CN114805888A

CN114805888A - Method for improving surface bioactivity and osseointegration performance of polyether-ether-ketone substrate

Info

Publication number: CN114805888A
Application number: CN202210319005.5A
Authority: CN
Inventors: 杨鹏; 张旭; 高颖涛; 逄艳云
Original assignee: Shaanxi Normal University
Current assignee: Shaanxi Normal University
Priority date: 2022-03-29
Filing date: 2022-03-29
Publication date: 2022-07-29
Anticipated expiration: 2042-03-29
Also published as: CN114805888B

Abstract

The application relates to a method for improving surface bioactivity and osseointegration performance of a polyether-ether-ketone substrate, which is sequentially carried out according to the following steps: (1) soaking the polyether-ether-ketone substrate in a mixed solution of a protein solution and a disulfide bond reducing agent to obtain a polyether-ether-ketone substrate modified by a nano film; (2) and (2) soaking the polyether-ether-ketone base material modified by the nano film obtained in the step (1) in a calcium-phosphorus solution, and carrying out closed incubation to obtain the polyether-ether-ketone base material modified by the hydroxyapatite coating. The method for improving the surface bioactivity and the osseointegration performance of the PEEK base material solves the problem that induced mineralization cannot be carried out on the surface of the PEEK base material in the prior art, so that the modified PEEK base material can be used as a tissue repair material to induce the formation of new tissues and the mineralization of bones, and is expected to become a novel hard tissue substitute material.

Description

Method for improving surface bioactivity and osseointegration performance of polyether-ether-ketone substrate

Technical Field

The invention mainly relates to the field of materials, in particular to a method for improving the surface bioactivity and osseointegration performance of a polyether-ether-ketone substrate.

Background art:

as a semi-crystalline linear polycyclic aromatic polymer, Polyetheretherketone (PEEK) material was first used in clinical implants in 1998. Due to their excellent chemical resistance, good mechanical strength and radiation permeability, have gained increasing popularity in recent years as a replacement for titanium and its alloys in orthopedics. The main advantage of PEEK as an implant material is that the elasticity and strength thresholds are very close to that of cortical bone. The elastic modulus of PEEK is in the range of 3-4GPa, and the elastic modulus of human cortical bone is about 18 GPa. Furthermore, it has been reported that carbon fiber reinforced PEEK composites have a modulus of elasticity close to that of cortical bone (about 18-25GPa), avoiding the risk of stress shielding-induced osteoporosis and bone resorption. However, the bioinert and hydrophobic nature of PEEK makes it poorly osseointegrative, preventing its widespread clinical use. Therefore, it is highly desirable to modify the PEEK substrate surface to improve its bioactivity and osteointegration. The method for inducing biomimetic mineralization, which is proposed in patent CN107115565A, can perform biomimetic induced mineralization on the surfaces of various substrates, but the method still cannot perform mineralization on the surface of a polyetheretherketone substrate.

Therefore, the invention is especially provided.

Disclosure of Invention

The invention aims to provide a simple, efficient, mild and long-term stable method for modifying the surface of a PEEK base material so as to improve the bioactivity and osseointegration performance of the surface of the PEEK base material.

In order to achieve the aim, the invention provides a method for improving the surface bioactivity and the osseointegration performance of a polyether-ether-ketone substrate, which sequentially comprises the following steps:

(1) soaking the polyether-ether-ketone substrate in a mixed solution prepared from a protein solution and a disulfide bond reducing agent solution in a ratio of 1: 10-10: 1 to obtain a nano-film modified polyether-ether-ketone substrate;

(2) and (2) soaking the polyether-ether-ketone base material modified by the nano film obtained in the step (1) in a calcium-phosphorus solution, and carrying out closed incubation to obtain the polyether-ether-ketone base material modified by the hydroxyapatite coating.

Preferably or alternatively, the protein in the protein solution in step (1) is any one or more of bovine serum albumin, human serum albumin, whey albumin, insulin, alpha-lactalbumin, fibrinogen, beta-lactoglobulin, ribonuclease A, cytochrome c, alpha-amylase, pepsin, myoglobin, albumin, collagen, keratin, soy protein, lactoferrin, hemoglobin, DNA polymerase, casein, lysozyme.

Preferably or alternatively, the concentration of protein in the protein solution in step (1) is 0.05-500 mg/mL.

Preferably or optionally, the disulfide bond reducing agent in step (1) is any one or more of dithiothreitol, beta-mercaptoethanol, tris (2-carboxyethyl) phosphine hydrochloride, cysteine, and reduced glutathione.

Preferably or alternatively, the concentration of the disulfide bond reducing agent in step (1) is from 0.05 to 600 mM.

Preferably or alternatively, the soaking time of the polyetheretherketone substrate in step (1) is 1-24 h.

Preferably or alternatively, the calcium-phosphorus solution in the step (2) is formed by mixing a solution 1 and a solution 2 in a volume ratio of 1:1, wherein the solution 1 comprises 20mM HEPES and 5mM Ca ²⁺ (ii) a The solution 2 included 20mM HEPES, 3mM PO ₄ ^3- ，30PPm NaF。

Preferably or alternatively, the incubation period in step (2) is 3 to 5 days.

Preferably or alternatively, the incubation temperature in step (2) is 25-80 ℃.

The method for improving the bioactivity and the osseointegration performance of the PEEK base material surface provided by the invention has the following advantages:

the invention provides a specific and feasible method for modifying the surface of a PEEK base material by adopting a hydroxyapatite/protein two-dimensional nano film composite coating, the composite coating has good mechanical stability, and also well improves the biocompatibility, osteoinduction performance and osteoconduction performance of the PEEK base material, so that the modified PEEK base material can be used as a tissue repair material to induce the formation of new tissues and the mineralization of bones, and is expected to become a novel hard tissue substitute material.

Description of the drawings:

FIG. 1 is a SEM representation of a hydroxyapatite coating prepared on the surface of a PEEK substrate in example 2;

FIG. 2 is a SEM characterized hydroxyapatite coating prepared on the surface of a PEEK substrate in example 5;

FIG. 3 is a SEM representation of a hydroxyapatite coating prepared on the surface of a PEEK substrate in example 6;

FIG. 4 is a SEM characterized hydroxyapatite coating prepared on the surface of a PEEK substrate in example 8;

FIG. 5 is a SEM characterized hydroxyapatite hybrid coating prepared on the surface of a PEEK substrate in example 15;

FIG. 6 is a SEM characterized hydroxyapatite coating prepared on the surface of a PEEK substrate in example 18;

FIG. 7 is a SEM characterized hydroxyapatite coating prepared on the surface of a PEEK substrate in example 19;

FIG. 8 is a SEM characterized hydroxyapatite coating prepared on the surface of a PEEK substrate in example 27;

FIG. 9 is a comparative graph of the surface topography of SEM-characterized protein two-dimensional nanofilm-modified PEEK substrates after soaking in the mineralization solutions described in example 1 and comparative example 1 for different periods of time;

FIG. 10 is a SEM representation of mineralized coatings on the surfaces of two substrates, namely an intervertebral fusion cage made of PEEK/carbon fiber composite material and a bone nail made of PEEK material;

FIG. 11 is an X-ray diffraction spectrum of a HAp-modified PEEK substrate prepared in example 1;

FIG. 12 is an infrared spectrum of a protein two-dimensional Nanoflayer (PTL), HAp-modified PEEK substrate prepared in example 1;

FIG. 13 is a Raman spectrum of Native Lysozyme, PTL, HAp modified PEEK substrate prepared in example 1;

FIG. 14 is an energy spectrum of a HAp-modified PEEK substrate material prepared in example 1;

FIG. 15 is a transmission electron micrograph of HAp on the surface of a PEEK substrate modified with HAp prepared in example 1;

FIG. 16 is a high resolution TEM image of the HAp-modified PEEK substrate surface prepared in example 1;

FIG. 17 is a scanning electron micrograph of the HAp-modified PEEK substrate prepared in example 1 before and after being peeled off with 3M tape and subjected to water bath ultrasonic treatment;

FIG. 18 is a graph showing the results of the cell activity assay (CCK-8 test) of the HAp-modified PEEK substrate prepared in example 1;

FIG. 19 is a cytoimmunofluorescence staining of the surface of the HAp-modified PEEK substrate prepared in example 1;

FIG. 20 is a Micro-ct scan image of the HAp-modified PEEK substrate prepared in example 1 after cranial defect repair in SD rats;

fig. 21 is a hematoxylin-eosin (HE) stain image and Massson stain image of tissue sections after the HAp-modified PEEK substrate prepared in example 1 was subjected to skull defect repair in rats.

Detailed Description

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

Example 1

The embodiment of the invention provides a method for improving the surface bioactivity and the osseointegration performance of a PEEK base material.

Adjusting the pH value of a tris (2-carboxyethyl) phosphine solution with the concentration of 50mmol/L to 4-6 by using NaOH, and mixing the tris (2-carboxyethyl) phosphine solution with the concentration of 2mg/mL and a bovine serum albumin solution according to the proportion of 1: 10-10: 1 to prepare a mixed solution. It should be noted that the concentration of tris (2-carboxyethyl) phosphine in the tris (2-carboxyethyl) phosphine solution can be any concentration within the range of 0.05-600mM, the concentration of bovine serum albumin in the bovine serum albumin solution can be any concentration within the range of 0.05-500mg/mL, the higher the concentration of each reactant, the thicker the protein two-dimensional nano-film is, and the too low protein concentration can make the protein two-dimensional nano-film not compact.

And (3) soaking the PEEK base material with the specification of 1cm multiplied by 0.1cm in the mixed solution, and standing for 12 hours at room temperature to obtain the PEEK base material modified by the protein two-dimensional nano film.

Washing PEEK substrate modified by protein two-dimensional nano film with ultrapure water, and completely immersing in Ca with concentration of 3-5mM ²⁺ ,1.8-3mM PO ₄ ^3- _, And 3-30PPm NaF in 20mM HEPES calcium phosphorus solution, incubating for three days under sealed condition at 37 ℃, taking out, cleaning and drying to obtain the PEEK base material modified by the Hydroxyapatite (HAP) coating.

The ratio of the calcium ion concentration to the phosphate concentration in the calcium-phosphorus solution of this example was 1.67: 1.

Example 2

The invention provides a method for improving the surface bioactivity and the osseointegration performance of a PEEK base material.

The steps of the embodiment are basically the same as those of embodiment 1, except that a mixed solution used in preparing the PEEK base material modified by the protein two-dimensional nano film is formed by mixing 2mg/mL lysozyme and 7mg/mL cysteine in a ratio of 1: 10-10: 1, and the standing time is 1 h.

The hydroxyapatite coating on the surface of the PEEK substrate prepared in this example is characterized by SEM as shown in fig. 1.

Example 3

The steps of the embodiment are basically the same as those of the embodiment 1, except that the mixed solution used for preparing the PEEK base material modified by the protein two-dimensional nano film is formed by mixing 2mg/mL lysozyme and 50mM TCEP in a ratio of 1: 10-10: 1, and the standing time is 1 h.

Example 4

The steps of the embodiment are basically the same as those of the embodiment 1, except that the mixed solution used for preparing the PEEK base material modified by the protein two-dimensional nano film is formed by mixing 2mg/mL lysozyme and 10mg/mL reductive glutathione in a ratio of 1: 10-10: 1, and the standing time is 12 hours.

Example 5

The steps of the embodiment are basically the same as those of the embodiment 1, except that the mixed solution used for preparing the PEEK base material modified by the protein two-dimensional nano film is prepared by mixing 2mg/mL fibrinogen and 2mg/mL beta-mercaptoethanol in a ratio of 1: 10-10: 1, and the standing time is 1 h.

The hydroxyapatite coating on the surface of the PEEK substrate prepared in this example is characterized by SEM as shown in fig. 2.

Example 6

The procedure of this example is basically the same as example 1, except that the mixture used in the preparation of the PEEK substrate modified by the two-dimensional protein nano-film is a mixture of myoglobin 2mg/mL and reduced glutathione 10mg/mL in a ratio of 1:10 to 10:1, and the standing time is 2 hours.

The hydroxyapatite coating on the surface of the PEEK substrate prepared in this example is characterized by SEM as shown in fig. 3.

Example 7

The steps of the embodiment are basically the same as those of embodiment 1, except that the mixed solution used for preparing the PEEK substrate modified by the protein two-dimensional nano film is formed by mixing 2mg/mL lysozyme and 2mg/mL dithiothreitol in a ratio of 1: 10-10: 1, and the standing time is 1 h.

Example 8

The procedure of this example is substantially the same as example 1, except that the mixture used in the preparation of the PEEK substrate modified by the two-dimensional protein nanofilm is 2mg/mL of insulin and 50mM of TCEP mixed at a ratio of 1:10 to 10:1, and the standing time is 1 hour.

The hydroxyapatite coating on the surface of the PEEK substrate prepared in this example is characterized by SEM as shown in fig. 4.

Example 9

The procedure of this example is basically the same as example 1, except that the mixture used in the preparation of the PEEK substrate modified with the two-dimensional protein nanofilm is prepared by mixing 2mg/mL of cytochrome C and 10mg/mL of glutathione at a ratio of 1:10 to 10:1, and the standing time is 1 hour.

Example 10

The procedure of this example is substantially the same as example 1, except that the mixture used in the preparation of the PEEK substrate modified by the two-dimensional protein nano-film is 2mg/mL bovine serum albumin and 10mg/mL cysteine mixed at a ratio of 1: 10-10: 1, and the standing time is 12 hours.

Example 11

The procedure of this example is basically the same as example 1, except that the mixed solution used in the preparation of the PEEK substrate modified by the protein two-dimensional nano-film is prepared by mixing 2mg/mL fibrinogen and 10mg/mL cysteine at a ratio of 1: 10-10: 1, and the standing time is 12 hours.

Example 12

The steps of the embodiment are basically the same as those of the embodiment 1, except that the mixed solution used for preparing the PEEK base material modified by the protein two-dimensional nano film is formed by mixing 2mg/mL of insulin and 10mg/mL of cysteine in a ratio of 1: 10-10: 1, and the standing time is 6 hours.

Example 13

The procedure of this example is basically the same as example 1, except that the mixed solution used in the preparation of the PEEK substrate modified by the two-dimensional protein nano-film is prepared by mixing 2mg/mL of insulin and 10mg/mL of glutathione at a ratio of 1:10 to 10:1, and the standing time is 1 hour.

Example 14

The steps of the embodiment are basically the same as those of embodiment 1, except that the mixed solution used for preparing the PEEK base material modified by the protein two-dimensional nano film is formed by mixing 2mg/mL insulin and 10mg/mL dithiothreitol in a ratio of 1: 10-10: 1, and the standing time is 1 h.

Example 15

The procedure of this example is substantially the same as that of example 1, except that a mixture of 2mg/mL lactoferrin and 50mM TCEP at a ratio of 1:10 to 10:1 is used in the preparation of the PEEK substrate modified with the protein two-dimensional nano film, and the standing time is 2 hours.

The hydroxyapatite coating on the surface of the PEEK substrate prepared in this example is characterized by SEM as shown in fig. 5.

Example 16

The steps of the embodiment are basically the same as those of the embodiment 1, except that the mixed solution used for preparing the PEEK base material modified by the protein two-dimensional nano film is prepared by mixing 2mg/mL alpha-amylase and 10mg/mL cysteine in a ratio of 1: 10-10: 1, and the standing time is 2 hours.

Example 17

The steps of the embodiment are basically the same as those of the embodiment 1, except that the mixed solution used for preparing the PEEK base material modified by the protein two-dimensional nano film is formed by mixing 2mg/mL ribonuclease A and 10mg/mL glutathione in a ratio of 1: 10-10: 1, and the standing time is 2 hours.

Example 18

The procedure of this example is substantially the same as example 1, except that the mixture used in the preparation of the PEEK substrate modified with the two-dimensional protein nanofilm was 2mg/mL human serum albumin and 50mM TCEP in a ratio of 1:10 to 10:1, and the standing time was 2 hours.

The hydroxyapatite coating on the surface of the PEEK substrate prepared in this example is characterized by SEM as shown in fig. 6.

Example 19

The procedure of this example is basically the same as example 1, except that the mixture used in the preparation of the PEEK substrate modified by the two-dimensional protein nano-film is 2mg/mL β -lactoglobulin and 50mM TCEP mixed at a ratio of 1:10 to 10:1, and the standing time is 2 hours.

The hydroxyapatite coating on the surface of the PEEK substrate prepared in this example is characterized by SEM as shown in fig. 7.

Example 20

The procedure of this example is substantially the same as example 1, except that the mixture used in the preparation of the PEEK substrate modified by the two-dimensional protein nano-film is a mixture of myoglobin and 50mM TCEP in a ratio of 1:10 to 10:1, and the standing time is 2 hours.

Example 21

The procedure of this example is substantially the same as example 1, except that the mixture used in the preparation of the PEEK substrate modified by the two-dimensional protein nano-film is 2mg/mL collagen and 50mM TCEP mixed at a ratio of 1:10 to 10:1, and the standing time is 2 hours.

Example 22

The procedure of this example is substantially the same as example 1, except that the mixture used in the preparation of the PEEK substrate modified by the two-dimensional protein nano-film is a mixture of 2mg/mL keratin and 50mM TCEP in a ratio of 1:10 to 10:1, and the standing time is 2 hours.

Example 23

The steps of the embodiment are basically the same as those of the embodiment 1, except that the mixed solution used for preparing the PEEK base material modified by the protein two-dimensional nano film is formed by mixing 2mg/mL of horseradish peroxidase and 50mM of TCEP in a ratio of 1: 10-10: 1, and the standing time is 2 hours.

Example 24

The procedure of this example is substantially the same as example 1, except that the mixture used in the preparation of the PEEK substrate modified by the two-dimensional protein nano-film is 2mg/mL of soy protein and 50mM of TCEP mixed at a ratio of 1:10 to 10:1, and the standing time is 2 hours.

Example 25

Example 26

The procedure of this example is substantially the same as that of example 1, except that the mixture used in the preparation of the PEEK substrate modified with the two-dimensional protein nanofilm is 2mg/mL casein and 50mM TCEP in a ratio of 1:10 to 10:1, and the standing time is 2 hours.

Example 27

The steps of the embodiment are basically the same as those of the embodiment 1, except that the mixed solution used for preparing the PEEK base material modified by the protein two-dimensional nano film is formed by mixing 2mg/mL lysozyme grafted with PEG and 50mM TCEP in a ratio of 1: 10-10: 1, and the standing time is 1 h.

The hydroxyapatite coating on the surface of the PEEK substrate prepared in this example is characterized by SEM as shown in fig. 8.

Comparative example 1

Adjusting the pH value of a tris (2-carboxyethyl) phosphine solution with the concentration of 50mmol/L to 5.8 by using NaOH, and mixing the solution with lysozyme with the concentration of 2mg/mL in a ratio of 1: 10-10: 1 to prepare a mixed solution.

The PEEK base material with the specification of 1cm multiplied by 0.1cm is soaked in the mixed liquid and stands for 12 hours at room temperature.

The substrate is fished out, washed by ultrapure water, and then completely immersed in 0.02mol/L calcium chloride aqueous solution, and immersed for 48 hours at normal temperature, and the calcium chloride aqueous solution is replaced every 12 hours.

Taking out the substrate again, cleaning with ultrapure water, drying with nitrogen, completely soaking in Simulated Body Fluid (SBF), incubating at 70 deg.C for 7 days under sealed condition, cleaning with ultrapure water after incubation, and vacuum drying at normal temperature.

Referring to fig. 9, HAp cannot induce mineralization and crystallization well on the surface of the PEEK substrate treated by the technical scheme in comparative example 1, and HAp can induce mineralization and crystallization well on the surface of the treated PEEK substrate by the technical scheme in example 1 of the present application.

Effect example 1

The samples obtained in examples 1 to 27 were characterized by an X-ray diffractometer, an infrared spectrometer, a raman spectrometer, a scanning electron microscope, an energy spectrometer, and a transmission electron microscope, and the results are shown in fig. 11 to 16.

The detection method of the X-ray diffractometer comprises the following steps: the surface-induced HAP mineralized coatings of PTL nanofilms were analyzed by powder x-ray diffraction (d8 Advance, Bruker, Cu target, 2.2kW,45kV,200mA) with a scan rate of 3 degrees/min.

The detection method of the infrared spectrometer comprises the following steps: FTIR spectra were obtained using a Vertex 70V spectrometer (Bruker Inc, Germany). FTIR spectra ranged from 400 to 4000cm-1 with a resolution of 1cm-1 using an Alpha-T spectrometer (Bruker) using KBr pellet method.

The detection method of the Raman spectrometer comprises the following steps: the scanning wave number is between 400 and 3000cm < -1 > by using a Raney Raman spectrometer (semiconductor laser 532,785 nm).

The scanning electron microscope detection method comprises the following steps: the FEI quan 200 was subjected to scanning electron microscopy testing. A field emission scanning electron microscope (FE-SEM) observation was performed on SU8020 (Hitachi). The sample was placed on a conductive adhesive and gold sprayed. Cross-section of the sample was wetted brittle in liquid nitrogen and the cross-section of the sample was examined using a field emission scanning electron microscope to obtain an energy dispersive x-ray (EDX) spectrum.

The transmission electron microscope detection method comprises the following steps: the sample was lyophilized with liquid nitrogen and dispersed with absolute ethanol. Then, the alcohol solution containing the HAp powder on the sample surface was transferred to a commercially available porous carbon-coated copper mesh surface and allowed to dry naturally for TEM measurement. The transient electromagnetic method test of 200kV was carried out on JEM-2100(JEOL Ltd). A field emission Transmission Electron microscopy (FE-TEM) study was performed at 200kV for FEI Tecnai G2F 20. The prepared TEM copper grids with HAp samples were placed in a desiccator prior to TEM testing to ensure moisture removal.

The X-ray diffraction analysis of fig. 11 shows that the crystals grown on the surface of the protein two-dimensional nano-film on the surface of the PEEK substrate are hydroxyapatite and not other calcium phosphate.

In the IR spectrum of FIG. 12, the protein is two-dimensional nanoAmide bonds of the films appeared at 1536 and 1670cm ^-1 The prepared mineralized coating material is at 566, 602 and 1034cm ^-1 A new peak appears, mainly the bending vibration of the O-P-O bond.

In the Raman spectrum of FIG. 13, several new peaks, mainly at 586cm, appear comparing with the original, non-grown crystal, protein two-dimensional nano-film ^-1 Bending vibration of O-P-O bond and 962cm ^-1 P-O stretching vibration of 962cm ^-1 The peak at (b) is the most representative sign of HAp crystallization.

The energy spectrum data in FIG. 14 also demonstrate that the Ca/P ratio of the induced Hap of the two-dimensional nano-film protein in the example of the present invention is 1.67, which is similar to the composition of natural bone.

As can be seen from fig. 15 and 16, after incubation, a layer of uniform and supported hydroxyapatite aggregate is formed on the surface of the PEEK substrate, the hydroxyapatite aggregate is formed by needle-shaped nano crystal clusters which are arranged along the axial direction, the structure of the hydroxyapatite vertically arranged is similar to the appearance of the hydroxyapatite of natural bones and teeth, and electron diffraction also confirms that the nano hydroxyapatite has typical (002), (210) and (211) crystal faces, wherein the (002) crystal face shows that the hydroxyapatite crystal induced by the BSA two-dimensional nano film grows directionally mainly along the c axis. The above characterization results prove that the components and the structure of the hydroxyapatite induced and mineralized by the lysozyme two-dimensional nano film are similar to those of natural bone tissues.

Effect example 2

The mechanical properties of the HAp-modified PEEK substrate obtained in example 1 were tested.

The HAp-modified PEEK base material obtained in example 1 was subjected to water bath ultrasonic treatment for 1 hour under conditions of 50kHz and 200W.

After the ultrasonic treatment is finished, the HAp attachment rate is measured, the measurement method is to obtain a surface SEM image of a sample and then analyze and calculate by using imageJ software, and the result shows that 98.34 percent of HAp can still be stably attached to the surface of the base material.

The HAp-modified PEEK substrate obtained in example 1 was subjected to a peel test using 3M Scotch tape.

The specific test method comprises the following steps: the sample was adhered to the surface using a commercial 3M scotch tape, and a 1Kg weight was loaded thereon, left for 5min, and then torn. SEM images of the sample surface after tearing were determined and calculated by analysis using imageJ software.

Test results show that the bonding strength of the HAp layer on the surface of the PEEK base material is higher than 1.23N cm ^-1 98.13 percent of HAp can be stably attached to the surface of the base material after being torn.

Fig. 17 is a scanning electron microscope image of the HAp-modified PEEK substrate before and after the 3M tape tearing test and the water bath ultrasonic treatment in the working example.

The results show that the HAp modified PEEK base material provided by the invention has good interface bonding stability.

Effect example 3

By adopting the technical scheme in the embodiment 1, the HAp modified PEEK base material is prepared.

HAp modified PEEK and blank PEEK substrates were sterilized by uv irradiation and 75% ethanol. After sterilization, the solution was washed repeatedly with PBS buffer solution having pH of 7.4 to remove ethanol remaining on the surface. Each sample was then plated in 24-well plates at 1X 10 ⁴ Bone marrow mesenchymal stem cells (rBMSCs) were seeded at a density of one/cm 2. After inoculation, the 24-well culture plate is placed at 37 ℃ and 5% CO ₂ The culture medium is replaced every two days. The proliferation of the cells was measured at 1, 3, 5 and 7 days of culture. The old medium in the well plate was aspirated at each time point with a sterile pipette, and then gently rinsed with PBS buffer, 900. mu.L of fresh medium and 90. mu.L of CCK-8 solution were added to each well of a 24-well plate, respectively, and the plate was left at 37 ℃ with 5% CO ₂ After culturing for 2 hours in the incubator, the culture medium was aspirated. After the aspirated culture solution was shaken well, the absorbance of the culture solution at 450nm was measured by spectrophotometry using a microplate reader (Bio-Rad 680). All samples were assayed in duplicate 3 times and the results averaged.

As can be seen from fig. 18, the optical density values of cells on the three materials did not significantly change after 1 day of culture, and there was no significant difference in OD values between the three materials. However, by day 5 and day 7, although the cellular absorbance values of the surfaces of the three materials are remarkably improved compared with that of day 1, the cellular optical density of the surface of the HAp modified PEEK base material is obviously higher than that of the blank PEEK base material.

The above results show that cell proliferation on the PEEK substrate surface is more favored by inducing a mineralized HAp coating on the surface.

Effect example 4

HAp-modified PEEK substrates were sterilized by uv irradiation and 75% ethanol. After sterilization, the solution was washed repeatedly with PBS buffer solution having pH of 7.4 to remove ethanol remaining on the surface. Then, the cells were planted in 24-well plates at 3X 10 ⁴ The mesenchymal stem cells are inoculated at the density of 2 per cm.

The cytoskeleton is stained by rhodamine-labeled phalloidin, the cell nucleus is stained by DAPI, and then the cell adhesion and extension conditions of the surface of each sample after 24h of culture are observed by an inverted fluorescence microscope, and the result is shown in FIG. 19.

As can be seen from fig. 19, the number of mesenchymal stem cells on the surface of the HAp-modified PEEK substrate is significantly increased, the number of filamentous pseudo-feet of the cells is also significantly increased, and the mesenchymal stem cells are connected into a sheet, which indicates that the HAp-modified PEEK substrate can better support the in vitro culture of the mesenchymal stem cells, has good cell compatibility, and can promote the proliferation and differentiation of the bone cells.

Effect example 5

HAp modified PEEK and blank PEEK substrates were sterilized by uv irradiation and 75% ethanol. After sterilization, the solution was washed repeatedly with PBS buffer solution having pH of 7.4 to remove ethanol remaining on the surface.

Each sample was individually restrained to the skull region of rats, and after 4 weeks and 12 weeks of implantation, the entire skull with the sample was removed and soaked in 4% paraformaldehyde solution overnight. And then carrying out Micro-CT scanning, and placing the probe in EDTA decalcification liquid for decalcification after the scanning is finished until the pin passes through the probe without resistance. The Micro-CT scan results are shown in FIG. 20.

After completion of decalcification, the skull was subjected to a conventional tissue section with a microtome, and after the tissue section was subjected to hematoxylin-eosin staining and Masson staining, it was observed under an optical microscope and photographed, and the results are shown in fig. 21.

The above results demonstrate that HAp modified PEEK substrates exhibit good tissue regeneration performance after implantation. A large amount of new bones and fibrous tissues are formed at the edge of the HAp modified PEEK base material, and the generated new bones are similar to host bones in shape.

Further, Micro-CT scans were combined with the two staining described above, and the implanted HAp-modified PEEK substrate was found to have significant new bone staining. Indicating that the collagen maturation degree in the bone tissue and the new bone ability are obviously improved within 12 weeks after the operation.

In conclusion, the HAp modified PEEK base material can well promote the proliferation and differentiation of osteoblasts and further realize the repair of bone defects.

The method for improving the surface bioactivity and the osseointegration performance of the polyether-ether-ketone base material overcomes the problem that the traditional method is difficult to form a protein two-dimensional nano-film on the surface of the polyether-ether-ketone base material, so that HAp modification cannot be further carried out. Meanwhile, the final product HAp modified PEEK also has good mechanical properties, biocompatibility and osteointegration capability, and has good market prospect.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Claims

1. A method for improving the surface bioactivity and osseointegration performance of a polyether-ether-ketone substrate is characterized by comprising the following steps in sequence:

2. The method for improving surface bioactivity and osseointegration performance of PEEK substrate according to claim 1, wherein the protein in the protein solution of step (1) is any one or more of bovine serum albumin, human serum albumin, whey albumin, insulin, alpha-lactalbumin, fibrinogen, beta-lactoglobulin, ribonuclease A, cytochrome c, alpha-amylase, pepsin, myoglobin, albumin, collagen, keratin, soy protein, lactoferrin, hemoglobin, DNA polymerase, casein, and lysozyme.

3. The method for improving surface bioactivity and osseointegration performance of a PEEK substrate according to claim 1, wherein the protein concentration in the protein solution in step (1) is 0.05-500 mg/mL.

4. The method for improving surface bioactivity and osseointegration performance of a polyetheretherketone substrate according to claim 1, wherein the disulfide bond reducing agent in step (1) is any one or more of dithiothreitol, β -mercaptoethanol, tris (2-carboxyethyl) phosphine hydrochloride, cysteine, and reduced glutathione.

5. The method for improving surface bioactivity and osseointegration properties of a polyetheretherketone substrate according to claim 1, wherein the concentration of the disulfide bond reducing agent in step (1) is 0.05 to 600 mM.

6. The method for improving surface bioactivity and osseointegration performance of a PEEK substrate according to claim 1, wherein the soaking time of the PEEK substrate in step (1) is 1-24 h.

7. The method for improving surface bioactivity and osseointegration performance of a PEEK substrate of claim 1, wherein the Ca/P solution of step (2) is Ca at a concentration of 3-5mM ²⁺ ,1.8-3mM PO ₄ ^3- And 3-30PPm NaF in 20mM HEPES.

8. The method for improving surface bioactivity and osseointegration properties of a PEEK substrate of claim 7, wherein Ca is present in the Ca/P solution ²⁺ And PO ₄ ^3- The concentration ratio of (A) to (B) is 1.67: 1.

9. The method for improving surface bioactivity and osseointegration performance of a polyetheretherketone substrate according to claim 1, wherein the incubation period in step (2) is 3-5 days.

10. The method for improving surface bioactivity and osseointegration performance of a polyetheretherketone substrate according to claim 1, wherein the incubation temperature in step (2) is 25-80 ℃.