 st, 3 th and 7 th days being selected for the evaluation of cell proliferation.

FIG. 16a shows the relative adhesion rates of Hacat when cultured for 6,12 and 24h on PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC 40. The results show that: comparing with PNC40, the relative adhesion rate of cell on the surface of material treated by 0.2M and 0.5M NaOH solution is increased, wherein 0.5Na-PNC40 is optimal; ② 5.0Na-PNC40 surface has no cell adhesion.

FIG. 16b shows the OD values of Hacat when cultured on PNC40,0.2Na-PNC40,0.5Na-PNC40 and 5.0Na-PNC40 for 1,3 and 7d (. p <0.05, vs PNC 40). The results show that: the OD values of the cells on the surfaces of 0.2Na-PNC40 and 0.5Na-PNC40 are obviously higher than that of PNC40 in all the culture time, wherein 0.5Na-PNC40 is the highest; ② 5.0Na-PNC40 surface has no cell proliferation. The specific data are shown in Table 5.

TABLE 5

Group of	PNC40	0.2Na-PNC40	0.5Na-PNC40	5.0Na-PNC40
					Cell adhesion Rate/% (6h)	100.00±4.91	107.78±10.13	141.21±10.21	0.00
Cell adhesion Rate/% (12h)	161.42±8.10	169.41±15.71	242.41±15.09	0.00
					Cell adhesion Rate/% (24h)	213.42±10.37	245.73±18.19	302.71±29.97	0.00
Absorbance (1 day)	0.25±0.01	0.28±0.02	0.36±0.03	0.00
					Absorbance (3 days)	0.44±0.02	0.48±0.03	0.65±0.05	0.00
Absorbance (7 days)	0.62±0.04	0.69±0.03	0.95±0.09	0.00

In summary, the following steps: to further improve the cellular responsiveness of PNC40, PNC40 surface was modified with 0.2M, 0.5M and 5.0M NaOH solutions. Effect examples 1-4 the results of the experiments show that: firstly, an amorphous Na-Nb-O nanowire structure is constructed on the surface of a material by the treatment of 0.2M and 0.5M NaOH solutions, wherein the surface of 0.2Na-PNC40 is partially covered by an amorphous Na-Nb-O nanowire layer, and the surface of 0.5Na-PNC40 is almost completely covered by an amorphous Na-Nb-O nanowire layer; ② the treatment of 5.0M NaOH solution can generate Na-Nb-O crystal particles with high crystallinity on the surface of the material.

Higher roughness favors cellular response. In the invention, the amorphous nano-wire structure and the high-crystallization particle structure generated by NaOH treatment improve the roughness of the material surface; in short, the higher the concentration of the NaOH treatment solution, the greater the roughness.

Better hydrophilicity may also promote cellular response. In the invention, the amorphous nano-wire structure and the high-crystallization particle structure generated by NaOH treatment improve the hydrophilicity of the surface of the material; in short, the higher the concentration of the NaOH treatment solution, the better the hydrophilicity.

The effect of the material on the pH of the surrounding liquid is also not negligible. In the present invention, after soaking the treated material in SBF, the pH of the 0.2Na-PNC40 and 0.5Na-PNC40 soaks were both in the normal range; dissolution of the 5.0Na-PNC40 particles, however, significantly increased the pH of the soaking solution, which was detrimental to both protein adsorption and cell growth.

Protein adsorption also affects cellular response as the first event to occur at the implant/body fluid interface. In the invention, the adsorption of BSA and Fn to the sample is improved along with the increase of amorphous Na-Nb-O nano-wires on the surface of the material. It is believed that the presence of amorphous Na-Nb-O nanowires roughens and hydrophilizes the material and builds up nanowire structures on the surface, ultimately improving its protein adsorption capacity.

The ability of biological materials to promote cell adhesion is of critical importance. In the invention, with the increase of the amorphous Na-Nb-O nano-wire on the surface of the material, the adhesion of three cells on the surface of the material is increased, and the material presents a more extensive flat state, which shows that the amorphous Na-Nb-O nano-wire structure on the surface of the material plays a key role in promoting the cell adhesion.

Cell proliferation is closely related to tissue regeneration capacity. In the invention, with the increase of the amorphous Na-Nb-O nano-wire on the surface of the material, the proliferation capacity of three cells on the composite material is enhanced, which shows that the amorphous Na-Nb-O nano-wire structure on the surface of the material plays a key role in promoting cell proliferation of the material.

For bone repair materials, osteogenic differentiation of BMSCs on the surface of the material is of no particular significance. In the invention, with the increase of the amorphous Na-Nb-O nano-wire on the surface of the material, the ALP activity of the BMSC on the composite material is obviously increased, which shows that the amorphous Na-Nb-O nano-wire structure on the surface of the material plays a considerable role in promoting osteoblast differentiation.

The biological performance of the biomaterial is strongly influenced by roughness, hydrophilicity and morphology (amorphous Na-Nb-O nanowire structure surface), and protein adsorption is taken as a ligament, so that the cell response is regulated and controlled. Research results show that the PNC40 is treated by using 0.2M and 0.5M NaOH solutions, so that the roughness and the hydrophilicity of the material are increased, an amorphous Na-Nb-O nano-wire structure is formed on the surface of the material, and the adsorption performance of protein is remarkably improved, wherein 0.5Na-PNC40 is optimal. Compared with PNC40, the materials treated with 0.2M and 0.5M NaOH solutions all had significant effects in promoting osteoblast, fibroblast, and epithelial cell responses, with 0.5Na-PNC40 being the best. Therefore, the 0.5Na-PNC40 has good cell responsiveness and has great application potential as a percutaneous bone implant.

Effect example 5 characterization of physical and chemical properties of the sample of example 2

XRD analysis

The chemical compositions and crystal structures of PNC40, Na-PNC40 and Ca-PNC40 were analyzed by XRD (range: 10-80 ℃).

FIG. 17 is an XRD pattern of PNC40, Na-PNC40 and Ca-PNC 40. As can be seen from the figure: after PNC40 is treated by 0.5M NaOH solution (namely Na-PNC40), the peak intensity of the material at the corresponding position is obviously reduced, which indicates that a layer of amorphous substance is generated on the surface of the material; ② Na-PNC40 in 0.2M CaCl₂After the solution treatment (i.e., Ca-PNC40), no new crystallization peak appeared, and the surface material still remained a layer of amorphous material.

SEM and EDS analysis

The surfaces of PNC40, Na-PNC40, Ca-PNC40 were observed using SEM, and elemental analysis was performed on the surfaces of the samples using EDS.

FIG. 18 is EDS maps of PNC40 (FIG. 18a), Na-PNC40 (FIG. 18b), and Ca-PNC40 (FIG. 18 c). As can be seen from the figure: firstly, after PNC40 is treated by 0.5M NaOH solution (namely Na-PNC40), Na elements appear on the surface of the material; ② Na-PNC40 through 0.2M CaCl₂After the solution treatment (i.e., Ca-PNC40), the Na element on the surface of the material was completely replaced by Ca element.

FIG. 19 is an SEM image of PNC40 (FIG. 19a, FIG. 19d, FIG. 19g), Na-PNC40 (FIG. 19b, FIG. 19e, FIG. 19h) and Ca-PNC40 (FIG. 19c, FIG. 19f, FIG. 19 i). As can be seen from the figure: PNC40 is treated by 0.5M NaOH solution (namely Na-PNC40), Nb particles are almost completely converted into nanowires, the surfaces of the materials are covered, and the diameters of the nanowires are about 50nm and are uniform; ② Na-PNC40 through 0.2M CaCl₂After the solution treatment (i.e., Ca-PNC40), no significant surface change occurred.

3. Berberine sustained release assay

A solution of 100. mu.g/mL of Ber-PBS was prepared and the maximum absorption wavelength was optimized by full sweep. Gradient Ber-PBS solutions were prepared, OD was measured at the preferred wavelength (344nm), and a standard curve was drawn. The Ca-PNC40@ Ber sample was placed in 10mL PBS and subjected to a Ber release assay at 37 ℃ and 60 r/min. At 1,3, 5, 7,10, 15, 20, 25d, respectively, 100. mu.L of the supernatant was taken, the OD value was measured at the preferred measurement wavelength (344nm), and the cumulative release amount was obtained according to the standard curve.

FIG. 20a shows the UV absorption curve for Ber, which shows a maximum absorption peak at 344nm, so 344nm can be selected as the measurement wavelength of the standard curve. FIG. 20b is the measured Ber standard curve. FIG. 20c shows the Ber release profile of Ca-PNC40@ Ber in PBS with a faster early release of Ber followed by a gradual decrease in release rate to 56.76% at 25 d. The result shows that the Ber can be released from the Ca-PNC40@ Ber continuously, and has better release rule.

4. Roughness analysis

The roughness of the surface of the sample was measured by performing roughness analysis on the surfaces of PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber using a three-dimensional topography measuring microscope.

FIG. 21 shows TMM images and roughness values for the surfaces of PNC40 (FIG. 21a), Na-PNC40 (FIG. 21b), Ca-PNC40 (FIG. 21c) and Ca-PNC40@ Ber (FIG. 21 d). As can be seen from the figure: roughness of PNC40 is 2.50 μm; ② after the PNC40 is treated by 0.5M NaOH solution (namely Na-PNC40), the surface roughness of the material is improved (3.31 μ M); ③ Na-PNC40 in 0.2M CaCl₂After the solution treatment (namely Ca-PNC40), the surface roughness of the material is not obviously changed (3.24 mu m); after the Ca-PNC40 is loaded with the Ber (namely Ca-PNC40@ Ber), the surface roughness of the material is not obviously changed (3.29 mu m).

5. Hydrophilicity assays

The surface water contact angles were measured using a contact angle meter for PNC40, Na-PNC40, Ca-PNC40, Ca-PNC40@ Ber. When the contact angle was measured, the volume of the liquid was 10. mu.L.

FIG. 22 shows the results of water contact angle measurements (/ p) for PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber surfaces<0.05, vs PNC 40). As can be seen from the figure: contact angle of PNC40 is 80.75 degree; ② after the PNC40 is treated by 0.5M NaOH (namely Na-PNC40), the hydrophilicity of the material is improved (41.50 degrees); ③ Na-PNC40 in 0.2M CaCl₂After treatment (i.e., Ca-PNC40), the hydrophilicity of the material decreased slightly (48.50 °); ③ after Ca-PNC40 is loaded with the Ber (namely Ca-PNC40@ Ber), the hydrophilicity of the material is slightly improved (44.75 degrees).

6. In vitro immersion experiment

PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber were soaked in 10mL of SBF and placed in a constant temperature shaking box. The pH of the SBF was measured at 1,3, 5, 7,10 and 14d, respectively.

FIG. 23 is a graph showing the change in pH of SBF after immersion in SBF of PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber. The data obtained show that: PNC40 is soaked in SBF, so that the pH value of the soaking solution cannot be changed; ② after the PNC40 is treated by 0.5M NaOH solution (namely Na-PNC40), the pH of the soaking solution will gradually rise, and is 7.89 at 14 days; ③ Na-PNC40 in 0.2M CaCl₂After the solution is treated (namely Ca-PNC40), the pH of the soaking solution is basically not changed; after the Ca-PNC40 is loaded with the Ber (namely Ca-PNC40@ Ber), the pH of the soak solution is slightly increased and is 7.54 at 14 d.

7. Protein adsorption assay

And (3) analyzing the protein adsorption capacity of the surfaces of PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber by using BSA and Fn as model proteins.

The experimental method was the same as that of the protein adsorption assay of item 6 of effect example 1, and will not be described herein.

FIG. 24 shows the results of protein adsorption assays (. about.p.) on the surfaces of PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber<0.05, vs PNC 40). As can be seen from the figure: the protein adsorption rate of PNC40 is BSA: 25.41% and Fn: 21.49 percent; ② after PNC40 is treated by 0.5M NaOH solution (namely Na-PNC40), the protein adsorption rate is increased to BSA: 38.78% and Fn: 37.15 percent; ③ Na-PNC40 in 0.2M CaCl₂After solution treatment (i.e., Ca-PNC40), the protein adsorption rate was further increased to BSA: 53.45% and Fn: 49.86 percent; after the Ca-PNC40 is loaded with the Ber (namely Ca-PNC40@ Ber), the protein adsorption rate is BSA: 51.42% and Fn: 48.74 percent.

Effect example 6 example 2 sample in vitro osteoblast response

1. Cell culture

The experimental procedure was the same as in the 1 st cell culture of effect example 2, and will not be described herein.

2. Morphology of cells

After culturing BMSCs on the surfaces of PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber for different times, the shapes, adhesion states and quantities of the BMSCs on the surfaces of the materials are observed by using SEM (at 1 and 5d) and CLSM (at 1,3 and 5d), respectively.

After culturing BMSCs on the surface of PNC40, Na-PNC40, Ca-PNC40, Ca-PNC40@ Ber for 3d and 7d, the morphology, adhesion state and amount of BMSCs on the surface of the material were observed using SEM and confocal laser scanning microscope (CLSM, A1R, Nikon, Japan), respectively.

FIG. 25 is an SEM image of BMSCs cultured on the surface of PNC40 (FIG. 25a, FIG. 25e), Na-PNC40 (FIG. 25b, FIG. 25f), Ca-PNC40 (FIG. 25c, FIG. 25g) and Ca-PNC40@ Ber (FIG. 25d, FIG. 25h) for 1d (FIG. 25a, FIG. 25b, FIG. 25c, FIG. 25d) and 5d (FIG. 25e, FIG. 25f, FIG. 25g, FIG. 25 h). At 1d, cells were present on the surface of all four materials, with Ca-PNC40 and Ca-PNC40@ Ber surface cells being the most abundant and spreading the best. At 5d, there was significant proliferation of cells on all four materials, in particular Ca-PNC40 and Ca-PNC40@ Ber, with the cells almost completely covering the surface of the material. The results show that: firstly, replacement of Na element by Ca element up-regulates osteoblast response; ② the load of the Ber does not bring adverse effect to osteoblasts.

FIG. 26 is a CLSM image of BMSCs cultured on the surface of 1d (FIG. 26a, FIG. 26b, FIG. 26c, FIG. 26h, FIG. 26d),3d (FIG. 26e, FIG. 26f, FIG. 26g, FIG. 26h) and 5d (FIG. 26i, FIG. 26j, FIG. 26k, FIG. 26l) at PNC40 (FIG. 26a, FIG. 26e, FIG. 26i), Na-PNC40 (FIG. 26b, FIG. 26f, FIG. 26j), Ca-PNC40 (FIG. 26c, FIG. 26g, FIG. 26k) and Ca-PNC40@ Ber (FIG. 26d, FIG. 26h, FIG. 26 l). The number of cells on all four materials increased gradually as the number of days of culture increased. Meanwhile, compared with Na-PNC40, BMSC has better adhesion and spreading on the surface of Ca-PNC40 and more quantity, which shows the beneficial effect of Ca element. In addition, after being loaded with the Ber (i.e., Ca-PNC40@ Ber), the spreading of the cells on the surface of the material is not deteriorated, and the number of the cells is not obviously reduced, which indicates that the load of the Ber is not adversely affected.

3. Cell adhesion and proliferation

The CCK-8 method was used to determine the BMSC viability of the PNC40, Na-PNC40, Ca-PNC40, Ca-PNC40@ Ber surfaces at different culture times, with 6 th, 12 th, and 24 th hours selected for evaluation of cell adhesion, and 1 st, 3 th, and 5 th days selected for evaluation of cell proliferation.

The CCK-8 method was used to determine the BMSC viability of the PNC40, Na-PNC40, Ca-PNC40, Ca-PNC40@ Ber surfaces at different culture times, with 6 th, 12 th, and 24 th hours selected for evaluation of cell adhesion, and 1 st, 3 th, and 7 th days selected for evaluation of cell proliferation.

samples

7,10 and 14d (ip <0.05, vs PNC 40).

FIG. 27a shows the relative adhesion rates of BMSCs cultured for 6,12 and 24h on PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber. At all incubation times, the results show that: the relative adhesion rate of Na-PNC40 is higher than that of PNC 40; compared with Na-PNC40, the relative cell adhesion rate of Ca-PNC40 is higher, which proves that the replacement of Na element by Ca element has favorable influence on cell adhesion; ③ compared with Ca-PNC40, the relative adhesion rate of Ca-PNC40@ Ber has no obvious change, which proves that the load of the Ber has no adverse effect on the cell adhesion.

FIG. 27b shows the OD values of BMSCs cultured for 1,3, and 5d on PNC40, Na-PNC40, Ca-PNC40, and Ca-PNC40@ Ber. At all incubation times, the results show that: the OD value of Na-PNC40 cells is obviously higher than that of PNC 40; OD values of cells on the surface of the Ca-PNC40 are obviously higher than those of Na-PNC40, so that the replacement of Na elements by Ca elements is proved to generate favorable influence on cell proliferation; ③ compared with Ca-PNC40, the cell OD value of Ca-PNC40@ Ber has no obvious change, which proves that the load of the Ber has no adverse effect on the cell proliferation.

The specific data are shown in Table 6.

TABLE 6

Effect example 7 example 2 sample cell differentiation

The ability of PNC40, Na-PNC40, Ca-PNC40, Ca-PNC40@ Ber to promote osteogenic differentiation of BMSCs was assessed by assaying the activity of alkaline phosphatase (ALP) in cells.

The specific experimental method was the same as that of the 4 th cell differentiation method in Effect example 2, and the details thereof are not repeated herein.

FIG. 27c shows ALP activity of BMSCs cultured on PNC40, Na-PNC40, Ca-PNC40, and Ca-PNC40@ Ber for 7,10, and 14 d. At all incubation times, the results show that: the ALP activity of cells of Na-PNC40 is obviously higher than that of cells of PNC 40; ALP activity of cells on the surface of the Ca-PNC40 is obviously higher than that of Na-PNC40, and the Ca element replacement on the Na element is proved to generate favorable influence on osteogenic differentiation of the cells; ③ compared with Ca-PNC40, the ALP activity of Ca-PNC40@ Ber cells has no obvious change, which proves that the load of the Ber does not cause negative effect on the osteogenic differentiation of the cells.

Effect example 8 example 2 sample in vitro fibroblast response

1. Cell culture

The specific experimental method was the same as that of the 1 st cell culture method in effect example 3, and details thereof are omitted.

2. Morphology of cells

After L929 was cultured on the surfaces of PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber for different times, the appearance, adhesion state and quantity of L929 on the material surface were observed by using SEM (at 3d and 5d) and CLSM (at 1d and 3d), respectively. After culturing BMSCs on the surface of PNC40, Na-PNC40, Ca-PNC40, Ca-PNC40@ Ber for 3d and 7d, the morphology, adhesion state and amount of BMSCs on the surface of the material were observed using SEM and confocal laser scanning microscope (CLSM, A1R, Nikon, Japan), respectively.

FIG. 28 is an SEM image of L929 cultured on the surface of PNC40 (FIG. 28a, FIG. 28e), Na-PNC40 (FIG. 28b, FIG. 28f), Ca-PNC40 (FIG. 28c, FIG. 28g) and Ca-PNC40@ Ber (FIG. 28d, FIG. 28h) for 3d (FIG. 28a, FIG. 28b, FIG. 28c, FIG. 28d) and 5d (FIG. 28e, FIG. 28f, FIG. 28g, FIG. 28 h). At 3d, cells were present on the surface of all four materials, with the most surface cells being Ca-PNC40 and Ca-PNC40@ Ber. At 5d, there was significant proliferation of cells on all four materials, in particular Ca-PNC40 and Ca-PNC40@ Ber, with the cells almost completely covering the surface of the material. The results show that: firstly, the replacement of Na element by Ca element up-regulates the response of fibroblast; ② the load of the Ber does not bring adverse effect to the fibroblasts.

FIG. 29 is a CLSM image of L929 cultured on the surface of 1d (FIG. 29a, FIG. 29b, FIG. 29c, FIG. 29d) and 3d (FIG. 29e, FIG. 29f, FIG. 29g, FIG. 29h) in PNC40 (FIG. 29a, FIG. 29e), Na-PNC40 (FIG. 29b, FIG. 29f), Ca-PNC40 (FIG. 29c, FIG. 29g) and Ca-PNC40@ Ber (FIG. 29d, FIG. 29 h). The number of cells on all four materials increased gradually as the number of days of culture increased. Meanwhile, adhesion and proliferation of L929 on the surface of Ca-PNC40 were improved compared with Na-PNC40, indicating the advantageous effect of Ca element. Furthermore, after loading of the Ber (i.e., Ca-PNC40@ Ber), there was no significant decrease in spreading and proliferation of the cells on the surface of the material, indicating that the loading of the Ber did not adversely affect.

3. Cell adhesion and proliferation

The CCK-8 method was used to determine the L929 viability of the surfaces of PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber at different culture times, with the 6 th, 12 th and 24 th hours being selected for the evaluation of cell adhesion and the 1 st, 3 th and 5 th days being selected for the evaluation of cell proliferation. The CCK-8 method was used to determine the BMSC viability of the PNC40, Na-PNC40, Ca-PNC40, Ca-PNC40@ Ber surfaces at different culture times, with 6 th, 12 th, and 24 th hours selected for evaluation of cell adhesion, and 1 st, 3 th, and 7 th days selected for evaluation of cell proliferation.

FIG. 30a shows the relative adhesion rates of L929 when cultured on PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber for 6,12 and 24 h. At all incubation times, the results show that: the relative adhesion rate of Na-PNC40 is higher than that of PNC 40; compared with Na-PNC40, the relative cell adhesion rate of Ca-PNC40 is higher, which proves that the replacement of Na element by Ca element has favorable influence on cell adhesion; ③ compared with Ca-PNC40, the relative adhesion rate of Ca-PNC40@ Ber has no obvious change, which proves that the load of the Ber has no adverse effect on the cell adhesion.

FIG. 30b shows OD values of L929 when cultured on PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber for 1,3 and 5d (. p <0.05, vs PNC 40). At all incubation times, the results show that: the OD value of Na-PNC40 cells is obviously higher than that of PNC 40; OD values of cells on the surface of the Ca-PNC40 are obviously higher than those of Na-PNC40, so that the replacement of Na elements by Ca elements is proved to generate favorable influence on cell proliferation; ③ compared with Ca-PNC40, the cell OD value of Ca-PNC40@ Ber has no obvious change, which proves that the load of the Ber has no adverse effect on the cell proliferation. The data are shown in Table 7.

TABLE 7

Group of	PNC40	Na-PNC40	Ca-PNC40	Ca-PNC40@Ber
					Cell adhesion Rate/% (6h)	100.00±6.97	132.71±6.13	141.20±6.22	149.20±15.20
Cell adhesion rate/% (12h))	141.12±8.11	185.23±14.79	238.20±20.19	236.21±18.61
					Cell adhesion Rate/% (24h)	187.31±14.56	258.19±18.89	319.47±21.87	324.90±23.31
Absorbance (1 day)	0.22±0.01	0.31±0.02	0.38±0.02	0.39±0.02
					Absorbance (3 days)	0.39±0.02	0.57±0.05	0.64±0.04	0.65±0.04
Absorbance (5 days)	0.48±0.03	0.68±0.04	0.93±0.05	0.91±0.05

Effect example 9 example 2 sample in vitro epithelial cell response

1. Cell culture

The specific experimental method was the same as that of the 1 st cell culture method in effect example 4, and details thereof are omitted.

2. Morphology of cells

After culturing the Hacat on the surfaces of PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber for different times, observing the morphology, adhesion state and quantity of the Hacat on the surface of the material by using SEM (at 3 and 5d) and CLSM (at 1,3 and 5d), respectively. After culturing BMSCs on the surface of PNC40, Na-PNC40, Ca-PNC40, Ca-PNC40@ Ber for 3d and 7d, the morphology, adhesion state and amount of BMSCs on the surface of the material were observed using SEM and confocal laser scanning microscope (CLSM, A1R, Nikon, Japan), respectively.

FIG. 31 is an SEM image of Hacat surface cultured on PNC40 (FIG. 31a, FIG. 31e), Na-PNC40 (FIG. 31b, FIG. 31f), Ca-PNC40 (FIG. 31c, FIG. 31g) and Ca-PNC40@ Ber (FIG. 31d, FIG. 31h) for 3d (FIG. 31a, FIG. 31b, FIG. 31c, FIG. 31d) and 5d (FIG. 31e, FIG. 31f, FIG. 31g, FIG. 31 h). At 3d, cells were present on the surface of all four materials, with the most surface cells being Ca-PNC40 and Ca-PNC40@ Ber. At 5d, there was significant proliferation of cells on all four materials, in particular Ca-PNC40 and Ca-PNC40@ Ber, with the cells almost completely covering the surface of the material. The results show that: the replacement of Na element by Ca element up-regulates the response of epithelial cells; ② the load of Ber has no adverse effect on epithelial cells.

FIG. 32 is a CLSM image of Hacat on the surface of PNC40 (FIG. 32a, FIG. 32e, FIG. 32i), Na-PNC40 (FIG. 32b, FIG. 32f, FIG. 32j), Ca-PNC40 (FIG. 32c, FIG. 32g, FIG. 32k) and Ca-PNC40@ Ber (FIG. 32d, FIG. 32h, FIG. 32l) for 1d (FIG. 32a, FIG. 32b, FIG. 32c, FIG. 32d),3d (FIG. 32e, FIG. 32f, FIG. 32g, FIG. 32h) and 5d (FIG. 32i, FIG. 32j, FIG. 32k, FIG. 32 l). The number of cells on all four materials increased gradually as the number of days of culture increased. Meanwhile, Hacat showed an increased adhesion, spreading and proliferation on the surface of Ca-PNC40 compared to Na-PNC40, indicating the beneficial effect of Ca element. Furthermore, after loading of the Ber (i.e., Ca-PNC40@ Ber), there was no significant decrease in spreading and proliferation of the cells on the surface of the material, indicating that the loading of the Ber did not adversely affect.

3. Cell adhesion and proliferation

The CCK-8 method was used to determine the Hacat activity of the surface of PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber at different culture times, with 6 th, 12 th and 24 th hours being selected for the evaluation of cell adhesion and 1 st, 3 th and 5 th days being selected for the evaluation of cell proliferation. The CCK-8 method was used to determine the BMSC viability of the PNC40, Na-PNC40, Ca-PNC40, Ca-PNC40@ Ber surfaces at different culture times, with 6 th, 12 th, and 24 th hours selected for evaluation of cell adhesion, and 1 st, 3 th, and 7 th days selected for evaluation of cell proliferation.

FIG. 33a shows the relative adhesion rates of Hacat when cultured for 6,12 and 24h on PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber. At all incubation times, the results show that: the relative adhesion rate of Na-PNC40 is higher than that of PNC 40; compared with Na-PNC40, the relative cell adhesion rate of Ca-PNC40 is higher, which proves that the replacement of Na element by Ca element has favorable influence on cell adhesion; ③ compared with Ca-PNC40, the relative adhesion rate of Ca-PNC40@ Ber has no obvious change, which proves that the load of the Ber has no adverse effect on the cell adhesion.

FIG. 33b shows the OD values of Hacat when cultured on PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber for 1,3 and 5 d. At all incubation times, the results show that: the OD value of Na-PNC40 cells is obviously higher than that of PNC 40; OD values of cells on the surface of the Ca-PNC40 are obviously higher than those of Na-PNC40, so that the replacement of Na elements by Ca elements is proved to generate favorable influence on cell proliferation; ③ compared with Ca-PNC40, the cell OD value of Ca-PNC40@ Ber has no obvious change, which proves that the load of the Ber has no adverse effect on the cell proliferation.

The data are shown in Table 8.

TABLE 8

4.Western Blot

Antibody dilution information is as follows: laminin-gamma 2 antibody (1: 100), Integrin-alpha 6 antibody (1: 100), Plectin antibody (1: 100), beta-Actin antibody (1: 5000), goat anti-mouse IgG secondary antibody (1: 5000), all from ProteinTech. Hacat is cultured on the surface of PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber for 5 days, cell whole protein extraction and protein sample preparation are carried out, and after glue preparation, electrophoresis, membrane transfer, sealing, antibody incubation and chemiluminescence, a gel imager is adopted for development.

FIG. 33c is a western blot of cohesin proteins from Hacat after incubation on PNC40, Na-PNC40, Ca-PNC40, and Ca-PNC40@ Ber. The results show that: the expression of cell adhesion protein of Na-PNC40 is higher than that of PNC40, and the amorphous Na-Nb-O nano-wire structure on the surface of the material is proved to generate favorable influence on epithelial cell adhesion; compared with Na-PNC40, the cell adhesion protein expressions of Ca-PNC40 are all higher, which proves that the replacement of Na element by Ca element has favorable influence on epithelial cell adhesion; ③ compared with Ca-PNC40, the cell adhesion protein expression of Ca-PNC40@ Ber has no obvious change, which proves that the load of the Ber has no adverse effect on the adhesion of the epithelial cells.

Effect example 10 example 2 sample in vitro macrophage response

1. Cell culture

The macrophage selected from RAW 264.7, purchased from the chinese compound denier IBS cell bank. Cells were plated at 3X 10 per well⁵The density of (A) was inoculated on the surface of the sterilized (ethylene oxide) material, and the medium was DMEM (Thermo Fisher Scientific Co., U.S.A.) containing serum and double antibody of cyan and catenary antibodies.

ELISA method for detecting TNF-alpha and IL-6

After 12h inoculation of RAW 264.7 on the surface of PNC40, Na-PNC40, Ca-PNC40, Ca-PNC40@ Ber, the standard medium was replaced with Lipopolysaccharide (LPS) -containing medium and incubated for 2 h. Next, the samples were washed thoroughly with PBS, placed in new well plates, and incubated in serum starved medium for 6 h. Subsequently, the supernatant culture was extracted and the amount of inflammatory factor expression was measured by using ELISA kit (R & D, USA) corresponding to IL-6 and TNF-. alpha..

3.Western Blot

Antibody dilution information is as follows: TNF-alpha antibody (1: 1000), IL-6 antibody (1: 1000), GAPDH antibody (1: 5000), goat anti-rabbit IgG secondary antibody (1: 5000), all purchased from ProteinTech. The cells were cultured as in 4.2.5.2. After the serum starvation incubation is finished, western blot is carried out, and antibody dilution information is as follows: laminin-gamma 2 antibody (1: 100), Integrin-alpha 6 antibody (1: 100), Plectin antibody (1: 100), beta-Actin antibody (1: 5000), goat anti-mouse IgG secondary antibody (1: 5000), all from ProteinTech. Hacat is cultured on the surface of PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber for 5 days, cell whole protein extraction and protein sample preparation are carried out, and after glue preparation, electrophoresis, membrane transfer, sealing, antibody incubation and chemiluminescence, a gel imager is adopted for development.

FIG. 34a shows inflammatory factor expression after RAW 264.7 culture on PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber. The results show that the load of Ber significantly reduced the expression of TNF-alpha and IL-6.

FIG. 34b is a western blot of inflammatory proteins after incubation of RAW 264.7 on PNC40, Na-PNC40, Ca-PNC40 and Ca-PNC40@ Ber ([ p ] <0.05, vs PNC 40). The results show that the load of Ber significantly reduced the expression of TNF-alpha and IL-6. The data are shown in Table 9.

TABLE 9

Protein concentration (pg/mL)	PNC40	Na-PNC40	Ca-PNC40	Ca-PNC40@Ber
					TNF-α	2690±280	2970±300	2360±260	820±120
IL-6	3570±280	3980±380	2950±260	1210±110

EXAMPLES 11 EXAMPLE 2 in vitro antibacterial assay

The materials were evaluated for in vitro antibacterial performance using the colony assay, and the bacteria were selected for e.coli (ATCC 25922) and s.aureus (ATCC 25923). Specifically, 10mL of the bacterial solution (1X 10)⁵CFU/mL) was inoculated on the surface of the sterilized (ethylene oxide method) material and incubated at 37 ℃ for 24 hours. Then, 0.1mL of the culture broth was uniformly applied to the plate and the culture was continued. After 24h, the plates were counted and converted according to the following equation:

xs represents the bacteriostasis rate, A represents the average colony number of the PEEK surface, and B represents the average colony number of the other material surface.

FIGS. 35 a-35 h are photographs of colonies of E.coli and S.aureus isolated from the surface of PNC40 (FIG. 35a, FIG. 35e), Na-PNC40 (FIG. 35b, FIG. 35f), Ca-PNC40 (FIG. 35c, FIG. 35g), Ca-PNC40@ Ber (FIG. 35d, FIG. 35h) grown on agar for 24 h; bacterial inhibition of e.coli (i) with s.aureus (j) (. p <0.05, vs PNC 40). The results showed that a large number of colonies were present in all of the PNC40, Na-PNC40, and Ca-PNC40 groups, while a very small number of colonies were present in the Ca-PNC40@ Ber group. Fig. 35i and fig. 35j are quantitative statistics, and the inhibition rates of Ca-PNC40@ Ber on e.coli and s.aureus are up to 93.53% and 89.75%, respectively. The result proves that the load of the Ber endows the material with better antibacterial performance.

In summary, the following steps: in order to further improve the cell responsiveness of 0.5Na-PNC40 (Na-PNC 40 for short), CaCl is adopted₂The solution further processes it. Effect examples 6-11 the results show that: firstly, CaCl₂The solution treatment does not change the amorphous nanowire structure on the surface of the material; na on the surface of the material⁺Is replaced by Ca²⁺. Briefly, the reaction mixture was washed with 0.5M NaOH solution and 0.2M CaCl₂The solutions are sequentially treated, and an amorphous Ca-Nb-O nanowire layer is generated on the surface of the material. And then, natural antibacterial and anti-inflammatory small-molecule berberine (Ber) is loaded by utilizing the loose and porous shape of the surface of the material, and a release curve proves that the berberine has a better release rule.

Higher surface roughness favors cellular response. In the present invention, CaCl₂The roughness of the material was not significantly affected by the treatment of (2) and the loading of the Ber, and both Ca-PNC40 and Ca-PNC40@ Ber maintained higher roughness than PNC40, which should be attributed to the amorphous nanowire structure at the surface of the material.

Higher hydrophilicity favors cellular response. In the present invention, CaCl₂The treatment of (a) slightly reduces the hydrophilicity of the material, but remains a very hydrophilic surface in nature; and the load of the Ber slightly improves the hydrophilicity of the material. In short, the presence of amorphous nanowire structures maintains the high hydrophilicity of the material.

The effect of the material on the pH of the surrounding liquid is also not negligible. In the present invention, the pH of the Ca-PNC40 and Ca-PNC40@ Ber baths after soaking the treated material in SBF was closer to 7.4 than Na-PNC40, which is advantageous for cell growth.

Protein adsorption also affects cellular response as the first event to occur at the implant/body fluid interface. In the present invention, Na in the nano-filaments is not formed along with the surface of the material⁺Substitution with Ca²⁺The protein adsorption capacity of the material is obviously improved; and the load of the beer does not have a significant influence on the protein adsorption capacity of the material. It is believed that the presence of amorphous nanowires roughens and hydrophilizes the material, constructs nanowire structures on the surface, improves the adsorption of the material to proteins, and simultaneously, Ca²⁺As a typical positive site, the adsorption of the material to the protein is greatly enhanced, which is a result of the synergistic effect.

The ability of biological materials to promote cell adhesion is of critical importance. In the present invention, Na in the nano-filaments is not formed along with the surface of the material⁺Substitution with Ca²⁺The adhesion of the three cells on the surface of the material is increased, and the material has a more extensive flat shape; while the load of the beer does not have a significant effect on the adhesion of the cells on the surface of the material. In addition, epithelial cells, unlike osteoblasts and fibroblasts, adhere to the surface of materials via a hemidesmosome-basement structure, which is mainly divided into intracellular cytoplasmic plaques (mainly containing Plectin), transmembrane connexin (mainly containing Integrin- α 6 β 4) and basement membrane (mainly containing lamin 332, subunit composition α 3 β 3 γ 2)^[101,102]In the present invention, Na in amorphous nanowires following the material surface was confirmed by Western Blot⁺Substitution with Ca²⁺The expression of three adhesion proteins of the epithelial cells is increased, and the load of the Ber does not have a significant influence on the expression of the adhesion proteins. It is considered that the material surface has an amorphous nano-filament structure and Ca²⁺The positive sites exert a synergistic effect in promoting cell adhesion.

Cell proliferation is closely related to tissue regeneration capacity. In the present invention, Na in the nano-filaments is not formed along with the surface of the material⁺Substitution with Ca²⁺The proliferation capacity of the three cells on the composite material is enhanced; and the load of the Ber does not have a significant influence on the proliferation of cells on the surface of the material. It is considered that the surface of the material is indefiniteType nanowire Structure and Ca²⁺The positive sites play a synergistic role in promoting cell proliferation.

For bone repair materials, osteogenic differentiation of BMSCs on the surface of the material is of no particular significance. In the present invention, Na in the nano-filaments is not formed along with the surface of the material⁺Substitution with Ca²⁺ALP activity of BMSC on composite was significantly increased; and the load of the Ber does not have a significant influence on the proliferation of cells on the surface of the material. It is considered that the material surface has an amorphous nano-filament structure and Ca²⁺The positive sites play a synergistic role in promoting osteogenic differentiation of BMSCs.

Anti-inflammatory and antibacterial capabilities are critical to percutaneous bone implants. In the invention, with the load of the Ber, the anti-inflammatory and antibacterial abilities of the material are greatly improved.

The biological performance of biomaterials is strongly influenced by roughness, hydrophilicity and surface energy and morphology (amorphous nanowire structure surface), and protein adsorption is taken as a ligament, thereby regulating and controlling cell response. Research results show that an amorphous nano-wire structure is constructed on the surface of PNC40, so that the roughness and the hydrophilicity of the material are improved, and the adsorption performance of protein is obviously improved. Furthermore, the positive sites on the surface of the material have a non-negligible effect on the cell behaviour, Ca-PNC40 and Ca-PNC40@ Ber surface Ca²⁺Greatly up-regulates the response of osteogenic, fibroblast and epithelial cells. Therefore, the mechanism by which Ca-PNC40 promotes cellular response should depend largely on two related factors: (1) physical properties: roughness, hydrophilicity and amorphous nanowire structure; (2) chemical characteristics: ca on the surface of the Material²⁺Positive site. The cellular response is stimulated, possibly as a result of a synergistic effect of the two. In addition, the load of the Ber endows the material with dual functions of anti-inflammation and antibiosis. In conclusion, Ca-PNC40@ Ber has remarkable promoting effect on the response of osteogenesis, fibroblasts and epithelial cells, has anti-inflammatory and antibacterial functions, and has great application potential as a percutaneous bone implant.

Remarking: statistical analysis, effect all experiments in examples 1-11 were repeated at least 3 times, and the results were expressed in Mean ± SD format, and statistical comparisons of differences between data groups were performed by analysis of variance (ANOVA), with p <0.05 indicating significant differences in the data obtained from the experiments.

Claims

1. A raw material composition, characterized in that it comprises the following components: the niobium powder and the polyether-ether-ketone powder are mixed according to a volume ratio of 1: 4-2: 3, and the particle size of the niobium powder is 10-120 nm.

2. The raw material composition according to claim 1, wherein the polyetheretherketone powder has a particle size of 10 to 40 μm;

the polyether-ether-ketone has a melting point of 330 to 340 ℃, a glass transition temperature of 140 to 150 ℃, and a density of 1.0 to 1.5g/cm³The polymerization degree is 150-250, and the molecular weight is 40000-60000; preferably, the polyetheretherketone has a melting point of 334 ℃, a glass transition temperature of 143 ℃ and a density of 1.30g/cm³A degree of polymerization of 200 and a molecular weight of 50000;

the particle size of the niobium powder is 20 nm-100 nm;

in the raw material composition, the volume ratio of the niobium powder to the polyether-ether-ketone powder is 20:80, 30:70, 40:60 or 50: 50; preferably, the niobium powder is 20 to 50% by volume, the polyetheretherketone powder is 50 to 80% by volume, for example, the niobium powder is 40% by volume, and the polyetheretherketone powder is 60% by volume.

3. The preparation method of the PEEK-based composite material is characterized by comprising the following steps: and (3) carrying out cold pressing, sintering and molding on the raw material composition.

4. The method for preparing a PEEK-based composite material according to claim 3, characterized in that the cold-pressure sintering comprises the following steps: pressing and molding the raw material composition, heating, and sintering and molding;

the sintering forming temperature is preferably 340-380 ℃, for example 350 ℃;

the time for the sintering and forming is preferably 2h to 5h, for example 3 h.

5. The method for preparing a PEEK-based composite material according to claim 3 or 4, wherein the PEEK-based composite material is dipped in an alkali solution;

the concentration of the alkali liquor is preferably 0.1mol/L to 1mol/L, such as 0.2mol/L, 0.3mol/L, 0.5mol/L, 0.7mol/L, 0.8mol/L or 0.9 mol/L.

6. The process for the preparation of PEEK-based composite materials according to claim 5, characterized in that the impregnation treatment in alkaline solution is carried out by the following steps: soaking the PEEK-based composite material in NaOH solution at the temperature of 55-65 ℃;

wherein, the dipping is preferably static dipping;

the soaking time is preferably 22-26 h, such as 24 h;

the dipping temperature is preferably 58-62 ℃, for example 60 ℃;

after the dipping treatment in the alkali solution, the obtained product is preferably washed and dried.

7. The method for preparing PEEK-based composite material of claim 6, wherein the PEEK-based composite material after the alkali impregnation treatment is in the presence of Ca⁺²Dipping again in the solution to prepare Ca-PEEK-based composite material;

the Ca is contained⁺²The solution is preferably CaCl₂A solution;

the Ca is contained⁺²The concentration of the solution is preferably 0.1-1 mol/L, such as 0.2mol/L, 0.3mol/L, 0.5mol/L, 0.7mol/L, 0.8mol/L or 0.9 mol/L;

the secondary impregnation is preferably static impregnation;

the time for re-dipping is preferably 22-26 h, such as 24 h;

the temperature of the second impregnation is preferably 58-62 ℃, for example 60 ℃;

after the re-impregnation, the resulting product is preferably washed and dried.

8. The method for preparing a PEEK-based composite material according to claim 7, wherein an antibacterial and anti-inflammatory molecule is loaded on the surface of the Ca-PEEK-based composite material;

the antibacterial and anti-inflammatory molecule is preferably berberine;

the loading operation is preferably to dropwise add the solution containing the antibacterial and anti-inflammatory molecules for several times and dry the solution;

when the antibacterial and anti-inflammatory molecule is berberine, the solution of the antibacterial and anti-inflammatory molecule is preferably berberine-ethanol solution;

when the antibacterial and anti-inflammatory molecules are berberine, the berberine loading is preferably 150-250 mug/379 mm²Composite materials, e.g. 200. mu.g/379 mm²A composite material.

9. A PEEK-based composite material prepared by the method of any one of claims 3 to 8.

10. Use of a PEEK-based composite material according to claim 9 in a bone prosthesis; wherein, the bone repair body is preferably a spine bone repair body or a dental implant.